Cell organelles
The general architecture of an eukaryotic cell (karyon in Greek means nucleus) is
quite complex (Fig. 1): the cytoplasm, filling the space between the nucleus and
cell membrane, contains a variety of organelles, many of them membranedelimited. Their density and degree of development varies according to the
specialization and functional processes occurring in that particular cell. In
addition, various filamentous structures form the cell cytoskeleton, where most of
these organelles are attached.
Fig. 1 General architecture of an eukaryotic cell, showing the membranedelimited and filamentous organelles.
Mitochondria (Gr. mitos = thread) are elongated organelles covered by a double
membrane bilayer. The inner membrane is folded to the interior, generating a
number of protrusions called cristae, which ensure a large surface area. The
inner volume of a mitochondrion, called matrix, contains a number of dense
granules, representing a store of intracellular calcium. It also contains
mitochondrial DNA and RNA, encoding a few dozens of mitochondrial proteins,
which prove that mitochondria were originally independent organisms that have
chosen at a certain moment during evolution to live in symbiosis with other cells,
being incorporated and providing a vital function: completion of aerobic
intermediate metabolism of glucose, fatty acids and some aminoacids and their
coupling to the respiratory chain and oxidative phosphorylation. Thus,
mitochondria function as a main power plant of the cell, generating the largest
amount of ATP, the “energy currency” in living systems.
Fig. 2 Reactions of the Krebs cycle and their coupling via coenzymes NAD+ and
FAD to the respiratory electron transport chain, located in the inner mitochondrial
membrane. The proton gradient created across this membrane drives ATP
synthesis.
The Krebs-Henseleit or tricarboxylic acid cycle (Fig. 2) accomplishes the final
degradation of glucose and fatty acids. The end product of anaerobic glycolysis,
pyruvate, enters the mitochondrion, where it is converted to acetyl-CoA
(coenzyme A), thus beginning the cycle by coupling two carbon units to
oxaloacetate and generating citrate. In cells where fatty acid degradation
represents a second energy source, like cardiomyocytes, these fatty acids are
processed in the mitochondrion as well, by progressive detachment of two
carbon units via acetyl-CoA in the Lynen spiral. Degradation of each pyruvate
molecule in the Krebs cycle produces 4 reduced nicotinamide-adeninedinucleotide molecules (NADH + H+) and one reduced flavinamide-adeninedinucleotide molecule (FADH2). These oxido-reduction coenzymes are later
reoxidized in the respiratory chain, located in the inner mitochondrial membrane
(for this reason, the protein content of this membrane is unusually high,
approximately 70 %). The respiratory chain (Fig. 2) is composed of a series of
metaloproteins called cytochromes, structurally related to hemoglobin, the
oxygen carrier. Their cofactor is often heme. This similarity explains the toxic
effects of cyanides, carbon monoxide, azides, etc., which complex the heme iron.
Other compounds like rotenone and barbiturates inhibit coenzyme Q
(ubiquinone). These oxidoreduction enzymes are grouped in 4 complexes (I –
IV), and they achieve the task of transferring electrons from a high to a low
energy level. During these electron transfers, protons are released in the outer
mitochondrial space at well-defined points, as shown in Fig. 2. Thus, a proton
gradient is achieved across the inner mitochondrial membrane. According to the
chemiosmotic theory of Mitchell (1961), the energy of this proton gradient is
converted into ATP by a special proton pump, the f1/f0 ATPase, located in the
inner mitochondrial membrane as well. This pump functions on the reverse cycle,
i.e. instead of hydrolyzing ATP to extrude protons, it uses proton gradient to
synthesize ATP. One ATP molecule is produced for each proton that reenters the
matrix. Compounds that increase proton permeability of the inner mitochondrial
membrane, like 2,4-dinitrophenol, cancel this proton gradient and uncouple
electron transport from oxidative phosphorylation, increasing the production of
free oxygen radicals, like the superoxide ion, O22-, an extremely reactive and
therefore toxic molecular species. To avoid free radical overproduction, electron
transport and oxidative phosphorylation have to be perfectly coupled, producing
ATP according to the energy requirements of the cell.
Mitochondria are attached to the microtubule network and mobile. They
concentrate in areas of increased energy consumption, like the baso-lateral end
of tubular renal cells, where they provide ATP for active Na+ transport. Other
metabolically active cells, and therefore rich in mitochondria, are neurons,
hepatocytes, skeletal muscle fibers and cardiomyocytes. Mitochondria can divide
longitudinally, like any bacteria, adapting their density to ATP requirements.
During mitotic cell division, each daughter cell receives approximately half of the
mitochondria within the parental cell. During fecundation, the egg cell (zygote)
keeps the maternal mitochondria within the oocyte.
Rybosomes are tiny dense granules, first described using electron microscopy
techniques in the years 1950 by George Emil Palade (they were formerly called
Palade’s granules). With the panoptic staining method (May-Grünwald – Giemsa
or Wright), used on peripheral blood or bone marrow smears, rybosomes appear
basophilic. Therefore, cells with increased protein synthesis, like B lymphocytes,
which secrete antibodies, or basophilic erythroblasts, which synthesize
hemoglobin, have a light-blue appearance of the cytoplasm, resembling a clear,
cloudless sky.
The detailed structure of ribosomal subunits has been recently revealed using Xray diffraction techniques. It is composed mainly of ribosomal RNA (rRNA), with
several enzymes attached in appropriate positions. The large ribosomal subunit
features two sites that can adapt transfer RNAs (tRNA). Each tRNA binds
specifically an aminoacyl residue at one end, and on a distant loop presents a
triplet of nucleotides called anticodon, specified according to the genetic code,
identical to the triplet encoding an aminoacyl residue on the leading (3’-5’) DNA
strand in the nucleus, and complementary to the triplet on messenger RNA
(mRNA). As shown in Fig. 3, mRNA attaches at a specific sequence on the small
ribosomal subunit, and thus the translation process begins. The first tRNA
attaches to the peptidyl locus, the second on the aminoacyl locus, then specific
enzymes detach the first residue and bind it to the second. Further, the small
subunit advances like the chariot of a typewriter, carrying the mRNA and the two
tRNAs attached to it. The free tRNA of the first residue detaches, and a new
tRNA, that of the third residue, binds on the free aminoacyl locus, near the
second tRNA having attached a dipeptide. Thus, the process goes on and on,
and the polypeptide chain elongates, until the entire gene sequence is translated.
Fig. 3 The process of mRNA sequence translation into a polypeptide chain
sequence is carried out by ribosomes, composed of two rRNA subunits. The
large subunit features two tRNA binding sites: a peptidyl and an aminoacyl site.
Rough endoplasmic reticulum (RER) is built of flattened membrane-delimited
cisternae communicating between them and with the nuclear envelope. They
have a high density of ribosomes attached on their outer surface, and carry out
posttranslational processing of export and transmembrane proteins. It is well
known that the polypetide sequence of many export proteins begins with a short
hydrophobic leader signal sequence (LSS), which is later detached by
appropriate proteases. This sequence penetrates the membrane of the rough
endoplasmic reticulum and triggers attachement of the ribosome to a polypeptide
chain transport protein called translocon. Translocons feature a large hydrophilic
channel that allows passage of the elongating polypeptide chain. When new
hydrophobic regions of the chain are synthesized (the transmembrane  helices
of integral proteins), translocons temporarily detach, allowing their insertion into
the bilayer, then reattach when the freshly translated sequence becomes again
hydrophylic. Thus, several transmembrane-spanning (TM) domains can develop,
linked in a specific topology by cytoplasmic and intra-cisternae loops.
Post-translational processing of integral and export proteins begins in the RER,
as the chain elongates, and continues in the Golgi apparatus. It comprises the
extremely important process of folding, which renders the adequate threedimensional shape of the mature protein. A class of specialized proteins called
chaperonins (formerly known as heat shock proteins – hsp) achieve a correct
folding. The 3D structure is consolidated by disulphide bridges between distant
cysteine residues, created by disulphidisomerase. N and C glycosylation follows
in the cisternae, via a tunicamycin-inhibited pathway. Lipidation is another
important process, helping to anchor membrane proteins to lipid rafts. Several
brief sequences on either the cytyoplasmic or the cisternal side of a newly
synthesized protein function as retention signals – just a particular case of
trafficking signals. For example, the inward rectifier K+ channel subunit KIR6
contains a small C-terminal sequence, RKR, which triggers RER retention. When
this sequence is masked by another type of subunit, the sulphonylureea receptor
(SUR), the assembly can be trafficked in order to reach cell surface. Other
trafficking signals are KKXX (where X signifies any residue) on the cytoplasmic C
terminus, or KDEL on the lumen side.
Smooth endoplasmic reticulum does not contain ribosomes attached on its
surface. It is a branching network of tubules, involved in calcium storage,
cholesterol and steroid synthesis, and detoxifying processes. IP3 receptors
located on its surface can respond to soluble IP 3 released at the inner cell
membrane surface by receptor-mediated PIP2 hydrolysis, and release Ca2+ in the
cytoplasm, contributing to important intracellular signaling pathways. Ca 2+ ions
are later recaptured in the endoplasmic reticulum via active, ATP-driven
transport, by SERCA pumps. Steroid hormones synthesis occurs in specialized
endocrine cells, like those of the cortical adrenal gland, ovaris and testis. Drug
detoxification occurs predominantly in liver and kidney. For example, in cronic
alcohol consumption smooth endoplasmic reticulum becomes abundant in
hepatocytes. A specific detoxifying system present in hepatic smooth ER
involves cytochrome P450 (maximal absorption wavelength 450 nm).
The Golgi apparatus
continues
trafficking and export of proteins
synthesized in the rough endoplasmic
reticulum. Transport vesicles secreted
by the RER reach the inner (cys or
receiving) face of the Golgi apparatus.
Additional glycosylation, lipidation and
sorting processes occur in the Golgi,
then export vesicles are secreted on the
outer (trans or shipping) face. There are
three types of vesicles secreted by the
Golgi apparatus, according to their fate
(Fig. 4): 1. vesicles containing proteins
to be secreted in the extracellular
space; 2. vesicles containing plasma
membrane-forming phospholipids and
proteins; 3. vesicles containing acid
proteases which become lysosomes.
Fig. 4 Export protein synthesis and
processing begins in the rough ER and
continues in the Golgi apparatus, which
secretes 3 types of vesicles containing
export
proteins,
membrane
components, or acid proteases.
Lysosomes contain proteolytic enzymes like acid hydrolases, aryl-sulphatases,
5’-nucleotidases, activated at a pH around 5. They fuse with phagocytosis
vesicles or phagosomes forming phagolysosomes. In certain immune system
cells, circulating monocytes or tissular macrophages, incomplete digestion of
foreign bodies is followed by exposure on cell surface of small fragments called
antigens, which trigger cellular or secretory immune responses via T and B
lymphocytes. The rich lysosomal contents of monocytes gives their cytoplasm a
grey colour at the panoptic staining, like a cloudy sky before a storm.
Peroxysomes are distinct membrane-delimited vesicles that achieve free
oxygen radicals scavenging. They contain superoxidedismutase and catalase.
The cell cytoskeleton is composed of three types of filaments: microtubules,
microfilaments, and intermediate filaments (Fig. 5).
Fig. 5 Three types of cytoskeletal filaments, their subunit architecture (upper
row) and distribution within a cell, as revealed by immunofluorescent staining.
Microtubules are tubular structures, 25 nm in diameter, composed of  and 
tubulin subunits.The microtubule system of a cell radiates from a central structure
called centrosome or cell center, containing a pair of centrioles, which are
symmetrical assemblies of microtubules (9 triads of microtubules around a
central core). Mitochondria, lysosomes, and secretory granules move along
microtubules using ATP-driven motor molecules (kinesin, dynein, etc.).
Microtubules assemble and disassemble continuously.
Microfilaments are composed of actin. Monomeric G (globular) actin
polymerizes into double helical F (filamentous) actin. This process is regulated by
a lot of factors. Microfilaments give the shape of the cell, and are involved in
active movements, using myosin molecules as engines.
Intermediate filaments are tissue specific: e.g. neurons contain neurofilaments,
gliasl cells glial filaments, stratified epithelia of the skin keratin filaments, etc.
They are less dynamic than microtubules and microfilaments. Generally they
strengthen cells. They form intercellular adhesion elements called desmosomes.
Detection of neural or glial intermediate filaments in the amniotic fluid by
immunofluorescent monoclonal antibodies can help early diagnosis of neural
tube defects.
The nucleus is the largest organelle of a cell. It stores the genetic information
represented by specific sequences of nucleotides within the non-repeating
regions called genes. Usually a gene specifies a single protein sequence. The
complete collection of genes of a species represents the genome. Prokaryotic
organisms (i.e. those lacking a nucleus, like bacteria or archaea) have a single
circular double-stranded DNA comprised of a few dozens of genes, while evolved
organisms show much more complex genomes (e.g. the human genome
contains an estimated 30,000 to 50,000 genes), arranged in several stretches of
DNA called chromosomes. The useful information within a gene is splitted in
several coding regions or exons, alternating with non-coding regions or introns.
According to the classical dogma of genetics, there is a unique sense of
circulation for genetic information: from DNA to messenger RNA (a process
called transcription), followed by RNA processing via removal of introns and endto-end joining of exons (splicing), and then by translation from mRNA to protein
sequence, according to the genetic code, by free cytoplasmic or RER-attached
ribosomes. In addition, chromosomal DNA replicates itself during cell division.
Most cells possess a single nucleus, although there are also multinucleate cells,
like skeletal muscle fibers, or anucleate cells, like erythrocytes. In order to protect
its valuable contents, the nucleus is surrounded by a nuclear envelope,
composed of a double bilayer, delimiting a narrow space – the perinuclear
cisterna, in continuity with the rough endoplasmic reticulum. Like RER, the
nuclear envelope has ribosomes attached to its outer membrane. Nucleotides
required for nucleic acid synthesis, as well as freshly synthetized mRNA, enter or
leave the nucleus via relatively large nuclear pores.
In either optical (e.g. using the classical Giemsa stain) or electron microscopy,
the nuclear contents appears as dense regions (heterochromatin) alternating with
sparse regions (euchromatin). Heterochromatin contains highly packed DNA, not
involved in active transcription processes, while euchromatin, abundant in
metabolically active cells, with high protein synthesis rates, contains unfolded
DNA representing the genes to be transcribed. Such active cells, like
hepatocytes or neurons, feature also one or several nucleoli inside the nucleus.
The nucleolus is the site of ribosomal RNA synthesis and assembly. Several
regions can be distinguished inside the nucleolus: pars amorpha, pars fibrosa,
pars granulosa, pars chromosoma.
The individual nuclear DNA stretches can be clearly distinguished only during cell
division, when they form highly packed assemblies called chromosomes. Each
chromosome contains two identical copies of the double-stranded DNA, packed
within the short and long arms (dubbed in genetics p and q). These two copies
are united in the middle region named kinetocore, which plays an important role
during cell division. Fig. 6 displays the various levels of DNA packing within
heterochromatin and chromosomes. The 2-nm diameter DNA double helix is
coiled around assemblies formed of eight monomers of specific basic proteins
called histones: two subunits of histones H2A, H2B, H3 and H4 – a structure called
nucleosome. Another histone subunit, H1, lies between two successive
nucleosomes. The next level of twisting is the tight helical fiber (30 nm in
diameter), followed by a supercoiled structure – the 200-nm fiber. This fiber
forms multiple loops giving rise
to chromatids (700-nm fibers)
that represent bands within a
chromosome.
They
give
specific alternating patterns,
which can be evidenced with
various stains (Giemsa, acridin
orange, etc.).
The life cycle of a cell
comprises two distinct periods:
an interphase, the time interval
between two divisions, and the
division period called mitotic
phase
or
mitosis
(M).
Interphase is in turn divided
into several periods. G1
(growth 1) follows immediately
after a cell division. Active
metabolic processes leading to
cell growth occur during this
time. It is followed by S
(synthesis), when DNA within
the nucleus is subjected to
semiconservative replication:
the double helix unfolds, and
each
strand
serves
as
template for the synthesis of a
complementary new strand.
Therefore, at the end of the S
period, the cell contains a
double amount of double
helical DNA. During the final
growth period (G2) final
preparations for cell division
are
performed.
The
centrosome
completes its
division into two identical
structures, each composed of
a pair of centrioles. The M
phase is in turn divided into
several
periods: prophase,
Fig. 6 Various levels of DNA folding within a
metaphase,
anaphase,
chromosome.
telophase, and cytokinesis,
which can be distinguished
with current microscopy techniques. During prophase several events occur in the
nucleus and cytoplasm. Nuclear chromatin condenses into diploid chromosomes,
each composed of two short and long arms united at the kinetocore – therefore
they have the shape of an H or X. The number of chromosomes is speciesspecific: for humans there are 23 pairs of chromosomes (one from the mother
and one from the father in each pair), 22 pairs of autosomal chromosomes plus
one pair of sex-determining chromosomes (XY in males and XX in females). The
nuclear envelope begins to fragment and chromosomes are released in the
cytoplasm. Meanwhile, the two centromeres separate from each other, migrating
to opposite poles of the cell. The system of microtubules disassembles and
reassembles, forming the mitotic spindle. In metaphase all chromosomes attach
to the mitotic spindle via their kinetochores, forming an equatorial plate. Then, in
anaphase, the centromeres of all chromosomes split, giving rise to haploid
chromosomes, which migrate to the opposite poles of the mitotic spindle, taking a
V-shape. In telophase two new nuclear envelopes grow from the ER, one around
each set of chormosomes, and in citokinesis the membrane narrows in the
equatorial plane, then the entire cell splits into two daughter cells.