SELF INSTRUCTIONAL MATERIAL
M.Sc. PREVIOUS (BOTANY)
PAPER –I CELL & MOLECULAR
BIOLOGY OF PLANTS
BLOCK -3
UNIT – 4
UNIT - 5
MADHYA PRADESH BHOJ ( OPEN)
UNIVERSITY, BHOPAL
1
UNIT- IV
Protein sorting , Cell shape and motility
4.0
Introduction of protein sorting
4.1
Objective
4.2
Protein translocation
4.3
Cell shape
Cytoskeleton
4.4
Flagella and motility
1.4.1
Flagellar ultra-structure
1.4.2
Mechanism of flagellar movement
4.5
Cell cycle and Apoptosis
4.6
Role of Cyclins and CDK
4.7
Retinoblastoma and E2F Protein
4.8
Cytokinesis
4.9
Cell plate formation
4.10
Programmed cell death
4.11
Let us sum up
4.12
Assignment/ Activity
4.13
References
2
4.0
Introduction of protein sorting
Protein targeting or protein sorting is the mechanism by which a cell transports proteins to the
appropriate positions in the cell or outside of it. Sorting targets can be the inner space of an
organelle, any of several interior membranes, the cell's outer membrane, or its exterior via
secretion. This delivery process is carried out based on information contained in the protein
itself. Correct sorting is crucial for the cell; errors can lead to diseases.
Targeting signals are the pieces of information that enable the cellular transport machinery to
correctly position a protein inside or outside the cell. This information is contained in the
polypeptide chain or in the folded protein. The continuous stretch of amino acid residues in
the chain that enables targeting are called signal peptides or targeting peptides. There are two
types of targeting peptides, the pre-sequences and the internal targeting peptides. The presequences of the targeting peptide are often found at the N-terminal extension and is
composed of between 6-136 basic and hydrophobic amino acids. In case of peroxisomes the
targeting sequence is on the C-terminal extension mostly. Other signals are composed by
parts which are separate in the primary sequence. To function these components have to
come together on the protein surface by folding. They are called signal patches. In addition,
protein modifications like glycosylation can induce targeting.
4.1
Objective
1.
This unit focus on molecular control of the cell cycle in higher plants and do
not deal with the developmental and environmental control of cell division.
2.
4.2
This unit emphasizes on the importance of protein targeting.
Protein translocation
In 1970, Günter Blobel conducted experiments on the translocation of proteins across
membranes. He was awarded the 1999 Nobel prize for his findings. He discovered that many
proteins have a signal sequence, that is, a short amino acid sequence at one end that functions
like a postal code for the target organelle. The translation of mRNA into protein by a
ribosome takes place within the cytosol. If the synthesized proteins "belong" in a different
organelle, they can be transported there in either of two ways, depending on the protein.
3
Co-translational translocation
The N-terminal signal sequence of the protein is recognized by a signal recognition particle
(SRP) while the protein is still being synthesized on the ribosome. The synthesis pauses while
the ribosome-protein complex is transferred to an SRP receptor on the endoplasmic reticulum
(ER), a membrane-enclosed organelle. There, the nascent protein is inserted into the
translocation complex that passes through the ER membrane. The signal sequence is
immediately cleaved from the polypeptide once it has been translocated into the ER by signal
peptidase in secretory proteins. This signal sequence processing differs for some ER
transmembrane proteins. Within the ER, the protein is first covered by another protein to
protect it from the high concentration of other proteins in the ER, giving it time to fold
correctly. Once folded, the protein is modified as needed (for example, by glycosylation),
then transported to the Golgi apparatus for further processing and goes to its target organelles
or is retained in the ER by various ER retention mechanisms.
Post translational translocation
Even though most proteins are co-translationally translocated, some are translated in the
cytosol and later transported to their destination. This occurs for proteins that go to a
mitochondrion, a chloroplast, or a peroxisome (proteins that go to the latter have their signal
sequence at the C terminus). Also, proteins targeted for the nucleus are translocated posttranslation. They pass through the nuclear envelope via nuclear pores.
Sorting of proteins to mitochondria
Most mitochondrial proteins are synthesized as cytosolic precursors containing uptake
peptide signals. The pre-protein with pre-sequence targeted for the mitochondria is bound by
receptors and the General Import Pore (GIP) (Receptors and GIP are collectively known as
Translocase of Outer Membrane or TOM) at the outer membrane. The pre-protein is
translocated through TOM as hairpin loops. The pre-protein is transported through the
intermembrane space by small TIMs (which also acts as molecular chaperones) to the TIM23
or 22 (Translocase of Inner Membrane) at the inner membrane. Within the matrix the
targeting sequence is cleaved off by mtHsp70.
Three mitochondrial outer membrane receptors are known: TOM20, TOM22 and TOM70
The pre-sequence translocase23 (TIM23) is localized to the mitochondrial inner membrane
and acts a pore forming protein which binds precursor proteins with its N-terminal. TIM23
4
acts a translocator for pre-proteins for the mitochondrial matrix, the inner mitochondrial
membrane as well as for the intermembrane space. TIM50 is bound to TIM23 at the inner
mitochondrial site and found to bind pre-sequences.
Mitochondrial matrix targeting sequences are rich in positively charged amino acids and
hydroxylated ones. Proteins are targeted to sub-mitochondrial compartments by multiple
signals and several pathways. Targeting to the outer membrane, intermembrane space, and
inner membrane often requires another signal sequence in addition to the matrix targeting
sequence.
Sorting of proteins to chloroplasts
The pre-protein for chloroplasts contain a stromal import sequence or a stromal and thylakoid
targeting sequence. The majority of pre-proteins are translocated through the Toc and Tic
complexes located within the chloroplast envelope. In the stroma the stromal import sequence
is cleaved off and intra-chloroplast sorting and folding continues.
Sorting of proteins to both chloroplasts and mitochondria
Many proteins are needed in both mitochondria and chloroplasts. In general the targeting
peptide is of intermediate character to the two specific ones. The targeting peptides of these
proteins have a high content of basic and hydrophobic amino acids, a low content of
negatively charged amino acids. They have a lower content of alanine and a higher content of
leucine and phenylalanine. The dual targeted proteins have a more hydrophobic targeting
peptide than both mitochondrial and chloroplastic ones.
Protein destruction
Defective proteins are occasionally produced, or they may be damaged later, for example, by
oxidative stress. Damaged proteins can be recycled. Proteins can have very different half life,
mainly depending on their N-terminal amino acid residue. The recycling mechanism is
mediated by ubiquitin.
4.3
Cell shape
The Cytoskeleton.
5
One distinguishing feature that separates the eukaryotes from prokaryotes is the presence of
the cytoskeleton in the former, and apparent absence of it in the latter. This suggests that the
cytoskeleton was an important factor in eukaryote evolution. The function of the cytoskeleton
is to maintain cellular shape, and is involved in intracellular organization, cell polarity, cell
adhesion, and in some cases, motility.
The cytoskeleton is composed of several different protein components. There are three
general classes of cytoskeletal fibers: (1) microtubules, (2) intermediate filaments, and (3)
actin filaments.
a)
Microtubules
Compared to the other cytoskeletal fibers, the microtubule is rather large (15 to 35 nm
diameter). Microtubules are composed of a globular protein, tubulin. The tubulin subunit is a
heterodimer of alpha- and beta-tubulin . The microtubule itself is made up of 13 "protofilaments", which are each composed of alternating alpha and beta subunits. These
protofilaments are cylindrically arranged to form a hollow tube. It is the arrangement of
proto-filaments that makes up the microtubule. Microtubules are polar molecules i.e. they
have a fast growing "plus end" and a slow growing "minus end". These strands are in a
constant state of flux, termed "dynamic instability" (i.e. they continuously grow and fall
apart). Free GTP (guanosine triphosphate) binds to the beta-tubulin, which alters its protein
structure and enables it to join to the growing end of the strand. Then, there is a delayed GTP
hydrolysis reaction to yield GDP (guanosine diphosphate). Thus, at the terminus of the
filament a GTP cap is present (i.e. tubulin subunits are bound to GTP), while further down
the strand, tubulin subunits are bound to GDP. The presence of GDP creates a weak bond
between tubulin subunits, and therefore the filament is more likely to de-polymerize. In other
words, if all the GTP is hydrolyzed in the filament, the filament is susceptible to falling apart
(i.e. under a microscope, the microtubules are observed to shrink or even disappear). There
are various proteins that are able to stabilize the microtubule, preventing de-polymerization
these are known as "cap-proteins" or MAPs (Microtubule-Associated Proteins). There are
also various other chemicals that can either stabilize or destabilize microtubules
6
Another group of cytoskeletal proteins are the intermediate filaments (IFs). IFs have an
intermediate size between microtubules and actin filaments (7 to 10 nm diameter). There are,
however, two general types of IFs: (1) cytoplasmic IFs, and (2) nuclear lamina. Cytoplasmic
IFs are for mechanical stress and cell-to-cell junctions. Nuclear lamina create a meshwork
beneath the inner nuclear membrane. One important difference to note between IF proteins
and proteins for microtubules or for microfilaments, is that most IF protein subunits are
filamentous, rather than globular (i.e. tubulin and actin subunits are globular).
b)
Microfilaments
Actin filaments (or "microfilaments" these terms are used interchangeably) are the smallest
of the cytoskeletal fibers (3 to 6 nm diameter). Microfilaments are flexible doubled-stranded
fibers composed of polymers of the protein actin (contrast this structure to microtubules,
which are hollow tubes composed of 13 protofilaments). Actin is present in all eukaryotes,
and microfilaments are typically found in the cell cortex (i.e. just beneath the cell membrane).
The actin subunit is globular and has a molecular mass of 43 kDa. As with microtubules,
actin filaments are also dynamic and polar molecules (i.e. they have a fast growing "plus end"
and a slow growing "minus end"). Free actin binds ATP (adenosine-triphosphate, as opposed
to GTP in tubulin subunits) which enables polymerization, while ATP hydrolysis to ADP
favours depolymerization. Unlike microtubules which undergo dynamic instability, actin
filaments may use a different dynamic process.
7
Actin Binding Proteins.
The actin-based cytoskeleton functions for bearing of tension and for compression resistance
(i.e. it is like a shock suspension system, which gives mechanical strength and maintains
structural integrity of a cell). In fact, there are different microfilament arrangements for a
variety of functional purposes. There are, however, two general classes of microfilament
arrangement: (1) bundles (parallel and contractile), and (2) gel-like networks. Different
proteins (i.e. actin-binding proteins) mediate the different arrangements. Parallel bundles are
structures where microfilaments are oriented with the same polarity (i.e. plus ends are all
"pointed" in the same direction) and which are closely spaced.
8
4.4
Flagella and Motility
Most motile move by use of flagella (flagellum) thread like locomotor appendages extending
outward from the plasma membrane and cell wall. They are slender, rigid structures, about 20
nm across and up to 15 or 20 pm long. Flagella are so thin they cannot be observed directly
with a bright-field microscope, but must be stained with special techniques designed to
increase their thickness. The detailed structure of a flagellum can only be seen in the electron
microscope.
Bacterial species often differ distinctively in their patterns of flagella distribution.
Monotrichous bacteria (trichous means hair) have one flagellum; if it is located at an end, it is
said to be a polar flagellum. Amphitrichous bacteria (amphi mean "on both sides") have a
single flagellum at each pole. In contrast, lophotrichous bacteria (lopho means tuft) have a
cluster of flagella at one or both ends. Flagella are spread fairly evenly over the whole
surface of peritrichours (peri means "around") bacteria. Flagellation patterns are very useful
in identifying bacteria.
4.4.1 Flagellar Ultra structure : Transmission electron microscope studies have shown that the bacterial flagellum is
composed of three parts. (1) The longest and most obvious portion is the filament, which
extends from the cell surface to the tip. (2) A basal body is embedded in the cell; and (3) a
9
short, curved segment, the hook, links the filament to its basal body and acts as a flexible
coupling. The filament is a hollow, rigid cylinder constructed of a single protein celled
flagellin, which ranges in molecular weight from 30,000 to 60,000. The filament ends with a
capping protein. Some bacteria have sheaths surrounding their flagella. For example Vibrio
cholerae has a lipopolysaccharide sheath.
The hook and basal body are quite different from the filament of different protein subunits.
The basal body is the most complex part of a flagellum. In E.coli and most gram-negative
bacteria, the body has four rings connected to a central rod. The outer L and P rings associate
with the lipopolysaccharide and peptidoglycan layers, respectively. The inner M ring contacts
the plasma membrane. Gram-positive bacteria have only two basal body rings, an inner ring
connected to the plasma membrane and an outer one probably attached to the peptidoglycan.
Flagellar Synthesis
The synthesis of flagella is a complex process involving at least 20 to 30 genes. Besides the
gene for flagellin, 10 or more genes code for hook and basal body proteins ; other genes are
concerned with the control of flagellar construction or function. It is not known how the cell
regulates or determines the exact location of flagella.
4.4.2 The Mechanism of Flagellar Movement
Procaryotic flagella operate differently from eukaryotic flagella. The filament is in the shape
of a rigid helix, and the bacterium moves when this helix rotates. Considerable evidence
shows that flagella act just like propellers on a boat. Bacterial mutants with straight flagella
or abnormally long hook regions (polyhook mutants) cannot swim. When bacteria are
tethered to a glass slide using antibodies to filament or hook proteins, the cell body rotates
rapidly about the stationary flagellum. If polystyrene-latex beads are attached to flagella, the
beads spin about the flagellar axis due to flagellar rotation. The flagellar motor can rotate
very rapidly. The E. coli motor rotates 270 revolutions per second ; Vibrio alginolyticus
averages 1,100 rps.
10
The direction of flagellar rotation determines the nature of bacterial movement.
Monotrichous, polar flagella rotate slowly clockwise. The rotating helical flagellar filament
thrusts the cell forward in a run with the flagellum trailing behind. Monotrichous bacteria
stop and tumble randomly by reversing the direction of flagellar rotation. Peritrichously
flagellated bacteria operate in a somewhat similar way to move forward, the flagella rotate
counterclockwise. As they do so, they bend at their hooks to form a rotating bundle that
propels them forward. Clockwise rotation of the flagella disrupts the bundle and the cell
tumbles.
Because bacteria swim though rotation of their rigid flagella, there must be some sort of
motor at the base. A rod or shaft extends from the hook and ends in the M ring, which can
rotate freely in the plasma membrane. It is believed that the S ring is attached to the cell wall
in gram positive cells and does not rotate . The P and L rings of gram negative bacteria would
act as bearings for the rotating rod. There is some evidence that the basal body is a passive
structure and rotates within a membrane embedded protein. The relationship of flagellar
rotation to bacterial movement. The rotor is like an electrical motor turns in the center of a
ring of electromagnets (the stator).
11
The exact mechanism that drives basal drives basal body rotation still is not clear
provides a more detailed depiction of the basal body in gram negative bacteria. The rotor
portion of the motor seems to be made primarily of a rod, the M ring, and C ring joined to it
on the cytoplasmic side of the basal body. These two rings are made of several proteins. The
two most important proteins in the stator part of the motor are Mot A and Mot B. These form
a proton channel thought the plasma membrane, and Mot B also anchors the Mot complex to
cell wall peptidoglycan. There is some evidence that Mot A and G directly interact during
flagellar rotation. This rotation is driven by proton or sodium gradients in prokaryotes, not
directly by ATP as is the case with eukaryotic flagella.
Bacteria can move by mechanisms other than falgellar rotations. Spriochetes are helical
bacteria that travel through viscous substances such as mucus or mud by flexing and spinning
movements caused by a special axial filament composed of periplasmic flagella. A very
different type of motility, gliding motility, is employed by many bacteria : cyanobacteria .
4.5
Cell cycle and apoptosis
The cell cycle, or cell-division cycle, is the series of events that take place in a eukaryotic cell
leading to its replication. These events can be divided in two brief periods: interphase during
which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA and
the mitotic (M) phase, during which the cell splits itself into two distinct cells, often called
"daughter cells". The cell-division cycle is a vital process by which a single-celled fertilized
egg develops into a mature organism, as well as the process by which hair, skin, blood cells,
and some internal organs are renewed.
Phases of the cell cycle
The cell cycle consists of four distinct phases: G1 phase, S phase, G2 phase (collectively
known as interphase) and M phase. M phase is itself composed of two tightly coupled
12
processes: mitosis, in which the cell's chromosomes are divided between the two daughter
cells, and cytokinesis, in which the cell's cytoplasm divides forming distinct cells. Activation
of each phase is dependent on the proper progression and completion of the previous one.
Cells that have temporarily or reversibly stopped dividing are said to have entered a state of
quiescence called G0 phase.
M phase
The relatively brief M phase consists of nuclear division (karyokinesis) and cytoplasmic
division (cytokinesis). In plants and algae, cytokinesis is accompanied by the formation of a
new cell wall. The M phase has been broken down into several distinct phases, sequentially
known as prophase, Prometaphase, metaphase, anaphase and telophase leading to cytokinesis.
Interphase
After M phase, the daughter cells each begin interphase of a new cycle. Although the various
stages of interphase are not usually morphologically distinguishable, each phase of the cell
cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation
of cell division.
G1 phase
The first phase within interphase, from the end of the previous M phase till the beginning of
DNA synthesis is called G1 (G indicating gap or growth). During this phase the biosynthetic
activities of the cell, which had been considerably slowed down during M phase, resume at a
high rate. This phase is marked by synthesis of various enzymes that are required in S phase,
mainly those needed for DNA replication. Duration of G1 is highly variable, even among
different cells of the same species.
S phase
The S phase starts when DNA synthesis commences; when it is complete, all of the
chromosomes have been replicated, i.e., each chromosome has two (sister) chromatids. Thus,
during this phase, the amount of DNA in the cell has effectively doubled, though the ploidy
of the cell remains the same. Rates of RNA transcription and protein synthesis are very low
during this phase. An exception to this is histone production, most of which occurs during the
S phase. The duration of S phase is relatively constant among cells of the same species.
13
G2 phase
The cell then enters the G2 phase, which lasts until the cell enters mitosis. Again, significant
protein synthesis occurs during this phase, mainly involving the production of microtubules,
which are required during the process of mitosis. Inhibition of protein synthesis during G2
phase prevents the cell from undergoing mitosis.
G0 phase
The term "post-mitotic" is sometimes used to refer to both quiescent and senescent cells.
Non-proliferative cells in multicellular eukaryotes generally enter the quiescent G0 state from
G1 and may remain quiescent for long periods of time, possibly indefinitely (as is often the
case for neurons). This is very common for cells that are fully differentiated. Cellular
senescence is a state that occurs in response to DNA damage or degradation that would make
a cell's progeny nonviable; it is often a biochemical alternative to the self-destruction of such
a damaged cell by apoptosis.
Cell cycle inhibitors
Two families of genes, the cip/kip family and the INK4a/ARF (Inhibitor of Kinase
4/Alternative Reading Frame) prevent the progression of the cell cycle. Because these genes
are instrumental in prevention of tumor formation, they are known as tumor suppressors.
The cip/kip family includes the genes p21, p27 and p57. They halt cell cycle in G1 phase, by
binding to, and inactivating, cyclin-CDK complexes. p21 is activated by p53 (which in turn is
triggered by DNA damage e.g. due to radiation). p27 is activated by Transforming Growth
Factor β (TGF β), a growth inhibitor. The INK4a/ARF family includes p16INK4a, which
binds to CDK4 and arrests the cell cycle in G1 phase, and p14arf which prevents p53
degradation. And the amount of chromosomes are able to double at the same rate as in phase
2
Checkpoints
Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell
cycle. Checkpoints prevent cell cycle progression at specific points, allowing verification of
necessary phase processes and repair of DNA damage. The cell cannot proceed to the next
phase until checkpoint requirements have been met.
14
Several checkpoints are designed to ensure that damaged or incomplete DNA is not passed on
to daughter cells. Two main checkpoints exist: the G1/S checkpoint and the G2/M
checkpoint. G1/S transition is a rate-limiting step in the cell cycle and is also known as
restriction point. An alternative model of the cell cycle response to DNA damage has also
been proposed, known as the post replication checkpoint. p53 plays an important role in
triggering the control mechanisms at both G1/S and G2/M checkpoints.
The development of the term apoptosis
Already since the mid-nineteenth century, many observations have indicated that cell death
plays a considerable role during physiological processes of multicellular organisms,
particularly during embryogenesis and metamorphosis . The term programmed cell death was
introduced in 1964, proposing that cell death during development is not of accidental nature
but follows a sequence of controlled steps leading to locally and temporally defined selfdestruction .
Eventually, the term apoptosis had been coined in order to describe the morphological
processes leading to controlled cellular self-destruction and was first introduced in a
publication by Kerr, Wyllie and Currie [Kerr, 1972]. Apoptosis is of Greek origin, having the
meaning "falling off or dropping off", in analogy to leaves falling off trees or petals dropping
off flowers. This analogy emphasizes that the death of living matter is an integral and
necessary part of the life cycle of organisms. The apoptotic mode of cell death is an active
and defined process which plays an important role in the development of multicellular
organisms and in the regulation and maintenance of the cell populations in tissues upon
physiological and pathological conditions. It should be stressed that apoptosis is a welldefined and possibly the most frequent form of programmed cell death, but that other, nonapoptotic types of cell death also might be of biological significance .
The significance of apoptosis
The development and maintenance of multicellular biological systems depends on a
sophisticated interplay between the cells forming the organism, it sometimes even seems to
involve an altruistic behaviour of individual cells in favour of the organism as a whole.
During development many cells are produced in excess which eventually undergo
programmed cell death and thereby contribute to sculpturing many organs and tissues .
Taken together, apoptotic processes are of widespread biological significance, being involved
in e.g. development, differentiation, proliferation/homoeostasis, regulation and function of
15
the immune system and in the removal of defect and therefore harmful cells. Thus,
dysfunction or deregulation of the apoptotic program is implicated in a variety of
pathological conditions. Defects in apoptosis can result in cancer, autoimmune diseases and
spreading of viral infections, while neurodegenerative disorders and AIDS diseases are
caused or enhanced by excessive apoptosis.
Due to its importance in such various biological processes, programmed cell death is a
widespread phenomenon, occurring in all kinds of metazoans such as in mammals, insects,
nematodes. Moreover, programmed cell death also might play a role in plant biology, and
apoptosis-like cell death mechanisms even have been observed and used as a model system in
yeast. Fascinating insights into the origin and evolution of programmed cell death might
possibly be given by the fact, that programmed cell death is also an integral part of the life
cycle of other unicellular eukaryotes and that even prokaryotes.
Morphological features of apoptosis
Apoptotic cells can be recognized by stereotypical morphological changes: the cell shrinks,
shows deformation and looses contact to its neighboring cells. Its chromatin condenses and
migrate at the nuclear membrane and finally the cell is fragmented into compact membraneenclosed structures, called 'apoptotic bodies' which contain cytosol, the condensed chromatin,
and organelles. The apoptotic bodies are engulfed by macrophages and thus are removed
from the tissue without causing an inflammatory response. Those morphological changes are
a consequence of characteristic molecular and biochemical events occurring in an apoptotic
cell, most notably the activation of proteolytic enzymes which eventually mediate the
cleavage of DNA into oligo-nucleosomal fragments as well as the cleavage of a multitude of
specific protein substrates which usually determine the integrity and shape of the cytoplasm
or organelles .
4.6
Role of Cyclins and CDKs
Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases (CDKs),
determine a cell's progress through the cell cycle. Leland H. Hartwell, R. Timothy Hunt, and
Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine for their discovery of
these central molecules. Many of the genes encoding cyclins and CDKs are conserved among
all eukaryotes, but in general more complex organisms have more elaborate cell cycle control
systems that incorporate more individual components. Many of the relevant genes were first
identified by studying yeast, especially Saccharomyces cerevisiae; genetic nomenclature in
16
yeast dubs many of these genes cdc (for "cell division cycle") followed by an identifying
number, e.g., cdc25.
Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated
heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a
partner cyclin. When activated by a bound cyclin, CDKs perform a common biochemical
reaction called phosphorylation that activates or inactivates target proteins to orchestrate
coordinated entry into the next phase of the cell cycle. Different cyclin-CDK combinations
determine the downstream proteins targeted. CDKs are constitutively expressed in cells
whereas cyclins are synthesised at specific stages of the cell cycle, in response to various
molecular signals.
Cyclin-dependent kinases (CDKs) are typical serine/threonine kinases that display the 11
subdomains shared by all kinases. Among the 19,099 predicted genes, there are 14 CDKs and
34 cyclins; nine CDKs and 11 cyclins have been identified in man, referred to as CDK1CDK9. This also allows the activating phosphorylation on Thr160 (by CDK7/cyclin
H/MAT1). The second conformational change induced by cyclin binding is found within the
ATP-binding site where a reorientation of the amino acid side chains induces the alignment
of the triphosphate of ATP necessary for phosphate transfer. The strong sequence homology
between the catalytic domains of different CDKs suggests that their tridimensional structures
will be similar. Progression through the G1, S, G2 and M phases of the cell division cycle is
directly controlled by CDKs. In early-mid G1, extracellular signals modulate the activation of
CDK4 and CDK6 associated with D-type cyclins. These complexes phosphorylate and
inactivate the retinoblastoma protein pRb, resulting in the release of the E2F and DP1
transcription factors which control the expression of genes required for the G1/S transition
and S phase progression. The CDK2/cyclin E complex, which is responsible for the G1/S
transition, also regulates centrosome duplication. During S phase, CDK2/cyclin A
phosphorylates different substrates allowing DNA replication and the inactivation of G1
transcription factors. Around the S/G2 transition, CDK1 associates with cyclin A. Later,
CDK1/cyclin B appears and triggers the G2/M transition by phosphorylating a large set of
substrates. Phosphorylation of the anaphase promoting complex (APC) by CDK1/cyclin B is
required for transition to anaphase and completion of mitosis
General mechanism of cyclin-CDK interaction
17
Upon receiving a pro-mitotic extracellular signal, G1 cyclin-CDK complexes become active
to prepare the cell for S phase, promoting the expression of transcription factors that in turn
promote the expression of S cyclins and of enzymes required for DNA replication. The G1
cyclin-CDK complexes also promote the degradation of molecules that function as S phase
inhibitors by targeting them for ubiquitination. Once a protein has been ubiquitinated, it is
targeted for proteolytic degradation by the proteasome. Active S cyclin-CDK complexes
phosphorylate proteins that make up the pre-replication complexes assembled during G1
phase on DNA replication origins. The phosphorylation serves two purposes: to activate each
already-assembled pre-replication complex, and to prevent new complexes from forming.
This ensures that every portion of the cell's genome will be replicated once and only once.
The reason for prevention of gaps in replication is fairly clear, because daughter cells that are
missing all or part of crucial genes will die. However, for reasons related to gene copy
number effects, possession of extra copies of certain genes would also prove deleterious to
the daughter cells.
Mitotic cyclin-CDK complexes, which are synthesized but inactivated during S and G2
phases, promote the initiation of mitosis by stimulating downstream proteins involved in
chromosome condensation and mitotic spindle assembly. A critical complex activated during
this process is a ubiquitin ligase known as the anaphase-promoting complex (APC), which
promotes degradation of structural proteins associated with the chromosomal kinetophore.
APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis
can proceed.
Specific action of cyclin-CDK complexes
Cyclin D is the first cyclin produced in the cell cycle, in response to extracellular signals (eg.
growth factors). Cyclin D binds to existing CDK4, forming the active cyclin D-CDK4
complex. Cyclin D-CDK4 complex in turn phosphorylates the retinoblastoma susceptibility
protein (RB). The hyperphosphorylated RB dissociates from the E2F/DP1/RB complex
(which was bound to the E2F responsive genes, effectively "blocking" them from
transcription), activating E2F. Activation of E2F results in transcription of various genes like
cyclin E, cyclin A, DNA polymerase, thymidine kinase, etc. Cyclin E thus produced binds to
CDK2, forming the cyclin E-CDK2 complex, which pushes the cell from G1 to S phase (G1/S
transition). Cyclin A along with CDK2 forms the cyclin A-CDK2 complex, which initiates
the G2/M transition. Cyclin B-CDK1 complex activation causes breakdown of nuclear
18
envelope and initiation of prophase, and subsequently, its deactivation causes the cell to exit
mitosis.
4.7
Retinoblastoma and E2F Protein.
Retinoblastoma is a human childhood disease, involving a tumor of the retina. It
occurs both as a heritable trait and sporadically (by somatic mutation). It is often associated
with deletions of band q 14 of human chromosome 13. The RB gene has been localized to
this region by molecular cloning.
Summarizes the situation. Retinoblastoma arises when both copies of the RB gene are
in-parental chromosome carries an alteration in this region. A somatic event in retinal cells
that causes loss of the other copy of the RB gene causes a tumor. In the sporadic form of the
disease, the parental chromosomes are normal, and both RB alleles are lost by (individual)
somatic events.
The cause of retinoblastoma is therefore loss of protein function. Loss of RB is
involved also in other forms of cancer, including osteosarcomas and small cell lung cancers.
RB is a nuclear phosphoprotein that influences the cell cycle. In resting (G0/G1) cells,
RB is not phosphorylated. RB is phoshorylated during the cell cycle by cyclin/cdk
complexes, most particularly at the end of G1; it is dephosphorylated during mitosis. The
non-phosphorylated form of RB specifically binds several protein, and these interactions
therefore occur only during part of the cell cycle (prior to S phase). Phosphorylation releases
these protein.
The target proteins include the E2F group of transcription factors, which activate
target genes whose products are essential for S phase. Binding to RB inhibits the ability of
E2F to activate transcription, Which suggests that RB may repress the expression of genes
dependent on E2F. In this way, RB indirectly prevents cells from entering S phase. Also, the
RB-E2F complex directly represses some target genes, so its dissociation allows them to be
expressed.
Certain viral tumor antigens bind specifically to the non-phosphorylated from of RB.
The best characterized are SV40 T antigen and adenovirus E1A. This suggests the model
shown in Non-phosphorylated RB prevents cell proliferation , this activity must be
suppressed in order to pass through the cell cycle, which is accomplished by the cyclicphosphorylation. And it may also be suppressed when a tumor antigen sequesters the nonphosphorylated bind E2F, the E2F is permanently free to allow entry into S phase (and the
RB-E2F complex is not available to repress its target genes.)
19
Over-expression of RB impedes cell growth. An indication of the importance of RB
for cell proliferation is given by the properties of an osteosarcoma cell line that lacks RB;
when RB is introduced into this cell line, its growth is impeded. However, the inhibition can
be overcome by expression of D cyclins, which form cdk-cyclin combinations that
phosphorylate RB. RB is not the only proteins of its type : proteins with related sequences,
called p107 and p130, have similar properties.
The connection between the cell cycle and tumor genesis is illustrated in several
regulators are identified as tumor suppressors by the occurrence of inactivating mutations in
tumors. In addition to RB itself, there are the small inhibitory proteins (most notably p16 and
possibly p21), and D cyclin. Although these proteins (most notable RB) play a role in the
cycle of a proliferating cell, the role that is relevant for tumor-genesis is more probably their
function in the quiescent (G0) state. In quiescent cells, RB is not phosphorylated, D cylin
levels are low or absent, and p16, p21 p27 ensure inactivity of cdk-cyclin complexes, The
loss of this circuit is necessary for unrestrained growth.
Check your progress : 1
1. Notes- Write your answer in the space given.
2. Compare your answer with the one given at the end of the unit.
1. What are the general classes of cytoskeletal fibers?
2. What are the phases of cell cycle?
3. What is Apoptosis?
4. What is Anaphase-promoting complex (APC)?
5. What is Retinoblastoma?
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------20
4.8
Cytokinesis
Mitosis is the process of separating the duplicates of each of the cell's chromosomes. It is
usually followed by division of the cell. However, there are cases (cleavage in the insect
embryo is an example) where the chromosomes undergo the mitotic process without division
of the cell. Thus a special term, cytokinesis, for the separation of a cell into two.
In animal cells, a belt of actin filaments forms around the perimeter of the cell, midway
between the poles. The interaction of actin and a myosin (not the one found in skeletal
muscle) tightens the belt, and the cell is pinched into two daughter cells.
In plant cells, a cell plate forms where the metaphase plate had been. The cell plate is
synthesized by the fusion of multiple membrane-bounded vesicles. Their fusion supplies new
plasma membrane for each of the two daughter cells. Synthesis of a new cell wall between
the daughter cells then occurs at the cell plate.
21
Cytokinesis is the process whereby the cytoplasm of a single eukaryotic cell is divided to
form two daughter cells. It usually initiates during the late stages of mitosis, and sometimes
meiosis, splitting a binucleate cell in two, to ensure that chromosome number is maintained
from one generation to the next. In plant cells, a dividing structure known as the cell plate
forms across the centre of the cytoplasm and a new cell wall forms between the two daughter
cells. [1]
4.9
Cell plate formation
Due to the presence of a cell wall, cytokinesis in plant cells is significantly different from that
in animal cells. Rather than forming a contractile ring, plant cells construct a cell plate in the
middle of the cell. The Golgi apparatus releases vesicles containing cell wall materials. These
vesicles fuse at the equatorial plane and form a cell plate. The cell plate begins as a fusion
tube network, which then becomes a tubule-vesicular network (TVN) as more components
are added. The TVN develops into a tubular network, which then becomes a fenestrated sheet
which adheres to the existing plasma membrane.
Phragmoplast and cell plate formation in a plant cell during cytokinesis. Left side:
Phragmoplast forms and cell plate starts to assemble in the center of the cell. Towards the
right: Phragmoplast enlarges towards the outside of the cell, leaving behind mature cell plate
in the center. The cell plate will transform into the new cell wall once cytokinesis is
complete.
Cytokinesis in terrestrial plants occurs by cell plate formation. This process entails the
delivery of Golgi-derived and endosomal vesicles carrying cell wall and cell membrane
components to the plane of cell division and the subsequent fusion of these vesicles within
this plane.
After formation of an early tubule-vesicular network at the center of the cell, the initially
labile cell plate consolidates into a tubular network and eventually a fenestrated sheet. The
cell plate grows outward from the center of the cell to the parental plasma membrane with
which it will fuse, thus completing cell division. Formation and growth of the cell plate is
dependent upon the phragmoplast, which is required for proper targeting of Golgi-derived
vesicles to the cell plate.
22
As the cell plate matures in the central part of the cell, the phragmoplast disassembles in this
region and new elements are added on its outside. This process leads to a steady expansion of
the phragmoplast, and concomitantly, to a continuous retargeting of Golgi-derived vesicles to
the growing edge of the cell plate. Once the cell plate reaches and fuses with the plasma
membrane the phragmoplast disappears. This event not only marks the separation of the two
daughter cells, but also initiates a range of biochemical modifications that transform the
flexible cell plate into a cellulose-rich, stiff primary cell wall.
The heavy dependence of cell plate formation on active Golgi stacks explains why plant cells,
unlike mammalian cells, do not disassemble their secretion machinery during cell division.
4.10
Programmed cell death
Programmed cell-death (PCD) is death of a cell in any form, mediated by an intracellular
program. In contrast to necrosis, which is a form of cell-death that results from acute tissue
injury and provokes an inflammatory response, PCD is carried out in a regulated process
which generally confers advantage during an organism's life-cycle. PCD serves fundamental
functions during both plant and metazoa (multicellular animals) tissue development.

Apoptosis or Type I cell-death

Autophagic or Type II cell-death ( cytoplasmic: characterized by the formation of
large vacuoles which eat away organelles in a specific sequence prior to the nucleus
being destroyed.)
Besides these two types of PCD, other pathways have been discovered. Called "non-apoptotic
programmed cell-death" (or "caspase-independent programmed cell-death" or "necrosis-like
programmed cell-death") these alternative routes to death are as efficient as apoptosis and can
function as either backup mechanisms or the main type of PCD. Plant cells undergo particular
processes of PCD which are similar to autophagic cell death. However, some common
features of PCD are highly conserved in both plants and metazoa.
History
The concept of "programmed cell-death" was used by Lockshin & Williams in 1964 in
relation to insect tissue development, around eight years before "apoptosis" was coined. Since
then, PCD has become the more general of these two terms. PCD has been the subject of
increasing attention and research efforts. This trend has been highlighted with the award of
23
the 2002 Nobel Prize in Physiology or Medicine to Sydney Brenner (United Kingdom), H.
Robert Horvitz (US) and John E. Sulston (UK).
The cell cycle, or cell-division cycle, is the series of events that take place in a eukaryotic cell
leading to its replication. In "APL regulates vascular tissue identity in Arabidopsis", Bonke
and colleagues state that one of the two long-distance transport systems in vascular plants,
xylem, consists of several cell-types "the differentiation of which involves deposition of
elaborate cell-wall thickenings and programmed cell-death." The authors emphasize that the
products of plant PCD play an important structural role.
Basic morphological and biochemical features of PCD have been conserved in both plant and
animal kingdoms. It should be noted, however, that specific types of plant cells carry out
unique cell-death programs. These have common features with animal apoptosis for instance,
nuclear DNA degradation but they also have their own peculiarities, such as nuclear
degradation being triggered by the collapse of the vacuole in tracheids elements of the xylem.
PCD in pollen prevents inbreeding
24
During pollination, plants enforce self-incompatibility (SI) as an important means to prevent
self-fertilization. Research on the corn poppy (Papaver rhoeas) has revealed that proteins in
the pistil on which the pollen lands, interact with pollen and trigger PCD in incompatible (i.e.
self) pollen. The researchers, Steven G. Thomas and Veronica E. Franklin-Tong, also found
that the response involves rapid inhibition of pollen-tube growth, followed by PCD.
Check your progress :2
1.Notes- Write your answer in the space given.
2.Compare your answer with the one given at the end of the unit.
1. What is programmed cell death?
2. What is cytokinesis?
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4.11
Let us sum up
1.
Protein targeting or protein sorting is the mechanism by which a cell transports
proteins to the appropriate positions in the cell or outside of it. Sorting targets can be the
inner space of an organelle, any of several interior membranes, the cell's outer membrane, or
its exterior via secretion. This delivery process is carried out based on information contained
in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases.
2.
The cytoskeleton is to maintain cellular shape, and is involved in intracellular
organization, cell polarity, cell adhesion, and in some cases, motility. The cytoskeleton is
composed of several different protein components. There are three general classes of
cytoskeletal fibers: (1) microtubules, (2) intermediate filaments, and (3) actin filaments.
3.
The cell cycle, or cell-division cycle, is the series of events that take place in a
eukaryotic cell leading to its replication. These events can be divided in two brief periods:
interphase during which the cell grows, accumulating nutrients needed for mitosis and
duplicating its DNA and the mitotic (M) phase, during which the cell splits itself into two
distinct cells, often called "daughter cells". The cell-division cycle is a vital process by which
25
a single-celled fertilized egg develops into a mature organism, as well as the process by
which hair, skin, blood cells, and some internal organs are renewed.
4.
Retinoblastoma is a human childhood disease, involving a tumor of the retina. It
occurs both as a heritable trait and sporadically (by somatic mutation). It is often associated
with deletions of band q 14 of human chromosome 13. The RB gene has been localized to
this region by molecular cloning.
5.
Cytokinesis is the process whereby the cytoplasm of a single eukaryotic cell is
divided to form two daughter cells. It usually initiates during the late stages of mitosis, and
sometimes meiosis, splitting a binucleate cell in two, to ensure that chromosome number is
maintained from one generation to the next.
6.
Programmed cell-death (PCD) is death of a cell in any form, mediated by an
intracellular program. In contrast to necrosis, which is a form of cell-death that results from
acute tissue injury and provokes an inflammatory response.
Check your progress 1 : The key
1.
Three general classes of cytoskeletal fibers: (1) microtubules, (2) intermediate
filaments, and (3) actin filaments.
2.
The cell cycle consists of four distinct phases: G1 phase, S phase, G2 phase
and M phase.
3.
The cell shrinks, shows deformation and looses contact to its neighboring
cells. Its chromatin condenses and migrate at the nuclear membrane and finally the
cell is fragmented into compact membrane-enclosed structures, called 'apoptotic
bodies' which contain cytosol, the condensed chromatin, and organelles which is a
form of cell death is called apoptosis.
4.
A critical complex activated during Cyclin-CDK Complex this process is a
ubiquitin ligase known as the anaphase-promoting complex (APC), which promotes
degradation of structural proteins associated with the chromosomal kinetophore.
5.
Retinoblastoma is a human childhood disease, involving a tumor of the retina.
It occurs both as a heritable trait and sporadically (by somatic mutation).
Check your progress 2 : The key
26
1.
Programmed cell-death (PCD) is death of a cell in any form, mediated by an
intracellular program. In contrast to necrosis, which is a form of cell-death that
results from acute tissue injury and provokes an inflammatory response, PCD
is carried out in a regulated process which generally confers advantage during
an organism's life-cycle
2.
Cytokinesis is the process whereby the cytoplasm of a single eukaryotic cell is
divided to form two daughter cells
4.12
Assignment/ Activity
1.
Study and explain the cell cycle, or cell-division cycle, is the series of
events that take place in a eukaryotic cell.
2.
Retinoblastoma is a human childhood disease, involving a tumor of the
retina, collect some more information on it.
4.13
References
1. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. (2003). Molecular
Biology of the Cell. Ch 17. Garland Science: New York. 4th ed
2. J. A. Smith and L. Martin (April 1, 1973). "Do Cells Cycle?". PNAS 70 (4): 12631267. doi:10.1073/pnas.70.4.1263. PMID 4515625.
3. Morgan DO. (2007) The Cell Cycle: Principles of Control. New Science Press:
London.
4. R.S. Prather, A.C. Boquest, B.N. Day (1999). "Cell Cycle Analysis of Cultured
Porcine Mammary Cells". Cloning 1 (1): 17-24. doi:10.1089/15204559950020067.
PMID 16218827.
5. Stephen J. Elledge (6 December 1996). "Cell Cycle Checkpoints: Preventing an
Identity
Crisis".
Science
274
(5293):
1664-1672.
doi:10.1126/science.274.5293.1664. PMID 8939848
6. Seydou Samaké, Lawrence C. Smith (1997 Oct 15). "Synchronization of cell
division in eight-cell bovine embryos produced in vitro: Effects of nocodazole".
Theriogenology 48 (6): 969-76. doi:10.1016/S0093-691X(97)00323-3. PMID
16728186.
27
7. Zuzarte-Luis, V and Hurle, JM (2002). "Programmed cell death in the developing
limb." Int J Dev Biol 46(7): 871-6.
28
UNIT- V
OTHER CELLULAR ORGANELLES
5.0
Introduction
5.1
Objective
5.2
Peroxisomes
5.3
Golgi apparatus
5.4
Lysosomes
5.5
Endoplasmic reticulum
5.6
Techniques in cell biology
5.7
FISH
5.8
Confocal microscopy
5.9
Let us sum up
5.10
Assignment/ Activity
5.11
Reference
29
5.0
Introduction
Microbodies : Structure and Types
Microbodies are spherical or oblate in form. They are bounded by a single
membrane and have an interior or matrix which is amorphous or granular Micrbodies
are most easily distinguished from other cell organelles by their content of catalase
enzyme. Catalase can be visualized with the electron microscope when cells are
treated with the stain DAB (i.e., 3,3'-diaminobenzidine). The product is electron
opaque and appears as dark regions in the cell where catalase is present
The technique of isolation of microbodies
of plant tissues includes the
following steps : (1) Tissues are group very carefully to save microbodies from
disruption. (2) The homogenate is with differential centrifugation to obtain a fraction
of the cell homogenate which is rich in microbodies. (3) The enriched fraction is
subjected to isopycnic ultra-centrifugation on discontinuous or continuous sucrose
density gradient. Recent biochemical studies have distinguished two types of
microbodies, namely peroxisomes and glycoxysomes. These two organelles differ
both in their enzyme complement and in the type of tissue in which they are found.
Peroxisomes are found in animal cells and the leaves of higher plants. They contain
catalases and oxidases (e.g., D-amino oxidase and urate oxidase). In both they
participate in the oxidation of substrates, producing hydrogen peroxide which is
subsequently destroyed by catalase activity.
In plant cells, peroxisomes remain associated with ER. chloroplasts and
mitochondria and are involved in photorespiration. Gloxysomes occur only in plant
cells and are particularly abundant in germinating seeds which store fats as a
reserve food material. They contain enzymes of glyoxylate cycle besides the
catalases and oxidases
5.1
Objectives
1.
This unit will fulfill the basic introduction , function, origin and chemical
composition of some organelles.
2.
Students will find the text useful as it will help in understanding some
molecular diagnostic technique.
5.2
Peroxisomes
30
Peroxisomes occur in many animal cells and in a wide range of plants. They
are present in all photosynthetic cells of higher plants in etiolated leaf tissue, in
coleoptiles and hypocotyls, in tobacco stem and callus, in ripening pear fruits and
also in Euglenophyta, Protozoa, brown algae, fungi liverworts, mosses and ferns.
Peroxisomes are variable in size and shape, but usually appear circular in cross
section having diameter between 0.2 and 1.5m (0.2 and 0.25 m diameter in most
mammalian tissues : 0.5m diameter in rat liver cells). They have a single limiting
unit membrane of lipid and protein molecules, which encloses their granular matrix.
In some cases (e.g., in the festuciod grasses) the matrix contains numerous threads
or fibrils, while in others they are observed to contain either an amorphous nucleoid
or a dense inner core which in many species shows a regular crystalloid structure
(e.g., tobacco leaf cell, Newcomb and Frederick, 1971). Little is known about the
function of the core, except that it is the site of the enzyme urate oxidase in rat liver
peroxisomes and much of the catalase in some plants (see Hall et al., 1974).
Recently, a possible relationship has been stressed between peroxides and free
radicals (such as superoxide anion -O2-) with the process of aging. These radicals
may act on DNA molecule to produce mutations altering the transcription into mRNA
and the translation into proteins. In addition, free radicals and peroxides can affect
the membranes by causing peroxidation of lipids and proteins. For these reasons
reducing compounds such as vitamin E or enzymes such as superoxide dismutase
could play a role in keeping the healthy state of a cell.
Glycolate cycle- Peroxisomes of plant leaves contain catalaze together with
the enzymes of glycolate pathway, as glycolate oxidase, glutamate glyoxylate,
serine-glyoxylate and aspirate--ketoglutarate aminotransferases, hydroxyl pyruvate
reductase and malic dehydrogenase. They also contain FAD, NAD and NADP
coenzymes. The glycolate cycle is throught to bring about the formation of the amino
acids-glycine
and
serine-from
the
non-phosphorylated
intermediates
of
phostosynthetic carbon reduction cycle, i.e., glycerate to serine, or glycolate to
glycine and serine in a sequence of reactions which involve chloroplasts,
peroxisomes, mitochondria and cytosol (Tolber. 1971). The glycolate pathway also
generates C1 compounds and serves as the generator of precursors for nucleic acid
biosynthesis.
Photorespiration- In green leaves, there are peroxisomes that carry out a
process called photorespiration which is a light - stimulated production of CO2 that is
31
different from the generation of CO2 by mitochondria in the dark. In photorespiration,
glycolic acid (glycolate), a two-carbon product of photosynthesis is released from
chloroplasts and oxidized into and H2O2 by a peroxisomal enzyme called glycolic
acid oxidase. Later on, glyoxylate is oxidized into CO2 and formate:
CH2OH.COOH + O2

CHO - COOH + H2O2
CHO - COOH + H2O2

HCOOH + CO2 + H2O
Photorepiration is so called because light induces the synthesis of glycolic
acid in chloroplasts. This process involves intervention of two organelles : cloroplasts
and peroxisomes. Lastly, photorespiration is driven by atmospheric conditions in
which the O2 tension is high and CO2 tension low. Apparently O2 competes with CO2
for the enzyme ribulose diphosphate carboxylase which normally is the key enzyme
in CO2 fixation during photosynthesis. When O2 is used by the enzyme, an unstable
intermediate
is
formed
which
breaks
down
into
3-phosphoglycerate
and
phosphoglycolate. The latter tends to increase the glycolate concentration by
removal of its phosphate group and, therefore, more glycolate is available for
additional oxidation and CO2 release .
Photorespiration is a wasteful process for the plant cell, since, it significantly
reduces the efficiency of the process of photosynthesis (i.e., it returns a portion of
fixed CO2 to the atmosphere). It is a particular problem in C3 plants that are more
readily affected by low CO2 tensions ; C4 plants are much more efficient in this
regard.
Biogenesis of Peroxisomes
At one time it was thought that the membrane 'shell' of the peroxisomes is
formed by building of the endoplasmic reticulum (ER), while the 'content' or matrix is
imported from the cytosol (cytoplasmic matrix). However, there is now evidence
suggesting that new peroxisomes always arise from pre-existing ones, being formed
by growth and fission of old organelles similar to mitochondria and chloroplasts.
Thus, peroxisomes are a collection of organelles with a constant membrane
and a variable enzymatic content. All of their proteins (both structural and enzymatic)
are encoded by nuclear genes and are synthesized in the cytosol (cytoplasmic
matrix) (i.e., on the free ribosomes). The proteins present in either lumen or
membrane of the peroxisome are taken up post-translationally from the cytosol
(cytoplasmic matrix) as the haeme-free monomer; the monomers are imported into
the lumen of peroxisomes, where they assemble into tetramers in the presence of
haeme./ Catalase and many peroxisomal proteins are found to have a signal
32
sequence (comprising of three amino acids) which is located near their carboxyl
ends and directs them to peroxisome (Gould, Keller and Subramani,1988).
Peroxisomes contain receptors exposed on their cytosolic surface to recognize the
signal on the imported proteins. All of the membrane proteins of the peroxisomes.
Including signal receptor proteins, are imported directly from the cytosol (cytoplasmic
matrix). The lipids required to make new peroxisomal membrane are also imported
from the cytosol (cytoplasmic matrix), possibly being carried by phospholipids
transfer proteins from sites of their synthesis in the DR membranes (Affe and
Kennedy, 1983).
Glyoxysomes
Glyoxysomes are found to occur in the cells of yeast, Neurospora, and oil rich
seeds of many higher plants. They resemble with peroxisomes in morphological
details, except that, their crystalloid core consists of dense rods of 6.0 m diameter.
They have enzymes for fatty acid metabolism and gluconeogenesis, i.e. conversion
of stored lipid molecules of spherosomes of germinating seeds into the molecules of
carbohydrates.
Functions
Glyoxysomes perform following biochemical activities of plants cells :
(1)
Fatty acid metabolism. During germination of oily seeds, the stored lipid
molecules of spherosomes are hydrolysed by the enzyme lipase (glycerol
ester hydrolase) to glycerol and fatty acids. The phospholipids molecules are
hydrolysed by the enzyme phospholipase. The long chain fatty acids which
are released by the hydrolysis are then broken down by the successive
removal of two carbon or C2 fragments in the process of -oxidation.
During -oxidation process, the fatty acid is first activated by enzyme fatty
acid thiokinase to fatty acyl-CoA which is oxidized by a FAD-linked enzyme fatty
acyl-CoA dehydrogenase into-2-enoyl-CoA. Trans-2-enoyl-CoA is hydrated by an
enzyme enoyl hydratase or crotonase to produce the L-3-hydroxyacyl-CoA, which is
oxidized a NAD Linked L-3-hydratase or crotonase to produce the L-3-hydroxyacylCoA, which is oxidized by a NAD linked L-3-hydroxyacyl-CoA dehydrogenase to
produce 3-Ketoacly-CoA. The 3-keto acyl-CoA looses a two carbon fragment under
the action of the enzyme thiolase to generate an acetyl-CoA and a new fatty acylCoA with two less carbon atoms thatn the original. This new fatty acyl-CoA is then
33
recycled thought the same series of reactions until the final two molecules of acetylCoA are produced.
In plant seeds -oxidation occurs in glyoxysomes (Cooper and Beevers,
1969). But in other plant cells -oxidation occurs in glyxysomes and mitochondria.
The glyoxysomal -oxidation requires oxygen for oxidation of reduced flavorprotien
produced as a result of the fatty-acyl-CoA dehydrogenase activity. In animal cells oxidation occurs in mitochondria.
In plant cells, the acetyl-CoA, the product of -oxidation chain is not oxidized
by Krebs cycle, because it remains spatially separated from the enzymes of Krebs
cycle, instead of it, acetyl-CoA undergoes the glyoxylate cycle to be converted into
succinate.
(2)
Glyoxylate cycle. The glyoxylate pathway occurs in glyoxysomes and it
involve some of the reactions of the Krebs cycle in which citrate is formed
from oxaloacetate and acetyl-CoA under the action of citrate synthetase
enzymes. The citrate is subsequently converted into isocitrate by aconitase
enzyme. The cycle then involves the enzymatic conversion of isocitrate to
glyoxylate and succinate by isocitratase enzyme :
Isocitratase
Isocitrate
Glyoxylate + Succinate
The glyoxylate and another mole of acetyl-CoA form a mole of malate by
malate synthesis ;
Malate synthetase
Acetyl CoA+Glyoxylate
Malate
This malate is converted to oxaloacetate by malate dehydrogenase for the
cycle to be completed. Thus, overall, the glyoxylate pathway involves :
2 Acetyl-CoA+NAD+
Succintiate + NADH+ H+
Succinate is the end product of the glyoxysomal metabolism of fatty acid and
is not further metabolized within this organelle.
The synthesis of hexose or gluconeogenesis involves the conversion of
succinate to oxaloacetate, which presumably takes place in the mitochondria, since
the glyoxsomes do not contain the enzymes fumarase and succinic dehydrogenase.
Two molecules of oxaloacetate are formed from four molecules of acetyl-CoA
34
without carbon loss. This oxaloacetate is converted to phosphoenol pyruvate in the
phosphoenol pyruvate caboxykinase reaction with the loss of two molecules of CO2 :
2 Oxaloacete + 2ATP
5.3

 2 Phosphoenol pyruvate + 2CO2 + 2ADP


Golgi Apparatus
For the performance of certain important cellular functions such as biosynthesis of
polysaccharides, packaging (compartmentalizing) of cellular synthetic products
(proteins), production of exocytotic (secretory) vesicles and differentiation of cellular
membranes, there occurs a complex organelle called Golgi complex or Golgi
apparatus in the in the cytoplasm of animal and plant cells. The Golgi apparatus, like
the endoplasmic reticulum, is a canalicular system with sacs, but unlike the
endoplasmic reticulum it has parallely arranged, flattened, membrane-bounded
vesicles which lack ribosomes and stainable by osmium tetraoxide and silver salts.
History
An Italian neurologist (i.e., physician) Camillo Golgi in 1873 discovered and
developed the silver chromate method (termed la reazione nera) for studying
histological details of nerve cells. of cerebral cortex of brain) of barn owl contained
and internal reticular network which stains black with the silver stain. He called this
structure apparato reticolare interno (internal reticular apparatus). By reporting the
existence of such an organelle inside cell, he inadvertently raised a storm of
controversy in the scientific world, which is commonly known as the Golgi
controversy.
Occurence
The Golgi apparatus occurs in all cells except the prokaryotic cells (viz.,
mycoplasmas, bacteria and blue green algae) and eukaryotic cells of certain fungi,
sperm cells of bryophytes and pteridiophytes, cells of mature sieve tubes of plants
and mature sperm and red blood cells of animals. Their number per plant cell can
vary from several hundred as in tissues of corn root and algal rhizoids (i.e., more
than 25,000 in algal rhizoids, Sievers, 1965), to a single organelle in some algae.
(Certain algal cells such as Pinularia and Microsterias, contain largest and most
complicated Golgi apparatuses. In higher plants, Golgi apparatuses are particularly
common in secretory cells and in young rapidly growing cells.
In animal cells, there usually occurs a single Golgi apparatus, however, its
number may vary from animal to animal and from cell to cell. Thus Paramoeba
35
species has two Golgi apparatus and nerve cells, liver cells and chordate oocytes
have multiple Golgi apparatuses, there being about 50 of them in the liver cells.
Distribution
In the cells of higher plants, the Golgi bodies or dictyosomes are usually found
scattered throughout the cytoplasm and their distribution does not seem to be
ordered or localized in any particular manner (Hall et al., 1974). However, in animal
cells the Golgi apparatus is a localized organelle. For example, in the cells of
ectodermal or endodermal origin, the Golgi apparatus remains polar and occurs in
between the nucleus and the periphery (e.g., thyroid cells, exocrine pancreatic cells
and mucus-producing goblet cells of intestinal epithelium) and in the nerve cells it
occupies a circum-nuclear position.
Morphology
The Golgi apparatus is morphologically very similar in both plant and animal
cells. However, it is extremely pleomorphic: in some cell types it appears compact
and limited, in others spread out and reticular (net-like). Its shape and form may very
depending on cell type. Typically, however, Golgi apparatus appears as a complex
array of interconnecting tubules, vesicles and cisternae. There has been much
debate concerning the terminology of the Golgi's parts. The classification given by
D.J. Morre (1977) is most widely used. In this scheme, the simplest unit of the Golgi
apparatus is the cisterna. This is a membrane bound space in which various
materials and secretions may accumulate. Numerous cisternae are associated with
each other and appear in a stack like (lamellar) aggregation. A group these cisternae
is called the dictyosome, and a group of dictyosomes makes up the cells Golgi
apparatus. All dictyosomes of a cell have a common function (see Berns, 1983).
1.
Flattened Sac or Cisternae
Cisternae
(aboute 1m in diameter) are central, flattened, plate-like or
saucer-like closed compartments which are help in parallel bundles or stacks
on above the other. In each stack, cisternae, are separated by a space of 20
to 30 nm which may contain rod-like elements or fibres. Each stack of
cisternae forms a dictyosome which may contain 5 to 6 Golgi cisternae in
animal cells or 20 or more cisternae in plant cells. Each cisterna is bounded
by a smooth unit membrane (7.5 nm thick), having a lumen varying in width
from about 500 to 1000 nm (see Sheeler and Bianchi, 1987).
The margins of each cisterna are gently curved so that the entire dictyosome
of Golgi apparatus takes on a bow like appearance. The cisternae at the
36
convex end of the dictyosome comprise proximal forming or cis-face and the
cisternae at the concave end of the dictyosome comprise the distal, maturing
or trans-face. The forming or cis-face of Golgi is located next to either the
nucleus or a specialized portion of rough ER that lacks bound ribosomes and
is called ''transitional'' ER. Trans face of Golgi is located near the plasma
membrane. This polarization is called cis-trans axis of the Golgi apparatus.
2.
Tubules
A complex array of associated vesicles and anastomosing tubules (30 to
50nm diameter) surround the dictyosome and radiate from it. In fact, the
peripheral area of dictyosome is fenestrated (lace-like) in structure.
3.
Vesicles
The vesicles (60 nm in diameter) are of three types :
(i)
Transitional vesicles are small membrane limited vesicles which are
through to form as blebs from the transitional ER to migrate and
converge to cis face of Golgi, where they coalesce to form new
cisternae.
(ii)
Secretory vesicles are varied-sized membrane-limited vesicles which
discharge from margins of cisternae of Golgi. They, often, occur
between the maturing face of Golgi and the plasma membrane.
(iii)
Clathrin-coated vesicles are spherical protuberances, about 50m in
diameter and with a rough surface. They are found at the periphery of
the organelle, usually at the ends of single tubules, and are
morphologically quite distinct from the secretory vesicles. The clathrincoated vesicles are known to play a role in intra-cellular traffic or
membranes and of secretory products. i.e., between ER and Golgi, as
well as between GELR region and the endosomal and lysosomal
compartments.
Isolation andChemical Composition
Initially, Golgi apparatus was isolated only from cells of the epididymis, however in
recent years, it has been isolated from number of plant and animal cells. The
isolation of Golgi apparatus brough about mainly by gentle homogenization following
by differential and gradient homogenization Gentle homogenization is preferred to
preserve the stacks of cisternae, Due to its low density, Golgi apparatuses tend to
form a distinct band in gradient centrifugation. The isolated Golgi apparatus is
37
apparatus tend to form a distinct band in gradient centrifugation. The isolated Golgi
apparatus washed with distilled water for purifying it, though, its secretory
components are lost (see Thorpe, 1984).
Chemically, Golgi apparatus of rat liver contains about 60 per cent lipid
material. The Golgi apparatus of animal cells contains phospholipids in the form of
phosphatidyl choline, whereas, that of plant cells contains phosphatidic acid and
phosphatidyl glycerol. The Golgi apparatus also contains a variety of enzyme (Table
7-1), some of which have been used as cytochemical markers.
Cytochemical Properties of Golgi Apparatus
Different parts of Golgi apparatus have been histochemically identified by specific
staining properties (Thorpe, 1984 Alberts et al., 1989) :
1. Osmium tetraxide (OsO4) selectively impregnates the outer face (cis-face) of the
Golgi apparatus. This stain adheres well to lipids, especially phospholipids and
unsaturated fats.
2. Phosphotungstic acid (H3PO4.12WO3.24H2O). selectively stain the maturing or
trans face of Golgi stack. This stain is an anionic stain having special affinity for
polysaccharides and proteins.
3. Glycosyl
tranferease
and
thiamine
pyrophosphatase
can
be
localized
cytochemically in the trans cisternae of Golgi apparatus. Transferase enzyme are
found to be located in the membrane of Golgi, not in the lumen of cisternae
(Thorpe, 1987).
4. Acide phosphatase enzyme is cytochemically marked in the GERL region.
Origin
Origin of Golgi apparatus involves the formation of new cisternae and there is
great variation in shape, number and size of cisternae in each stack (dictyosome).
The process of formation of new cisternae may be performed by any of the following
methods ; 1. Individual stacks of cisternae may arise from the pre-existing stacks by
division or fragmentation. 2. The alternative method of origin of Golgi is based on
denovo formation. In fact various cytological and biochemical envidences have
established that the membranes of the Golgi apparatus are originated from the
membranes of the smooth ER which in turn have originated from the rough ER. The
proximal Golgi saccules are formed by fusion of ER-derived vesicles, while distal
saccules "give their all" to vesicle formation and disappear. Thus, Golgi saccules are
constantly and rapidly renewed.
38
The cells of dormant seeds of higher plants generally lack Golgi apparatuses
but they do display zone of exclusion having aggregation of small transition vesicles.
Photomicrographs of cells in early stages of germination suggest progressive
development of Golgi bodies in these zones of exclusion ; and the development of
Golgi apparatuses coincides with the disappearance of the aggregation of vesicles
(see Sheeler and Bianchi, 1987).
Function
Golgi vesicles are often, referred to as the ''traffic police" of the cell (Dernell et
al., 1986. They play a key role in sorting many of cell's proteins and membrane
constituents, and in directing them to their proper destinations. To perform this
function, the Golgi vesicles contain different sets of enzymes in different types of
vesicles-cis, middle and trans cisternae- that react with and modify secretory
proteins passing through the Golgi lumen or membrane proteins and glycoprotein's
in the Golgi membranes as they are on route to their final destinations. For example
a Golgi enzyme may add a "signal" or "tag" such as a carbohydrate or phosphate
residues to certain proteins to direct them to their proper sites in the cell. Or, a
proteolytic Golgi enzyme may cut a secretory or membrane protein into two or more
specific segments (E.g., molecular processing involved in the formation of pancreatic
hormone insulin: preproinsulinproinsulininsulin).
Recently, in the function of Golgi apparatus, sub compartmentalization with a
division of labour has been proposed between the cis region (in which proteins of
RER are sorted and some of them are returned back possibly by coated vesicles),
and the trans region in which the most refined proteins are further separated for their
delivery to the various cell compartments (e.g., plasma membrane, secretory
granules and lysosomes).
39
Thus, Golgi apparatus is a centre of reception, finishing, packaging, and dispatch for
a variety of materials in animal and plant cells :
1.
Golgi Functions in Plants
In plants, Golgi apparatus is mainly involved in the secretion of mainly
involved in the secretion of materials of primary and secondary cell walls (e.g.,
formation and export of glycoprotein, lipids, pectins and monomers for
hemicellulose, cellulose, lignin, etc). During cytokinesis of mitosis or meiosis,
the vesicles originating from the periphery of Golgi apparatus, coalesce in the
phragmoplast area to form a semisolid layer, called cell plate. The unit
membrane of Golgi vesicles fuses during cell plate formation and becomes
part of plasma membrane of daughter cells .
5.4
Lysosomes
The lysosomes (Gr. lyso=digestive + soma = bodies) are tiny membrane-bound
vesicles involved in intracellular digestion. They contain a variety of hydrolytic
enzymes that remain active under acidic conditions. The lysosomal lumen is
maintained at an acidic pH (around 5) by an ATP-driven proton pump in the
membrane. Thus, these remarkable organelles are primarily meant for the digestion
of a variety of biological materials and secondarily cause aging and death of animal
cells and also a variety of human diseases such as cancer, gout Pompe's silicosis
and I-cell disease.
History
During
early electron microscopic studies, rounded dense bodies were
observed in rat liver cells. These bodies were initially described as "perinulclear
40
dense bodies", C. de Duve, in 1955, renamed these organelles as "lysosomes" to
indicate that the internal digestive enzymes only became apparent when the
membrane of these organelles was lysed (See Reid and Leech, 1980). However, the
term lysosome means lytic body having digestive enzymes capable of lysis (viz.,
dissolution of a cell or tissue ; (De Robertis and De Robertis, Jr., 1987).
Lysosomes were investigated according to following two schools (1) C, de
Duve and his coworkers (1963, 1964) worked in Belgium and their approach was
biochemical one . (2) Alex Novikoff and his research group (1962, 1964) worked in
United States and their approach was morphological and cytochemical. For the
discovery of lysosomes and a brilliant series of experiments on them, de Duve
shared the 1974 Nobel Prize for physicality with Palade andClaude, both were
pioneer cells biologists.
Occurence
The lysosomes occur in most animal and few plant cells. They are absent in
bacteria and mature mammalian ertythrocytes. Few lysosomes occur in muscle cells
or in cells of the pancreas. Leucocytes, especially granulocytes are a particularly rich
source of lysosomes. Their lysosomes are so large sized that they can be observed
under the light microscope. Lysosomes are also numerous in epithelial cells of
absorptive, secretory and excretory organs (e.g., intestine, liver, kidney, etc.) They
occur in abundance in the epithelial cells of lungs and uterus. Lastly phagocytic cells
and cells of reticuloendothelial system (e.g., bone marrow, spleen and liver) are also
rich in lysosomes.
Structure
The lysosomes are round vacuolar structure which remain filled with dense
material and are bounded by single unit membrane. Their shape and density vary
greatly. Lysosmes are 0.2 to 0.5m in size. Since, size and shape of lysosomes vary
from cell to cell and time to time (i.e. they are polymorphic), their identification
becomes difficult. However, on the basis of the following three criteria, a cellular
entity can be identified as a lysosome : (1) It should be bound by a limiting
membrane (2) It should contain two or more acid hydrolases ; and (3) It should
demonstrate the property of enzyme latency when treated in away that adversely
affects organelle's membrane structure.
41
Isolation and Chemical Composition
Lysosomes are very delicate and fragile organelles. Lysosomal fraction have
been isolated by sucrose-density centrifugation (or Isopycnic centrifugation) after
mild methods of homogenization. Since the original de Duve's isolated lysosomal
fractions were having contaminations of mitochondria, microsomes and microbodies,
so, 1960's it was investigated that rats injected with dextran or Triton WR-1339,
incorporated these compounds into their lysosomes, thereby altering their density
and making their cleaner separation possible by differential centrifugation and
density gradients (see Reid and Leech, 1980).
Lysosomes tend to accumulate certain dyes (vital stains such as Neutral red,
Niagara, Evans blue) and drugs such as anti-malarial drug chloroquine. Such
'loaded' lysosomes can be demonstreated by fluorescence microscopy.
The location of the lysosomes in the cell can also be pinpointed by various
histochemical or cytochemical methods. For example, lysosomes demonstrate the
property of metachromasia with toluidine blue and give a positive acid Schiff reaction
(see Chapter 2). Metachromasia is the property exhibited by certain pure dyestuffs,
chiefly basic stains, of colouring certain tissue elements in a different colour. Certain
lysosomal
enzymes
are
good
histochemical
markers.
For
example,
acid
phosphatase is the principal enzyme which is used as a marker for the lysosomes by
the use of Gomori staining technique (Gomori, 1952). Specific stains are also used
for other lysosomal enzymes such as B-glucuronidase, aryl sulphatatase, N-acetylB-glucosaminidase and 5-bromo-4-chloroindolacetate esterase.
Lysosomal Enzymes
According to a recent estimate, a lysosome may contain up to 40 types of
hydrolytic enzymes (see Alberts et al., 1989). they include proteases (e.g.,
cathespsin for protein digestion), nucleases, glycosidases (for digestion of
polysaccharides and glycosides ), lipase, phospholipases, phosphatases and
sulphatases (table 8-2). All lysosomal enzymes are acid hydrolases, optimally active
at the pH5 maintained within lysosomes. The lysosomal enzymes latent and out of
the cytoplasmic matrix or cytosol (whose pH is about ~ 7.2), but the acid dependency
of lysosmal enzymes protects the contents of the cytosol (cytoplasmic matrix)
against any damage even if leakage of lysosomal enzymes should occur.
The so-called latency of the lysosomal enzymes is due to the presence of the
membrane which is resistant to the enzymes that it encloses. Most probably this is to
42
the face that most lysosomal hydrolases are membrane-bound, which may prevalent
the active centre of enzymes to gain access to susceptible groups in the membrane (
Reid and Leech, 1980).
Kind of Lysosomes
Lysosomes are extremely dynamic organelles, exhibiting polymorphism in
their morphology. Following four types of lysosomes have been recognized in
different types of cells or at different time in the same cell. Of these, only the first is
the primary lysosome, the other three have been grouped together as secondary
lysosomes.
1.
Primary Lysosomes
These are also called storage granules, protolysosomes or virgin lysosomes.
Primary lysosomes are newly formed organelles bounded by a single
membrane and typically having a diameter of 100nm. They contain the
degradative enzymes which have not participated in any digestive process.
Each primary lysosome contains one type of enzyme or another and it is only
in the secondary lysosome that the full complement of acid hydrolases is
present.
2.
Heterophagosomes
They
are
also
called
heterophagic
vacuoles,
heterolysosomes
or
phagolysosomes. Heterophagosomes are formed by the fusion of primary
lysosomes with cytoplasmic vacuoles containing extracellular particles into the
cell by any of a variety of endocytic processes (e.g., pinocytosis, phagocytosis
or receptor mediated endocytosis. The digestion of engulfed substances takes
place by the enzymatic activities of the hydrolytic enzymes of the secondary
lysosomes.
The digested material has low molecular weight and readily
passes through the membrane of the lysosomes to become the part of the
matrix.
3.
Autophagosomes
They are also called autophagic vacuole, cytolysosomes or autolysosomes.
Primary lysosomes are able to digest intracellular structures including
mitochondria,
ribosomes, peroxisomes and
glycogen
granules.
Such
autodigestion (called autophagy) of cellular organelles is a normal event
during cell growth and repair and is especially prevalent in differentiating and
de-differentiating tissues (e.g., cells undergoing programmed death during
43
meta-morphosis or regeneration) and tissue under stress. Autophagy takes
several forms. In some cases the lysosome appears to flow around the cell
structure and fuse, enclosing it in a double membrane sac, the lysosomal
enzymes being initially confined between the membrane. The inner
membrane then breaks down and the enzymes are able to penetrate to the
enclosed organelle. In other cases, the organelle to be digested is first
encased by smooth ER, forming a vesicle that fuses with a primary lysosome.
Lysosomes also regularly engulf bits of cytosol (cytoplasmic matrix) which is
degraded by a process, called microautophagy.
As digestion proceeds, it becomes increasingly difficult to identify the nature
of
the
original
secondary
lysosome
(i.e.,
heterophagosome
or
autophagosome) and the more general term digestive vacuole is used to
describe the organelle at this stage.
4.
Residual Bodies
They are also called telolysosomes or dense bodies. Residual bodies are
forced if the digestion inside the food vacuole is incomplete. Incomplete
digestion may be due to absence of some lysosomal enzymes. The
undigested food is present in the digestive vacuole as the residues and may
take the form of whorls of membranes. grains, amorphouse masses, ferritinlike or myelin figures.
Residual bodies are large, irregular in shape and are usually quite electrondense. In some cells, such as Amoeba and other potozoa, these residual
bodies are eliminated defecation. In other cells, residual bodies may remain
for a long time and may load the cells to result in their aging. For example,
pigment inclusions (age pigment or lipofuscin granules) found in nerve cells
(also in liver cells, heart cells and muscle cells) of old animals may be due to
the accumulation of residual bodies.
Origin
The biogenesis (origin) of the lysosomes requires the synthesis of specialized
lysosomal hydrolases and membrane proteins. Both classes of proteins are
synthesized in the ER and transported through the Golgi apparatus, then transported
from the trans Golgi network to an intermediate compartment (an endolysosome) by
means of transport vesicles (which are coated by clathrin protein). The lysosmal
44
enzymes are glycol proteins, containing N-linked oligosaccharides that are
processed in a unique way in the cis Golgi so that their mannose residues are
phophorylated. These mannose 6-phosphate (M6P) groups are recognized by M6P
groups are recognized by M6P-receptors (which are trans membrane proteins) in the
trans Golgi network that segregates the hydrolases and helps to package them into
budding clathrin-coated vesicles which quickly lose their coats. These transport
vesicles containing the M6P-receptors act as shuttles that move the receptors back
and forth between the Golgi network and endolysosomes. They low pH in the
endolysosome dissociates the lysosomal hydrolases from this receptor, making the
transport of the hydrolases unidirectional.
Function of Lysosomes
The important functions of lysosomes are as follow :
1.
Digestion of large extracellular particles. The lysosomes digests the food
contents of the phoagosomes or pinosomes.
2.
Digestion of intracellular substance. During the starvation, the lysosomes
digest the stored food contents viz proteins, lipids and carbohydrates
(glycogen) of the cytoplasm and supply to the cell necessary amount of
energy.
3.
Autolysis. In certain pathological conditions the lysosomes start to digest the
various organelles of the cells and this process is known as autolysis or
cellular autophagy. When a cell dies, the lysosome membrane ruptures and
enzymes are liberated. These enzymes digest the dead cells. In the process
of metamorphosis of amphibians and / tunicates many embryonic tissues,
e.g., gills, fins, tail, etc., are digested by the lysosomes and utilized by the
other cells.
4.
Extracellular Digestion. The lysosomes of certain cells such as sperms
discharge their enzymes outside the cell during the process of fertilization.
The lysosomal enzymes digest the limiting membrane of the ovum and form
penetration path in ovum for the sperms. Acid hydrolases are released from
osteoclasts and break down bone for the reabsorption ; these cells also
secrete lactic acid which makes the local pH enough for optimal enzyme
activity. Likewise, preceding ossification (bone formation), fibroblasts release
cathepsin D enzyme to break down the connective tissue.
45
Lysosomes in plants
Plants contain several hydrolases, but they are not always as neatly
compartmentalized as they are in animal cells. Many of these hydrolases are found
bound to and functioning within the vicinity of the cells wall and are not necessarily
contained in membrane bound vacuoles at these sites. Many types of vacuoles and
storage granules of plants are found to contain certain digestive enzymes and these
granules are considered as lysosomes of plant cell (Gahan, 1972). According to
Matile (1969) the plant lysosomes can be defined as membrane bound cell
compartments containing hydrolytic digestive enzymes. Matile (1975) has divided
vacuoles of plants into following three types :
1.
Vacuoles
The vacuole of a mature plant cell is formed from the enlargement and fusion
of smaller vacuoles present in meristematic cells; these provacuolse, which
are believed to be derived from the ER and possibly the Golgi and contain
acid hydrolases. These lysosomal enzymes are associated with the tonoplast
of large vacuole of differentiating cells. Sometimes, mitochondria and plastids
are observed inside the vacuole suggesting autophagy in plants (Swanson
and Webster,1989).
2.
Spherosomes
46
The spherosomes are membrane bounded, spherical particles of 0.5 to 2.5
m diameter, occurring in most plant cells. They have a fine granular structure
internally which is rich in lipids and proteins. They originate from the
endoplasmic reticulum (ER). Oil accumulates at the end of a strand of ER and
a small vesicle is then cut off by contribution to form particles, called
prospherosomes. The prospherosomes grow in size to form spherosoms.
Basiocally, the spherosomes are involved in lipid synthesis and storage. But,
the spherosomes of maize root tips (Matile, 1968) and spherosome of tobacco
endosperm tissue (Spichiger, 1969) have been found rich in hydrolytic
digestive enzymes and so have been considered as lysosomes. Like
lysosomes they are not only responsible for the accumulation and mobilization
of reserve lipids, but also for the digestion of other cytoplasmic components
incorporated by phagocytosis.
3.
Aleurone Grain
The aleurone grains or protein bodies are spherical membrane-bounded
storage particle occurring in the cells of endosperm and cytoledons of seeds.
They are formed during the later stages of seed ripening and disappear in the
early stages of germination. They store protein (e.g., globulins) and
phosphate in the form of phytin. Matile (1968) has demonstrated that aleurone
grains from pea seed contain a wide range of hydrolytic enzymes including
protease and phosphatase which are required for the mobilization of stored
protein and phosphate, although the presence of other enzymes such as amylase and RNAase suggest that other cell constituents may also be
digested. Thus like spherosomes, aleurone grains store reserve materials,
mobilize them during germination and in addition form a compartment for the
digestion of other cell components (Hall et al., 1974). The aleurone grains are
derived from the strands of the endoplasmic reticulum.
During germinating of barley seed, the activity of hydrolases is found to be
controlled by hormones such as gibberellic acid. Gibberellic acid, a plant
growth hormone, is released by the embryo to the aleurone layer where, in
turn, the hyrolases are released to the endosperm. This hormone operates by
derepressing appropriate genes in the aleurone cells, which then begin to
crank out new hydrolytic proteins (see Thorpe, 1984).
5.5 Endoplasmic Reticulum
47
The cytoplasmic matrix is traversed by a complex network of inter-connecting
membrane bound vacuoles or cavities. often remain concentrated in the
endoplasmic portion of the cytoplasm ; therefore, known as endoplasmic reticulum,
a name derived from the fact that in the light microscope it looks like a "net in the
cytoplasm." (Eighteenth-century European ladies carried purses of netting called
reticules).
The name "endoplasmic reticulum" was coined in 1953 by Porter, who in 1945
had observed it in electron micrographs of liver cells. Fawcet and Ito (1958), Thiery
(1958) and Rose and Pomerat (1960) have made various important contributions to
the endoplasmic reticulum.
The occurrence of the endoplasmic reticulum varies from cell to cell. The
erythrocytes (RBC), egg and embryonic cells lack in endoplasmic reticulum.
The spermatocytes have poorly developed endoplasmic reticulum. The
adipose tissues, brown fat cells and adrenocortical cells, interstitial cells of testes
and cells of corpus luteum of ovaries, sebaceous cells and retinal pigment cells
contain only smooth endoplasmic reticulum (SER). The cells of those organs which
are actively engaged in the synthesis of proteins such as chinar cells of pancreas,
plasma cells, goblet cells and cells of some endocrine glands are found to contain
rough endoplasmic reticulum (RER) which is highly developed. The presence of
both SER and RER in the hepatocytes (liver cells) is reflective of the variety of the
roles played by the liver in metabolism.
Morphology
Morphologically, the endoplasmic reticulum may occur in the following three
forms : 1. Lamellar form or cisternae (A closed, fluid-filled sac, vesicle or cavity is
called cisternae) 2. vesicular form or vesicle and 3. tubular form or tubules.
1. Cisternae. The cisternae are long, flattened, sac-like, unbranched tubules
having the diameter of 40 to 50 m. They remain arranged parallely in
bundles or stakes. PER usually exists as cisternae which occur in those cells
of pancreas, notochord and brain.
2. Vesicles. The vesicles are oval, membrane bound vacuolar structures having
the diameter of 25 to 500m. They often remain isolated in the cytoplasm and
occur in most cells but especially abundant in the SER.
48
3. Tubules. The tubules are branched structures forming the reticular system
along with the cisternae and vesicles. They usually have the diameter from 50
to 190m and occur almost in all the cells. Tubular form of ER is often found
in SER and is dynamic in nature, i.e., it is associated with membrane
movements, fission and fusion between membranes of cytocavity network
(see Thorpe, 1984).
Ultra structure
The cavities of cisternae, vesicles and tubules of the endoplasmic reticulum
are bounded by a thin membrane of 50 to 60 A 0 thickness. The membrane of
endoplasmic reticulum is fluid-mosaic like the unit membrane of the plasma
membrane, nucleus, Golgi apparatus, etc. The membrane, thus, is composed of a
bimolecular layer of phospholipids in which 'float' proteins of various sorts. The
membrane of endoplasmic reticulum remains continuous with the membranes of
plasma membrane, nuclear membrane and Golgi apparatus. The cavity of the
endoplasmic reticulum is well developed and acts as a passage for the secretory
products. Palade (1956) has observed secretory granules in the cavity of
endoplasmic reticulum.
Sometimes, the cavity of RER is very narrow with two membranes closely
apposed and is much distended in certain cells which are actively engaged in protein
sysnthesis (e.g., acinar cells, plasma cells and goblet cells). Weibel et al. , 1969,
have calculated that the total surface of ER contained in 1ml of liver tissue is about
11 square metres, two-third of which is or rough types (i.e., RER).
Types of Endoplasmic reticulum
Two types of endoplasmic reticulum have been observed in same or different
types of cells which are as follows :
1.
Agranular or Smooth Endoplasmic Reticulum
This type of endoplasmic reticulum possesses smooth walls because the
ribosomes are not attached with its membranes. The smooth type of endoplasmic
reticulum occurs mostly in those cells, which are involved in the metabolism of lipids
(including steroids) and glycogen. The smooth endoplasmic reticulum is general
found in adipose cells, interstitial cells, glycogen storing cells of the liver, conduction
fibres of heart, spermatocytes and leucocytes. The muscle cells are also rich in
smooth type of endoplasmic reticulum and here it is known as sarcoplasmic
49
reticulum. In the pigmented retinal cells it exists in the form of tightly packed vesicles
and tubes known as myeloid bodies.
Glycosomes. Although the SER forms a continuous system with RER, it has
different morphology. For example, in liver cells it consists of a tubular network that
pervades major portion of the cytoplasmic matrix. These fine tubules are present in
regions rich in glycogen and can be observed as dense particles, called glycosomes,
in the matrix. Glycosomes measure 50 to 200 mm in diameter and contain glycogen
along with enzymes involved in the synthesis of glycogen (Rybicka, 1981). Many
glycosomes attached to the membranes of SER have been observed by electron
microscopy in the liver and conduction fibre of heart.
2.
Granular of Rough Endoplasmic Reticulum
The granular or rough type of endoplasmic reticulum possesses rough walls
because the ribosomes remain attached with its membranes. Ribosomes play a vital
role in the process of protein synthesis. The granular or rough type of endoplasmic
reticulum is found abundantly in those cells which are active in protein sysnthesis
such as pancreatic cells, plasma cells, goblet cells, and liver cells. The granular type
of endoplasmic reticulum takes basiophilic stain due to its RNA content of
ribosomes. The region of the matrix containing granular type of endoplasmic
reticulum takes basiophilic stain and is names as ergastoplasm, basiophilic bodies,
chromophilic substances or Nissl bodies by early cytologists. In RER, ribosomes are
often present as polysomes held together by mRNA and are arranged in typical,
"rosettes" of spirals. RER contains two transmembrane glycoproteins (called
ribophorins I and II of 65,000 and 64,000 dalton MW, respectively), to which are
attached the ribosomes by their 60S subunits.
Isolation and chemical composition
The membranes of the endoplasmic reticulum can be isolated by subjecting
homogenized tissues to differential centrifugation. Electron microscopy of such ER
preparations reveals that the membranes disrupt to form closed vesicles (~100 nm
diameter) of either a rough or a smooth form. These membranous entities were
coined the term "microsomes" by Claude in 1940, and the relationship between
microsomes and the elements of endoplasmic reticulum in the intact cell was
established by Palade and Siekevitz 1956.
Microsomes derived from rough ER are studded with and are called rough or
granular microsomes. The ribosomes are always found on the outside surface, the
interior beings biochemical equivalent to the luminal space of the ER. Homogenate
50
also contains smooth or agranular micro-somes which lack attached ribosomes.
They may be derived in part from smooth portion of the ER and in part from
fragments of plasma membrane, Golgi apparatus, endosomes and mitochondria.
Thus, while rough microsomes can be equated with rough portions of ER, the origin
of smooth microsomes cannot be so easily assigned. However, since the
hepatocytes of liver contain exceedingly large quantities of smooth ER, therefore,
most of the smooth microsomes in liver homogenates are derived from smooth ER
(see Alberts et al., 1989).
Enzymes of the ER membranes
The membranes of the endoplasmic reticulum are found to contain many
kinds of enzymes which are needed for various important synthetic activities. Some
of the most common enzymes are found to have different transverse distribution in
the ER membranes (Table 6-1). The most important enzymes are the stearases,
NADH-cytochrome C reductase, NADH diaphorase, glucose-6-phosphotase and
Mg++ activated ATPase. Certain enzymes of the endoplasmic reticulum such as
nucleotide diphosphate are involved in the biosynthesis of phospholipids, ascorbic
acid, glucuronide, steroids and hexose metabolism. The enzymes of the
endoplasmic reticulum perform the following important functions :
1.
Synthesis of glycerides, e.g., triglycerides, phospholipids, glycolipids and
plasmalogens.
2.
Metabolism of plasmalogens.
3.
Sythesis of fatty acids.
4.
Biosynthesis of the steroids, e.g., cholesterol biosynthesis, steroid
hydrogenation of unsaturated bonds.
5.
NADPH2+O2- requiring steroid transformations : Aromatization and
hydroxylation.
6.
NADPH2+O2-requireing
steroid
transformations
:
Aromatization
hydroxylation's side-chain oxidation, thio-ether oxidations, desulphuration.
7.
L-ascorbic acid metabolism.
8.
UDP-glucose dephosphorylation.
9.
Ary1- and steroid sulphatase.
Origin of Endoplasmic reticulum
51
The exact process of the origin of endoplasmic reticulum is still unknown. But
because membranes of ER resemble with the nuclear membrane and plasma
membrane and also at the telophase stage the ER membranes are found the nuclear
envelope. Therefore, it is normally assumed that the ER has originated by
evagination of the nuclear membranes. Teikevitz and Palade (1960) have reported
that the granular type of ER has originated first and later it synthesizes the agranular
or smooth type of endoplasmic reticulum.
The synthesis of membranes of ER is found to proceed in the following
direction : RERSER. In face, membrane biogenesis is a multi-step process
involving, first, the synthesis of a basic membrane of lipid and intrinsic proteins and
thereafter the addition of other constituents such as enzymes, specific sugars, or
lipids. The process by which a membrane is modified chemically and structurally is
called membrane differentiation. The insertion of proteins into ER membranes occurs
at the level of RER. Most of these proteins are formed on membrane-bound
ribosomes. However, some of these are synthesized by free ribosomes in the cytosol
(cytoplasmic matrix) and then are inserted into the membrane. For example, the
enzyme NAD-cytochrome-b5-reductase is synthesized in the cytosol (cytoplasmic
matrix) and then becomes incorporated in various parts of the endomembrane
system (i.e., RER, SER and Golgi apparatus) and in the outer mitochondrial
membrane (Borghese and Gaetani, 1980).
Function of Endoplasmic reticulum
The endoplasmic reticulum acts as secretory, storage, circulatory and nervous
system for the cell. performs following important functions :
A.
Common Functions of Granular and Agranular Endoplasmic Reticulum
1. The endoplasmic reticulum provides and ultrastructural skeletal framework
to the cell and gives mechanical support to the colloidal cytoplasmic
matrix.
2. The exchange of molecules by the process of osmosis, diffusion and
active transport occurs through the membranes of endoplasmic reticulum.
Like plasma membrane, the ER membrane has permeases and carries.
52
3. The endoplasmic membranes contain many enzymes which perform
various synthetic and metabolic activities. Further the endoplasmic
reticulum provides increase surface for various enzymatic reactions.
4. The endoplasmic reticulum acts as an intracellular circulatory or
transporting system. Various secretory products of granular endoplasmic
reticulum are transported to various organelles as follows : Granular
ERagranular ERGolgi membranelysosomes, transport vesicles or
secretory granules. Membrane flow many also be an important mechanism
for carrying particles, molecules and ions into and out of the cells. Export
of RNA and nucleoproteins from nucleus to cytoplasm may also occur by
this type of flow (see De Robertis and De Robertis, Jr., 1987).
5. The ER membranes are found to conduct intra-cellular impulses. For
example, the sarcoplasmic reticulum transmits impulses from the surface
membrane into the deep region of the muscle fibres.
6. The ER membranes form the new nuclear envelope after each nuclear
division.
7. The sarcoplasmic reticulum plays a role in releasing calcium when the
muscle is stimulated and actively transporting calcium back into the
sarcoplasmic reticulum when the stimulation stops and the muscle must
be relaxed.
B.
Functions of Smooth Endoplasmicreticulum
Smooth ER performs the following functions of the cell :
1.
Synthesis of lipids. SER perform synthesis of lipids (e.g., phospholipids,
cholesterol, etc.) and lipoproteins. Studies with radioactive precursors
have indicated that the newly synthesized phospholipids are rapidly
transferred to other cellular membranes by the help of specific cytosolic
enzymes, called phospholipids exchange proteins.
2.
Glycogenolysis and blood glucose homeostasis. This process of glycogen
synthesis (glycogenesis) occurs in the cytosol (in glycosomes). The
enzyme UDPG-glycogen transferase, which is directly involved in the
synthesis of glycogen by addition of uridine diphosphate glucose (UDPG)
to primer glycogen is bound to the glycogen particles or glycosomes.
53
SER is found related to glycogenolysis or breakdown of glycogen. An
enzyme, called glucose-6-phosphatase (a marker enzyme) exists as an
integral protein of the membrane of SER (e.g., liver cell). Generally, this
enzyme acts as a glycogenic phosphorhydrolase that catalyzes the
release of free glucose molecule in the lumen of SER from its
phosphorylated form in liver. Thus, this process operates to maintain
homeostatic levels of glucose in the blood for the maintenance of functions
of red blood cells and nerve tissues.
3.
Sterol metabolism. The SER contains several key enzymes that catalyze
the synthesis of cholesterol which is also a precursor substance for the
biosynthesis of two types of compounds- the steroid hormones and bile
acids :
(i)
Cholesterol biosynthesis. The cholesterol is synthesized from the
acetate and its entire biosynthetic pathway involve about 20 steps,
each step catalyzed by an enzyme. Out of these twenty enzymes,
eleven enzymes are bounded to SER membranes, rest nine
enzymes are the soluble enzymes located in the cytosol and
mitochondria. Examples of SER-bound enzyme include HMG-Co A
reductase and squalene synthetase (see Thorpe, 1984).
(ii)
Bile acid synthesis. The biosynthesis of the bile acids represents a
very complex pattern of enzymes and products. Enzymes involved
in the biosynthetic pathway of bile acids are hydroxylases, monooxygenases, dehydrogenases, isomerases and reductases. For
example, by the help of the enzyme cholesterol 7-hydroxylase, the
cholesterol is first converted into 7-hydroxyl cholesterol, which is
then converted into bile acids by the help of hydroxylase enzymes.
The latter reaction requires NADPH and molecular oxygen and
depends on the enzymes of Electron transport chains of SER such
as cytochrome P-450 and NADPH-cytochrome-c reductase .
(iii)
Steroid hormone biosynthesis. Steroid hormones are synthesized in
the cells of various organs such as the cortex of adrenal gland, the
ovaries, the testes and the placenta. For example, cholesterol is the
precursor for both types of sex hormones-estrogen and testosterone54
made in the reproductive tissues, and the adrenocorticoids (e.g.,
corticosterone, aldosterone and cortisol) formed in the adrenal glands.
Many enzymes (e.g., dehydrogenase,s isomerases and hydroxylases)
are involved in the biosynthetic pathway of steroid hormones, some of
which are located in SER membranes and some occur in the
mitochondria
4.
Detoxification. Protectively, the ER chemically modifies xenobiotics (toxic
materials of both endogenous and exogenous origin), making them more
hydrophilic, hence, more readily excreted. Among these materials are drugs,
aspirin (acetyl-salicylic-acid), insecticides, anaesthetics, petroleum products,
pollutant and carcinogens (i.e., inducers of cancer ; e.g., 3-4-benzophrene
and 3-methyl cholantherene).
The enzymes involved in the detoxification of aromatic hydrocarbonds are
aryhydraoxylases. It is now know that benzophyrene (found in charcoalbroiled meat) is not carcinogenic, but under the action of aryl hydroxylase
enzyme in the liver, it is converted into 5,6-epoxide, which is a powerful
carcinogen (see De Robertis and De Robertis, Jr., 1987)
A wide variety of drugs (e.g., Phenobarbital), when administrated to animals,
they bring about the proliferation of the ER membranes (first RER and then
SER) and /or enhanced activity of enzymes related to detoxification (Thorpoe,
1984).
C.
Functions of Rough Endoplasmic Reticulum
The major function of the rough ER is the synthesis of protein. It has long
been assumed that proteins destined for secretion (i.e., export) from the cell
or proteins to be used in the synthesis of cellular membranes are synthesized
on rough DR-bound ribosomes, while cytoplasmic proteins are translated for
the most part on free ribosomes. In fact, the array of the rough endoplasmic
reticulum provides extensive surface area for the association of metabolically
active enzymes, amino acids and ribosomes. There is more efficient
functioning of these materials to synthesize proteins when oriented on a
membrane surface than when they are simply in solution, mainly because
55
chemical combinations between molecules can be accomplished in specific
geometric patterns.
The membrane-bound ribosomes are attached with specific binding sites or
receptors of rough ER membrane by their large 60S subunit, with small or
40S subunit sitting on top like a cap. These receptors are membrane proteins
which extend well into and possibly through the lipid bilayer. The receptor
proteins with bound ribosomes can float laterally like other membrane proteins
and may facilitate formation of the polysome and probably translation which
requires that mRNA and ribosome move with respect to each other.
Further, the secretory proteins, instead of passing into the cytoplasm, appear
to pass instead into the cisternae of the rough ER and are, thus, protected
56
from protease enzymes of cytoplasm. It is calculated that about 40 amino acid
residues long segment at the - COOH end of the nascent protein remains
protected inside the tunnel of 'free' or 'bond' ribosomes and rest of the chain,
with-NH2 end, is protected by the lumen of RER. The passage of nascent
polypeptide chain into the ER cisterna take place during translation leaving
only a small segment exposed to the cytoplasm at any one time.
Protein glycosylation. The covalent addition of sugars to the secretory
proteins (i.e., glycosylation) is one of the major biosynthetic functions of rough
ER. Most of the proteins that are isolated in the lumen of RER before being
transported to the Golgi apparatus, lysosomes, plasma membrane or
extracellular space, are glycoproteins (A notable exception is albumin). In
contrast, very few proteins in the cytosol (Cytoplasmic matrix) are
glycosylated and those that carry them have a different sugar modification.
The process of protein glycosylation in RER lumen is one of the most well
understood cell biological phenomena. During this process, a single species
of loligosaccharide (Which comprises N-acetyl-glucosamine, mannose and
glucose, containing a total of 14 sugar residues) is transferred to proteins in
the ER.
Check your progress: 1
1. Notes- Write your answer in the space given.
2. Compare your answer with the one given at the end of the unit.
1. What is photo-respiration?
2. Explain Glycogenolysis?
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
57
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
5.6
Techniques in cell biology; Immunotechniques
Radioimmunoassay
On of the most sensitive techniques for detecting antigen or antibody is
radioimmunoassay (RIA). The technique was first developed in 1960 by two
endocrinologists, S.A. Berson and Rosalyn Yalow, to determine levels of insulin-antiinsulin complexes in diabetics. Although their technique encountered some
skepticism, it soon proved its value for measuring hormones, serum proteins, drugs,
and vitamins at concentrations of 0.001 micrograms per milliliter or less. In 1977,
some years after Berson's death, the significance of the technique was
acknowledged by the award of a Nobel Prize to Yalow.
The Principle of RIA involves competitive binding of radiolabeled antigen and
unlabeled antigen to a high-affinity antibody. The labeled antigen is mixed with antibody at a concentration that saturates the antigen-binding sites of the antibody. Then
test samples of unlabeled antigen of unknown concentration are added in
progressively larger amounts. The antibody does not distinguish labeled from
unlabeled antigen, so the two kinds of antigen compete for available binding sites on
the antibody. As the concentration of unlabeled antigen increases, more labeled
antigen will be displaced from the binding sites. The decrease in the amount of radio
labeled antigen bound to specific antibody in the presence of the test sample is
measured in order to determine the amount of antigen present in the test sample.
The antigen is generally labeled with a gamma-emitting isotope such as
125I,
but
beta-emitting isotopes such as tritium (3H) are also routinely used as labels. The
radio labeled antigen is part of the assay mixture; the test sample may be a complex
mixture, such as serum or other body fluids, that contains the unlabeled antigen. The
first step in setting up an RIA is to determine the amount of antibody needed to bind
50% to 70% of a fixed quantity of radioactive antigen in the assay mixture. This ratio
of antibody to Ag* is chosen to ensure that the number of epitope presented by the
labeled antigen always exceeds the total number of antibody binding sites.
58
Consequently, unlabeled antigen added to the sample mixture will complete with
radio labeled antigen for the limited supply of antibody. Even a small amount of
unlabeled antigen added to the assay mixture of labeled antigen and antibody will
cause a decrease in the amount of radioactive antigen bound, and this decrease will
be proportional to the amount of unlabeled antigen added. To determine the amount
of labeled antigen bound, the Ag-Ab complex is precipitated to separate it from free
antigen (antigen not bound to antibody), and the radioactivity in the precipitate is
measured. A standard curve can be generated using unlabeled antigen samples of
known concentration (in place of the test sample), and from this plot the amount of
antigen in the test mixture may be precisely determined.
Several methods have been developed for separating bound antigen from free
antigen in RIA. One method involves precipitating Ag-Ab complexes with a
secondary anti-isotope antiserum. For example, if the Ag-Ab complex contains rabbit
IgG antibody, then goat anti-rabbit IgG will bind to the rabbit IgG and precipitate the
complex. Another method makes use of the fact that protein A of Staphylococcus
aureus has high affinity for IgG. If the Ag-Ab complex contains an IgG antibody, the
complex can be precipitated by mixing with formalin-killed S. aureus. After removal
of the complex by either of these methods, the amount of free labeled antigen
remaining in the supernatant can be measured in a radiation counter; subtracting this
value from the total amount of labeled antigen added yields the amount of labeled
antigen bound.
Various solid-phase RIAs have been developed that make it easier to separate the
Ag-Ab complex from the unbound antigen. In some cases, the antibody is covalently
cross-linked to Sepharose beads. The amount of radiolabeled antigen bound to the
beads can be measured after the beads have been centrifuged and washed.
Alternatively, the antibody can be immobilized on polystyrene or poly-vinylchloride
wells and the amount of free labeled antigen in the supernatant can be determined in
a radiation counter. In another approach, the antibody is immobilized on the walls of
microtiter wells and the amount of bound antigen determined. Because the
procedure requires only small amounts of sample and can be conducted in small 96well microtiter plates (slightly larger than a 3 × 5 card), this procedure is well suited
for determining the concentration of a particular antigen in large numbers of
samples. For example, a microtiter RIA has been widely used to screen for donor
blood has sharply reduced the incidence of hepatitis B infections in recipients of
blood transfusions.
59
ELISA
Enzyme-Linked Immunosorbent assay, commonly known as ELISA (or EIA), is
similar in principle to RIA but depends on an enzyme rather than a radioactive label.
An enzyme conjugated with an antibody reacts with a colorless substrate to generate
a colored reaction product. Such a substrate is called a chromogenic substrate. A
number of enzymes have been employed for ELISA, including alkaline phosphatase,
horseradish peroxidase, and -galactosidase. These assays match the sensitivity of
RIAs and have the advantage of beings safer and less costly.
There are numerous variants of ELISA
A number of variations of ELISA have been developed, allowing qualitative detection
or quantitative measurement of either antigen or antibody. Each type of ELISA can
be used qualitatively to detect the presence of antibody or antigen. Alternatively, a
standard curve based on known concentrations of antibody or antigen is prepared,
from which the unknown concentration of a sample can be determined.
Indirect ELISA
Antibody can be detected or quantitatively determined with an indirect ELISA. Serum
or some other sample containing primary antibody (Ab 1) is added to an antigen
coated microtiler well and allowed to react with the antigen attached to the well. After
any free Ab1 is washed away, the presence of antibody bound to the antigen is
detected by adding an enzyme-conjugated secondary antibody (Ab2) that binds to
60
the primary antibody. Any free Ab2 is then washed away, and a substrate for the
enzyme is added. The amount of colored reaction product that forms is measured by
specialized spectrophotometric plate readers, which can measured the absorbance
of all of the wells of a 96-well plate in seconds.
Indirect ELISA is the method of choice to detect the presence of serum antibodies
against human immunodeficiency virus (HIV), the causative agent of AIDS. In this
assay, recombinant envelope and core proteins of HIV are adsorbed as solid-phase
antigens to microtiter wells. Individuals infected with HIV will produce serum
antibodies to epitopes on these viral proteins. Generally, serum antibodies to HIV
can
be
detected
by
indirect
ELISA.
Sandwich ELISA
Antigen can be detected or measured by a sandwich ELISA. In this technique, the
antibody (rather than the antigen) is immobilized on a microtiter well. A sample
containing antigen is added and allowed to react with the immobilized antibody. After
the well is washed, a second enzyme-linked antibody specific for a different epitope
61
on the antigen is added and allowed to react with the bound antigen. After any free
second antibody is removed by washing substrate is added, and the colored reaction
product is measured.
Competitive ELISA
Another variation for measuring amounts of antigen is competitive ELISA. In this
technique, antibody is first incubated in solution with a sample containing antigen.
The antigen antibody mixture is then added to an antigen coated microtiter well. The
more antigen present in the sample, the less free antibody will be available to bind to
the antigen-coated well. Addition of an enzyme-conjugated secondary antibody (Ab2)
specific for the isotype of the primary antibody can be used to determine the amount
of primary antibody bound to the well, as in an indirect ELISA. In the competitive
assay, however, then higher the concentration of antigen in the original sample, the
lower the absorbance.
Western Blotting
Identification of a specific protein in a complex mixture of proteins can be a
accomplished by a technique known as Western blotting, named for its similarity to
Southern blotting, which detects DNA fragments, and Northern blotting which detects
mRNA. In Western, a protein mixture is electrophoretically separated on an SDSpolyacrylamide gel (SDS-PAGE), a slab gel infused with sodium dodecyl sulfate
(SDS), a dissociating agent. The protein bands are transferred to a nitrocellulose
membrane by electrophoresis, and the individual protein bands identified by flooding
the membrane with radiolabeled or enzyme-protein of interest. The Ag-Ab
complexes that form on the band containing the protein recognized by the antibody
can was bound by a radioactive antibody, its position on the blot can be determined
by exposing the membrane to a sheet of x-ray film, a procedure called
autoradiography. However, the linked antibodies against the protein. After binding of
the enzyme antibody conjugate, addition of a chromogenic substrate that produces a
highly colored and insoluble product causes the appearance of a colored band at the
site of the target antigen. Even greater sensitivity can be achieved if a
chemicluminescent compound with suitable enhancing agents is used to produce
light at the antigen site.
Western blotting can also identify a specific antibody in a mixture. In this case,
known antigens of well defined molecular weight are separated by SDS-PAGE and
blotted onto nitrocellulose. The separated bands of known antigens are then probed
62
with the sample suspected of containing antibody specific for one or more of these
antigens. Reaction of an antibody with a band is detected by using either
radiolabeled or enzyme-linked secondary antibody that is specific for the spiciest of
the antibodies in the test sample. The most widely used application of this procedure
is in confirmatory testing for HIV, where western blotting is used to determine
whether the patient has antibodies that react with one or more viral proteins.
5.7
FISH-Fluorescent in situ hybridization
FISH (Fluorescent in situ hybridization) is a cytogenetic technique that can be used to detect
and localize the presence or absence of specific DNA sequences on chromosomes. It uses
fluorescent probes that bind to only those parts of the chromosome with which they show a
high degree of sequence similarity. Fluorescence microscopy can be used to find out where
the fluorescent probe bound to the chromosome. FISH is often used for finding specific
features in DNA. These features can be used in genetic counseling, medicine, and species
identification.
Probes are often derived from fragments of DNA that were isolated, purified, and amplified
for use in the Human Genome Project. The size of the human genome is so large, compared
to the length that could be sequenced directly, that it was necessary to divide the genome into
fragments. The fragments were added into a framework that made it possible to use bacteria
to replicate the fragments. The fragments were put into order by analyzing size-exclusion
separation of enzymatically-digested fragments. Clonal populations of bacteria, each
population maintaining a single artificial chromosome, are stored in various laboratories
around the world. The artificial chromosomes (BAC) can be grown, extracted, and labeled, in
any lab. These fragments are on the order of 100 thousand base-pairs, and are the basis for
most FISH probes.
Preparation and Hybridization Process
63
Scheme of the principle of the FISH Experiment to localize a gene in the
nucleus.
First, a probe is constructed. The probe must be large enough to hybridize specifically with
its target but not too large to impede the hybridization process. The probe is tagged directly
with fluorophores, with targets for antibodies or with biotin. Tagging can be done in various
ways, for example nick translation and PCR using tagged nucleotides.
Then, an interphase or metaphase chromosome preparation is produced. The chromosomes
are firmly attached to a substrate, usually glass. Repetitive DNA sequences must be blocked
64
by adding short fragments of DNA to the sample. The probe is then applied to the
chromosome DNA and incubated for approximately 12 hours while hybridizing. Several
wash steps remove all un-hybridized or partially-hybridized probes. The results are then
visualized and quantified using a microscope that is capable of exciting the dye and recording
images.
If the fluorescent signal is weak, amplification of the signal may be necessary in order to
exceed the detection threshold of the microscope. Florescent signal strength depends on many
factors such as probe labeling efficiency, the type of probe, and the type of dye.
Fluorescently-tagged antibodies or streptavidin are bound to the dye molecule. These
secondary components are selected so that they have a strong signal.
Variations on Probes and Analysis
FISH is a very general technique. It is often arbitrarily divided into more specific categories
based on application, however each category is similar in that, in a chemical sense, the
technique is the same; hybridization is the common denominator. The differences between
the various FISH techniques are usually due to the construction and content of the
fluorescently-labeled DNA probe. The size, overlap, colour, and mixture of the probes make
possible all FISH techniques.
Probe size is important because longer probes hybridize more specifically than shorter
probes. The overlap defines the resolution of detectable features. If the goal of an experiment
is to detect the breakpoint of a translocation, then the overlap of the probes — the degree to
which one DNA sequence is contained in the adjacent probes — defines the minimum
window in which the breakpoint occurs.
The mixture of probes determines the type of feature the probe can detect. Probes that
hybridize along an entire chromosome are used to count the number of a certain
chromosome, show translocations, or identify extra-chromosomal fragments of chromatin.
This is often called "whole-chromosome painting." If every possible probe is used, every
chromosome, (in essence the whole genome) would be marked fluorescently, which would
not be particularly useful for determining features of individual sequences. A mixture of
smaller probes can be created that are specific to a particular region (locus) of DNA; these
mixtures are used to detect deletion mutations. When combined with a specific colour, a
locus-specific probe mixture is used to detect very specific translocations. Special locusspecific probe mixtures are often used to count chromosomes, by binding to the centromeric
65
regions of chromosomes, which are unique enough to identify each chromosome (with the
exception of Chromosome 13, 14 21, 22.)
Because modern microscopes can detect a range of colours in fluorescent dyes each human
chromosome can be identified (M-FISH) using whole-chromosome probe mixtures and a
variety of colours. There are currently twice as many chromosomes than fluorescent dye
colours. However, ratios of probe mixtures can be used to create additional colours. As with
comparative genomic hybridization, the probe mixture for the secondary colours is created by
mixing the correct ratio of two sets of differently-labeled probes for the same chromosome.
Differently coloured probes can be used for the detection of translocations. Several
techniques exploit the resolution limitations of microscopes to resolve spatial distributions of
dye below a few hundred nanometers. Colours that are adjacent appear to overlap, and a
secondary colour is observed.
In reciprocal translocations, where both breakpoints are known, locus-specific probes are
made for it and part of the region one either side of breakpoint. In normal cells, two colours
will be visible; in diseased cells such as those found in BCR/ABL translocations, the two dye
colours overlap, and a third colour is observed. This technique is known as double-fusion
FISH or D-FISH. In translocations where only one of the breakpoints is known or constant,
locus-specific probes are made for one side of the breakpoint and the other intact
chromosome. In normal cells, the secondary colour is observed, but only the primary colour
is observed when the translocation occurs. This technique is known as "break-apart FISH".
Medical applications
Often parents of children with a developmental delay want to know more about their child's
conditions before choosing to have another child. These concerns can be addressed by
analysis of the parents' and child's DNA. In cases where the child's developmental delay is
not understood, the cause of it can be determined using FISH and cytogenetic techniques.
Examples of diseases that are diagnosed using FISH include Prader-Willi syndrome,
Angelman syndrome, 22q13 deletion syndrome, chronic myelogenous leukemia, acute
lymphoblastic leukemia, Cri-du-chat, Velocardiofacial syndrome, and Down syndrome.
In medicine, FISH can be used to form a diagnosis, to evaluate prognosis, or to evaluate
remission of a disease, such as cancer. Treatment can then be specifically tailored. A
traditional exam involving metaphase chromosome analysis is often unable to identify
features that distinguish one disease from another, due to subtle chromosomal features; FISH
66
can elucidate these differences. FISH can also be used to detect diseased cells more easily
than standard Cytogenetic methods, which require dividing cells and requires labor and timeintensive manual preparation and analysis of the slides by a technologist. FISH, on the other
hand, does not require living cells and can be quantified automatically, a computer counts the
fluorescent dots present. However, a trained technologist is required to distinguish subtle
differences in banding patterns on bent and twisted metaphase chromosomes.
Species identification
FISH is often used in clinical studies. If a patient is infected with a suspected pathogen,
bacteria, from the patient's tissues or fluids, are typically grown on agar to determine the
identity of the pathogen. Many bacteria, however, even well-known species, do not grow well
under laboratory conditions. FISH can be used to detect directly the presence of the suspect
on small samples of patient's tissue.
FISH can also be to used compare the genomes of two biological species, to deduce
evolutionary relationships. A similar hybridization technique is called a zoo blot. Bacterial
FISH probes are often primers for the 16s rRNA region.
FISH is widely used in the field of microbial ecology, to identify microorganisms. Bio-films,
for example, are composed of complex (often) multi-species bacterial organizations.
Preparing DNA probes for one species and performing FISH with this probe allows one to
visualize the distribution of this specific species within the bio-film. Preparing probes (in two
different colors) for two species allows to visualize/study co-localization of these two species
in the bio-film, and can be useful in determining the fine architecture of the bio-film.
5.8
Confocal Microscopy
In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded in
light from a light source. Due to the conservation of light intensity transportation, all parts of
the specimen throughout the optical path will be excited and the fluorescence detected by a
photo-detector or a camera. In contrast, a confocal microscope uses point illumination and a
pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus
information. Only the light within the focal plane can be detected, so the image quality is
much better than that of wide-field images. As only one point is illuminated at a time in
67
confocal microscopy, 2D or 3D imaging requires scanning over a regular raster (i.e. a
rectangular pattern of parallel scanning lines) in the specimen. The thickness of the focal
plane is defined mostly by the square of the numerical aperture of the objective lens, and also
by the optical properties of the specimen and the ambient index of refraction.
Types
Three types of confocal microscopes are commercially available: Confocal laser scanning
microscopes, spinning-disk (Nipkow disk) confocal microscopes and Programmable Array
Microscopes (PAM). Confocal laser scanning microscopy yields better image quality than
Nipkow and PAM, but the imaging frame rate was very slow (less than 3 frames/second)
until recently; spinning-disk confocal microscopes can achieve video rate imaging—a
desirable feature for dynamic observations such as live cell imaging. Confocal laser scanning
microscopy has now been improved to provide better than video rate (60 frames/second)
imaging by using MEMS based scanning mirrorsConfocal microscopy offers several
advantages over conventional optical microscopy, including controllable depth of field, the
elimination of image degrading out-of-focus information, and the ability to collect serial
optical sections from thick specimens. The key to the confocal approach is the use of spatial
filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of
focus. There has been a tremendous explosion in the popularity of confocal microscopy in
recent years, due in part to the relative ease with which extremely high-quality images can be
68
obtained from specimens prepared for conventional optical microscopy, and in its great
number of applications in many areas of current research interest.
Basic Concepts –
Current instruments are highly evolved from the earliest versions, but the principle of
confocal imaging advanced by Marvin Minsky, and patented in 1957, is employed in all
modern confocal microscopes. In a conventional wide field microscope, the entire specimen
is bathed in light from a mercury or xenon source, and the image can be viewed directly by
eye or projected onto an image capture device or photographic film. In contrast, the method
of image formation in a confocal microscope is fundamentally different. Illumination is
achieved by scanning one or more focused beams of light, usually from a laser or arcdischarge source, across the specimen. This point of illumination is brought to focus in the
specimen by the objective lens, and laterally scanned using some form of scanning device
under computer control. The sequences of points of light from the specimen are detected by a
photomultiplier tube (PMT) through a pinhole (or in some cases, a slit), and the output from
the PMT is built into an image and displayed by the computer. Although unstained specimens
can be viewed using light reflected back from the specimen, they usually are labeled with one
or more fluorescent probes.
Imaging Modes - A number of different imaging modes are used in the application of
confocal microscopy to a vast variety of specimen types. They all rely on the ability of the
technique to produce high-resolution images, termed optical sections, in sequence through
relatively thick sections or whole-mount specimens. Based on the optical section as the basic
image unit, data can be collected from fixed and stained specimens in single, double, triple,
or multiple-wavelength illumination modes, and the images collected with the various
illumination and labeling strategies will be in register with each other. Live cell imaging and
time-lapse sequences are possible, and digital image processing methods applied to sequences
of images allow z-series and three-dimensional representation of specimens, as well as the
time-sequence presentation of 3D data as four-dimensional imaging. Reflected light imaging
was the mode used in early confocal instruments, but any of the transmitted light imaging
modes commonly employed in microscopy can be utilized in the laser scanning confocal
microscope.
69
Refinements in design have simplified confocal microscopy to the extent that it has become a
standard research tool in cell biology. However, as confocal microscopes have become more
powerful, they have also become more demanding of their optical components. In fact,
optical aberrations that cause subtle defects in image quality in wide field microscopy can
have devastating effects in confocal microscopy. Unfortunately, the exacting optical
requirements of confocal microscopy are often hidden by the optical system that guarantees a
sharp image, even when the microscope is performing poorly.
Three-Color Imaging for Confocal Microscopy - The laser scanning confocal microscope
(LSCM) is routinely used to produce digital images of single-, double-, and triple-labeled
fluorescent samples. The use of red, green and blue (RGB) color is most informative for
displaying the distribution of up to three fluorescent probes labeling a cell, where any colocalization is observed as a different additive color when the images are colorized and
merged into a single three-color image.
Check your progress: 2
1. Notes- Write your answer in the space given.
2. Compare your answer with the one given at the end of the unit.
1.
What is FISH?
2.
What are the three types of confocal microscopes?
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------5.9
Let us sum up
Microbodies are spherical or oblate in form. They are bounded by a single
membrane and have an interior or matrix which is amorphous or granular
Microcopies are most easily distinguished from other cell organelles by their
content of catalase enzyme.
70
In green leaves, there are peroxisomes that carry out a process called
photorespiration which is a light - stimulated production of CO2 that is different
from the generation of CO2 by mitochondria in the dark.
The Golgi apparatus occurs in all cells except the prokaryotic cells (viz.,
mycoplasmas, bacteria and blue green algae) and eukaryotic cells of certain
fungi, sperm cells of bryophytes and pteridiophytes, cells of mature sieve tubes
of plants and mature sperm and red blood cells of animals. Their number per
plant cell can vary from several hundred as in tissues of corn root and algal
rhizoids (i.e., more than 25,000 in algal rhizoids, Sievers, 1965), to a single
organelle in some algae.
The lysosomes (Gr. lyso=digestive + soma = boy) are tiny membrane-bound
vesicles involved in intracellular digestion. They contain a variety of hydrolytic
enzymes that remain active under acidic conditions. The lysosomal lumen is
maintained at an acidic pH (around 5) by an ATP-driven proton pump in the
membrane. Thus, these remarkable organelles are primarily meant for the
digestion of a variety of biological materials and secondarily cause aging and
death of animal cells and also a variety of human diseases such as cancer, gout
Pompe's silicosis and I-cell disease.
The endoplasmic reticulum provides and ultrastructural skeletal framework to the
cell and gives mechanical support to the colloidal cytoplasmic matrix.
The exchange of molecules by the process of osmosis, diffusion and active
transport occurs through the membranes of endoplasmic reticulum. Like plasma
membrane, the ER membrane has permeases and carries.
The endoplasmic membranes contain many enzymes which perform various
synthetic and metabolic activities. Further the endoplasmic reticulum provides
increase surface for various enzymatic reactions.
RIA involves competitive binding of radiolabeled antigen and unlabeled antigen
to a high-affinity antibody. The labeled antigen is mixed with anti-body at a
concentration that saturates the antigen-binding sites of the antibody.
ELISA (or EIA), is similar in principle to RIA but depends on an enzyme rather
than a radioactive label. An enzyme conjugated with an antibody reacts with a
colorless substrate to generate a colored reaction product. Such a substrate is
called a chromogenic substrate. A number of enzymes have been employed for
71
ELISA, including alkaline phosphatase, horseradish peroxidase, and galactosidase. These assays match the sensitivity of RIAs and have the
advantage of beings safer and less costly.
FISH (Fluorescent in situ hybridization) is a cytogenetic technique that can be
used to detect and localize the presence or absence of specific DNA sequences
on chromosomes.
A confocal microscope uses point illumination and a pinhole in an optically
conjugate plane in front of the detector to eliminate out-of-focus information.
Only the light within the focal plane can be detected, so the image quality is
much better than that of wide-field images. As only one point is illuminated at a
time in confocal microscopy, 2D or 3D imaging requires scanning over a regular
raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen.
Check your progress 1: The key
1.
Photo-respiration is so called
because light induces the synthesis of
glycolic acid in chloroplasts. This process involves intervention of two
organelles : chloroplasts and peroxisomes.
2.
Glycogenolysis and blood glucose homeostasis. This process of glycogen
synthesis (glycogenesis) occurs in the cytosol (in glycosomes). The
enzyme UDPG-glycogen transferase, which is directly involved in the
synthesis of glycogen by addition of uridine diphosphate glucose (UDPG)
to primer glycogen is bound to the glycogen particles or glycosomes.
Check your progress 2 : The key
1.
FISH (Fluorescent in situ hybridization) is a cytogenetic technique that can be
used to detect and localize the presence or absence of specific DNA sequences on
chromosomes.
2.
Three types of confocal microscopes are commercially available: Confocal
laser scanning microscopes, spinning-disk (Nipkow disk) confocal microscopes and
Programmable Array Microscopes (PAM).
5.10
Assignment/ Activity
1.
Explain different molecular diagnostic techniques with their use.
2.
Do a activity in sandwich ELISA Technique.(Use Kit from Gene if required.)
72
5.11
Reference
Annelie Pernthaler, Jakob Pernthaler, Rudolf Amann (2002): Fluorescence In Situ
Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria,
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2002, p. 3094–3101 Vol. 68,
No. 6 DOI: 10.1128/AEM.68.6.3094–3101.2002
James B. Pawley - Department of Zoology, 1117 W. Johnson Dr., University of Wisconsin,
Madison, Wisconsin 53706.
Kenneth W. Dunn and Exing Wang - Department of Medicine, Indiana University, School of
Medicine, 1120 South Drive, FH115, Indianapolis, Indiana 46202-5116.
Matthew Parry-Hill, Thomas J. Fellers, and Michael W. Davidson - National High Magnetic
Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee,
Florida, 32310.
Michael Wagner, Matthias Horny and Holger Daimsz (2003): Fluorescence in situ
hybridisation for the identification and characterisation of prokaryotes. Current Opinion in
Microbiology 2003, 6:302–309 DOI:10.1016/S1369-5274(03)00054-7
Stephen W. Paddock, Eric J. Hazen, and Peter J. DeVries - Laboratory of Molecular Biology,
Howard Hughes Medical Institute, University of Wisconsin, Madison, Wisconsin 53706.
V. J. Sieben, C. S. Debes-Marun, P. M. Pilarski, G. V. Kaigala, L. M. Pilarski, and C.
Backhouse, "FISH and chips: chromosomal analysis on microfluidic platforms", IET
Nanobiotechnology, vol. 1(3), pp. 27-35, June 2007. DOI:10.1049/iet-nbt:20060021
73
74