Lecture 8
Cell and Tissue Basics
Cell: The functional
unit of all living
organisms
• Eukaryotes: group of
organisms whose
cells have a defined
nucleus surrounded
by a nuclear
membrane.
• Cells are divided into
at least two
components: the
nucleus and the
cytoplasm
(cytosol+organelles)
• Cell size on the order
of tens of microns.
Plasma membrane
•
•
•
Outer cell membrane, whose structure is not well known
Fluid mosaic model is the prevalent model for its architecture
Membrane consists of phospholipid bilayer (~10 nm); its major
components are:
-phospholipid molecules; amphipathic (polar, hydrophilic head; nonpolar, hydrophobic tail)
-cholesterol (1:1 cholesterol:phospholipids)
* regulate fluidity
* stability
-protein molecules (half of the total mass of membrane)
*intrinsic or integral proteins
*transmembrane proteins
*extrinsic or peripheral proteins
-Glycocalyx
*short chain of polysaccharides conjugated with membrane
proteins and some of the membrane lipids
Plasma membrane
• Major functions:
– Nutrient transport in/waste transport out
– Barrier to unwanted materials
– Maintenance of proper ionic composition, pH, osmotic
pressure
– Contact with other cells and extracellular matrix
– Cell signaling receptors
• Spectroscopic features:
– Refractive index (n=1.43-1.49) higher than water
(n=1.33) leads to scattering
• Detected only by highly sensitive methods such as lowcoherence interferometry
cytoskeleton
• Framework of minute filaments and tubules
• Provides structural support for PM, cellular organelles
• Responsible for locomotor mechanism for amoeboid
movements and specialized structures such as cilia and
flagella
• Responsible for contractility of cells in specialized tissues
such as muscle
• Includes:
– Microfilaments
• ~5nm in diameter; made out of actin
• Responsible for cell shape and motility; provide energy for
contractile processes
– Microtubules
• 25 nm in diameter; made up of globular protein subunits
• Can be readily assembled and disassembled
• Provide network onto which organelles such as mitochondria can
move
– Intermediate filaments
• 10-12 nm in diameter; stable fibrous structure
• Tough supporting network
• Distribute tensile forces across cell
• Spectroscopic features: Birefringence
Endoplasmic reticulum
• Rough ER
– Interconnecting network of membranes,
tubules and vesicles
– Surfaces studded with ribosomes, where
protein synthesis takes place
– Proteins synthesized here are destined
for lysosomes or export or incorporation
in the PM
– Continuous with outer lipid bilayer of
nuclear envelope
• Smooth ER
– Irregular network of membranes, tubules
and vesicles devoid of ribosomes
– Continuous with rough ER and Golgi
apparatus
– Function: lipid biosynthesis and
membrane synthesis and repair
Golgi apparatus
• System of stacked, saucer shaped cisternae, with concave surface
facing nucleus
• Function: glycosylation of proteins and packaging into secretory
products or lysosomes
• Spectroscopic properties: It is expected that the Golgi apparatus
along with the ER will contribute to light scattering from the cells
because they consist of membranes (lipids) which have higher
Golgi apparatus
refractive index than water
•
Lysosomes
Structure: Membrane bound organelles containing amorphous granular
material (primary lysosomes) or electron dense particulates (secondary
lysosomes)
•
Function: degradation of worn-out cell constituents and foreign materials
into monomeric units by acid hydrolases (e.g. proteases degrade proteins
into peptides)
•
Several hundred lysosomes may be present in eukoryotic cell
•
Vary in size and appearance
from hundreds of nm to a few mm.
•
pH of lysosome ~ 4.8
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Protein denaturation
Protection in case of release of
lysosomal contents
Spectroscopic properties: scattering
• Structure:
mitochondria
– Outer membrane
• Relatively permeable to small molecules
– Inner membrane
• Folds into cristae and is much less permeable
• Contains lots of proteins including the
cytochromes, enzymes involved
in ATP production
– Intermembrane space
– Matrix (gel like; 50% protein content)
• Location of mitochondrial DNA and
ribosomes
• Typical size: 200x600nm, but there is a
lot of variation
• Their number varies from very few in
metabolically inactive cells to a couple of
thousand in liver and skeletal muscle
cells
mitochondria
• Function: power-plant of the cell
– Site of ATP production
– ATP: energy currency for cells
• ATP is used for
– Movement
– Macromolecule synthesis
– Molecule transport against concentration
gradient
• Glucose metabolism: most efficient means
of generating ATP
Glucose metabolism
• Overall reaction:
C6H12O6+6O2+36Pi2-+36ADP3-+36H+→6CO2+36ATP4-+42H2O
• 1st stage: anaerobic glycolysis (in cytosol)
C6H12O6+2NAD+ + 2ADP3-+2Pi2- →C3H4O3+2NADH+2ATP4-
• 2nd stage: aerobic glycolysis
– Pyruvate transferred to mitochondria and oxidized to CO2 by O2. In
the process 34 ATP molecules produced
C6H12O6: glucose
Pi2-: inorganic phosphate
ADP: adenosine diphosphate
ATP: adenosine triphosphate
C3H4O3: pyruvate
NAD + : nicotinamide adenine dinucleotide (oxidized form)
NADH: nicotinamide adenine dinucleotide (reduced form)
Aerobic glycolysis
• 3 reaction groups:
– Oxidation of pyruvate and fatty acids to CO2,
coupled to reduction of NAD+ and FAD into
NADH and FADH2 (Kreb’s cycle or citric acid
cycle)
• Matrix of inner membrane
– Electron transfer from NADH/FADH2 to O2
coupled to generation of proton-motive force
• Inner membrane
– Production of ATP using electrochemical H+
gradient across inner membrane
Aerobic glycolysis
Electron transport chain
As electrons are transported via four enzyme complexes
from NADH and FADH2 to O2, protons are pumped across
the inner mitochondrial membrane, generating a protonmotive force (~220 mV), which is due to
– proton concentration gradient (~60 mV)
– Electric potential (matrix becomes negative with respect to
intermembrane space) -160 mV
Mitochondrial spectroscopic signatures
• Scattering
• Fluorescence
– NADH and FAD are fluorescent molecules
– NAD+ and FADH2 are essentially non-fluorescent
– Handle on metabolic activity of the cell through redox ratio: FAD/(NADH+FAD)
NADH image: exc:360 nm/em: 450 nm
Epithelial
Keratinocytes
From human
foreskins
FAD image: exc: 455 nm/em:520 nm
Nucleus: command center of the cell
• Contents:
– DNA (20% mass)-deoxyribonucleic acid
• Usually arranged as tangled strands
– Nucleoproteins
• DNA binding proteins
– Histones (DNA folding, regulation of DNA activity)
– Non-histones (regulation of gene activity)
• DNA/RNA synthesis proteins
– DNA usually arranged as tangled strands
• Heterochromatin: DNA and associated proteins not involved in RNA
synthesis (electron-dense)
• Euchromatin: DNA and associated proteins involved in RNA
synthesis (electron-lucent)
Structure of the nucleus
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•
heterochromatin
The nucleus consists of the nucleoplasm
and the nucleolus (may be more than one
per nucleus)
It is surrounded by two membranes,
comprising the nuclear envelope (NE)
– Each membrane has a phospholipid bilayer
structure
– the outer membrane is continuous with the
ER and is studded with ribosomes
– At the inner membrane there is a fibrillar
layer, the nuclear lamina, which links
membrane proteins and heterochromatin
– The envelope contains numerous nuclear
pores, at the margins of which the inner and
outer membranes become continuous
– Nuclear pores (NP) permit and regulate the
exchange of metabolites, macromolecules
and ribosomal subunits between the nucleus
and the cytoplasm
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•
The nuclear cytoskeleton is attached to the
nuclear lamina and provides support to
nuclear contents
The nucleolus is the site of ribosomal RNA
synthesis and ribosomal assembly
euchromatin
Nuclear spectroscopic signatures
• The nucleus doesn’t have any significant autofluorescence
• However, it has a refractive index of ~1.42, which is significantly
higher than the surrounding cytosol (n~1.36).
• Its high refractive index in combination with its large size (typically 510 mm) yield highly directional scattering properties
• This scattering is used in light scattering spectroscopic
measurements to assess nuclear morphology
• Changes in nuclear morphology constitute hallmarks that are used
routinely by histopathologists to determine the presence of precancerous and early cancerous changes. These changes include:
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Nuclear enlargement (increase in the overall nuclear size)
Nuclear pleomorphism (variation in the size of the nuclei among cells)
Hyperchromasia (increased staining density with standard DNA dyes)
Increase in the number of nuclei per unit area
Increased roughness in nuclear texture
Epithelial tissues
Basic properties of epithelia
• Epithelia cover or line all body surfaces, cavities and
tubes.
• They form continuous sheets of cells, comprising one or
more cell layers. Epithelial cells are closely bound to one
another
• All epithelia are supported by a basement membrane of
variable thickness, which separates them from
underlying supporting tissues and blood vessels.
• They function as interfaces between different biological
compartments. Thus, they mediate
– Selective diffusion, absorption, and/or secretion
– Physical protection
– Containment
Classification of epithelia
• Epithelia are classified according to three
morphological characteristics
– The number of cell layers
• Simple (single layer)
• Stratified (many layers)
– The shape of the component cells
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Squamous
Columnar
Cuboidal
transitional
– The presence of surface specializations
• Ciliated
• keratinizing
Simple epithelia
• Simple epithelia consist of a single layer of
cells
• Almost always found at interfaces involved
in selective diffusion, absorption or
secretion.
• They provide little protection against
mechanical abrasion
Simple squamous epithelia
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Composed of flattened, irregularly shaped cells
They are supported by basement membrane
Found lining surfaces involved in passive transport of either
gases or fluids
Cells frequently have specialized receptors that control
secretion of locally acting chemical messengers
Found in the lining of:
– Lungs
– Blood vessels (endothelium)
– Peritoneal, pleural and pericardial cavity (mesothelium)
endothelium
Mesothelium-peritoneum
Simple columnar epithelium
• Cells are tall and appear like columns in sections at right
angles to the basement membrane
• Height of the cells may vary depending on site and degree
of functional activity
• Nuclei are elongated and polar, i.e. they are located
towards the base, center or apex of the cell
• Most often found on highly absorptive or secretory
surfaces
• The luminal plasma membranes of highly absorptive
epithelial cells have a brush or striated border made up
of microvilli, minute finger like projections, mainly for the
purpose of increasing surface area, by as much as 30-fold.
– Small intestine
– Stomach
– Gall bladder
– Renal tubules
– colon
– Larger breast ducts
Simple columnar ciliated epithelium
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Simple columnar epithelium with surface specializations called cilia on the
majority of the cells
Among ciliated cells, non-ciliated cells can be found that usually have
secretory function
Cilia are much larger than microvilli and are visible with light microscopy.
Each cell may have 300 cilia beating in synchrony with cilia from
neighboring cells to propel fluid of minute particles over the epithelial
surface
Found in the human reproductive tract
– Fallopian tube (propel ovum and secretions from ovary to uterus)
Pseudostratified columnar ciliated
epithelium
• Just like simple columnar epithelium, with nuclei
disposed at different levels, but typically still
confined to the basal two-thirds of the epithelium
• Found in larger airways of respiratory system
• Often referred to as respiratory epithelium
• Cilia propel mucous towards the pharynx
Simple cuboidal epithelium
• Intermediate form between simple squamous and
simple columnar epithelium
• Cells appear square in section perpendicular to the
basement membrane
• Usually round nucleus in the center of the cell
• Cuboidal epithelium lines small ducts and tubules
– Kidney
– Salivary glands
– Pancreas
– Breast ducts
Stratified epithelia
• Consist of two of more layers of cells
• Mainly protective function
• Degree and nature of stratification related
to the types of stresses that epithelium is
exposed to
• Poorly suited for absorption and secretion
Stratified squamous epithelium
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Consists of a variable number of cell layers which exhibit transition from a
cuboidal basal layer to a flattened surface layer.
Basal cells divide continuously, with the offspring being pushed
progressively towards the free surface, where they are ultimately shed.
Nuclei become progressively condensed and flattened, before ultimately
disintegrating
Well adapted to withstand abrasion, since loss of surface cells doesn’t
compromise the underlying tissue
Poorly adapted to withstand desiccation
Found in sites subject to mechanical abrasion but kept moist by glandular
secretions, such as:
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Oral cavity
Pharynx
Esophagus
Anal canal
Uterine cervix
vagina
Stratified squamous keratinizing
epithelium
• During maturation, cells accumulate cross-linked
cytoskeletal proteins in a process called keratinization,
resulting in the formation of a tough, non-living surface
layer consisting of the protein keratin
• Keratin is highly fluorescent and scattering
• Squamous keratinizing epithelia are found in the
– Skin
– Oral cavity
Stratified cuboidal
• Thin stratified epithelium, consisting of two
or three layers of cuboidal or low columnar
cells.
• Found in the lining of excretory ducts of
glands, such as the salivary glands
Transitional epithelium
• Stratified epithelium almost exclusively confined to the urinary tract
• Highly specialized to accommodate a great degree of stretch and
to withstand the toxicity of urine.
• In relaxed (contracted) state, it appears to be about four to five cell
layers thick
• Basal cells are roughly cuboidal, intermediate cells are polygonal
and surface cells are large and rounded and may contain two nuclei
• In the stretched state, the epithelium appears only two or three
layers thick and top layers are extremely flattened
• Found in
– bladder
Basement membrane
• The basement membrane provides structural support for
the epithelium as well as binding to the underlying
supporting tissue.
• Involved in control of epithelial growth and differentiation,
forming an impenetrable barrier to downward epithelial
growth; this is breached during malignant transformation
• Controls flow of nutrients, metabolites and other
molecules to and from epithelium
• 50-300 nm thick
• Main constituents:
– Glycosaminoglycan: heparan sulfate
– Fibrous protein: collagen IV
– Structural glycoproteins: fibronectin,
laminin and entactin
Glycosaminoglycans (GAG): unbranched polysaccharide chains,
each composed of repeating disaccharide units
Supporting/connective tissues
• Basic type of tissue which provides structural and metabolic
support for other tissues and organs
• Connective tissues usually contain blood bessels and mediate the
exchange of nutrients, metabolites and waste products between
tissues and the circulatory system.
• In most organs, loose supportive tissues act as a biological
packing material between cells and other tissues with more specific
functions
• Dense forms of supporting tissue provide tough physical support
in the dermis of the skin, comprise the capsule of organs such as
the liver and spleen, and are the source of great tensile strength in
ligaments and tendons. Cartilage and bone are highly specialized
forms of supporting tissue
• Important metabolic roles in the context of fat storage and
temperature regulation
• Cells of the immune system enter supporting tissues to defend
against pathogens
• Tissue repair is largely a function of supporting tissues
Supporting/connective tissues
• Three major components:
– Cells
• Fibroblasts (synthesis and maintenance of extracellular material)
• Adipocytes (fat storage and metabolism)
• Immune system cells (macrophages, lymphocytes, all types of
white blood cells)
– Extracellular matrix
• Matrix of organic material called ground substance within which
are embedded a variety of fibers
• Ground substance composed of GAGs, which are entangled and
electrostatically linked to one another and their water of hydration to
form a flexible gel through which metabolites can diffuse
• Fibers include
– Collagen
– elastin
– Structural glycoproteins
• Fibrillin, fibronectin, laminin, entactin and tenascin
• Mediate interaction of cells with other constituents
• Fibronectin also plays a role in collagen deposition and orientation
Supportive tissue cells
• Fibroblasts
– Synthesis and maintenance of extracellular
material
• Mature fibroblasts
– Condensed, elongated nuclei
– Limited cytoplasmic volume
• active fibroblasts
– Large, round nuclei with prominent
nucleoli reflecting active
protein synthesis
– Extensive cytoplasm
Active fibroblasts are strongly
autofluorescent
Epithelial cells
fibroblasts
NADH fluorescence
Adipocytes
• Fat stored in adipocytes accumulates as lipid droplets
which fuse to form a single large droplet, which occupies
most of the cytoplasm
• The nucleus is compressed and diplaced to one side of
the lipid droplet
• Cytoplasm is reduced to a small rim around the
periphery
• In routine histological processing lipid content is
extracted, so that adipocytes have large unstained space
Lipids are rich in beta carotene
Molar extinction coef (M-1 cm-1)
• Beta carotene exhibits strong absorption
features in the visible spectrum.
160000
120000
80000
Series1
40000
0
380
480
580
Wavelength (nm)
680
Collagen fibers
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Collagen is the main fiber type found in
most supporting tissues
It is the most abundant protein in the
human body
Its major function is to provide tensile
strength
At least 19 different types of collagen have
been characterized
Type I collagen is the major collagen type
found in fibrous supporting tissue, the
dermis of the skin, tendon, ligaments and
bone
Collagen is secreted in the extracellular
matrix as tropocollagen (three polypeptide
chains bound together to form a helical
structure 300 nm long and 1.5 nm in
diameter.
Tropocollagen molecules are aggregated to
form fibrils
Parallel collagen fibrils are further arranged
into strong bundles 2-10 mm in diameter
Collagen has strong scattering and
autofluorescent features
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Collagen contributes to highly scattering nature of most epithelial tissues,
i.e. it diffuses the light
It is an asymmetric molecule and exhibits a strong second harmonic
generation signal
It has very strong fluorescence in the near UV and visible range of the
spectra
Multi-photon excitation images acquired with 840 nm excitation
TPEF: Two-photon excited fluorescence; SHG: second harmonic generation;
FAD: flavin adenine dinucleotide
TPEF+SHG
SHG
FAD TPEF
SHG+FAD TPEF
Elastin fibers
• Elastin is found in varying proportions in most supporting
tissues
• Its major function is to confer elasticity to enable
recovery of tissue shape following normal physiological
deformation
• Typically occurs in the form of short branching fibers with
no recognizable periodicity
• Present prominently in lung, skin, urinary bladder and
blood vessel walls
Skin dermis
elastin