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Cell biology is a complex fundamental science that studies the functional structures and
general biological phenomena that take place at the cell’s level. The knowledge about the cell
was brought by the studies made by the light microscope.
In 1590, Jansen brothers put in a tube some glass lens. They used this apparatus for
navigation and astronomy. In 1665, Robert Hooke modified this apparatus and observed some
vegetal cells. The Dutch Leeuwenhoeck improved the microscope so that it magnified 300
times. He described some microorganisms and spermatozoa. He also described in the middle of
the cell the presence of a spherical corpuscle (the nucleus). Malpighi described the cells and
named them utricule and saccule. He also described in the kidney the Malpighi’s corpuscle.
Preformist theory has persisted in the eighteen centuries. It supposed that the beings exist in a
preformated state in the shape of miniature - beings (homunculus) into sexual cells sad they
develop by growing processes. Spallanzani refuted this theory through the following
experiment: he collected spermatic fluid from frog and filtered it. Then he mixed the residuum
with frog eggs and new beings resulted. The filterable liquid mixed with frog eggs does not form
anything. So that one has concluded the followings:
1) The spermatozoon is the fecundating agent;
2) The spermatozoon combined with the ovule forms a new being;
3) There are not performed beings.
In the XIX century there were three theories:
1) Cell Theory
2) Evolution theory
3) Heredity laws
Cell Theory
In 1905, Oken in his work named ”The Generation” assumed that all the beings had
developed from cells. Schleiden and Schwann formulated the cell theory and the following laws:
a) Vegetal and animal bodies are formed by cells; b) Each ceH has its individual cell c) The
individual life of each cell influences the life of’ the whole body and vice- versa. At cellular
level there are two phenomena: - metabolic phenomena - plastic phenomenon at cellular
structures’ level.
Virchow applies the cellular theory in embryology: the union of two gametes forms all the
cells. The progressive development of the egg cell builds up the organism.
The Theory of Evolution
Darwin and Wallace gave the first modern scientific response to three central questions, in
their theory of evolution:
- How did life originate?
- Why does “like begged like”?
- How does an individual animal or plant develop from a fertilized?
Heredity Laws
Gregor Mendel formulated the heredity laws. The characters of each being are written on a
material particle in the gametes. The particle is named cellular factor. The method he used is
named experimental hybridization.
The use of staining from textile industry guided to the discovery of new cellular
components:
- Brown discovered the nucleus, a constant component of the cell;
- Bovery described the cellular center;
- Waldayer described the chromosomes;
- Flemming and Strasburger described mitosis;
- Altman described the mitochondrion;
- Nissl described the Tigroid substance (Nissl corpuscle) that is the granular (rough)
endoplasmic reticulum from central nervous system;
- Berg described the Berg corpuscles from liver cells.
Cellular biology was considered as a proper science when cell culture technique,
differential ultracentrifugation technique and electron microscopy appeared.
Cells make all living creatures. The cells are small membrane bounded compartments filled
with concentrated water solution of chemicals. Superior organisms, such as, are like cellular
cities in which groups of cells perform specialized functions and are halfway between molecules
and individual beings. We study them to learn how they are made by molecules and, on the
other hand, how they cooperate to make an organism as complex as the human being.
All organisms and all the cells that constitute them are assumed to descend from a common
ancestral cell by evolution. Evolution involves two essential processes:
1) The occurrence of random variations in the genetic information passed from an
individual to its descendants;
2) The selection of genetic information that helps its possessors to survive and
multiply.
Remarkable scientific progress in last 50 years made possible the pertinent but still
incomplete explanation concerning the appearance of the first living structure, the ancestral cell,
and at the same time its evolution in time up to the appearance of the impressive cell variety find
now on earth.
The conditions that existed on the earth in its first thousand million years are still a matter
of dispute. Everyone seems to agree, however, that the earth was a violent place with volcanic
eruptions, high temperatures and electrical discharges. There was little of any free oxygen and
no layer of ozone to absorb the harsh ultraviolet radiation from the sun. That atmosphere
contained N, H, NH 3 , CH 4 , CO 2 , and water vapors. From the moment when the planet
temperature downed, water vapors condensed, starting rains that involved the chemical elements
from atmosphere dissolving them and earthly. In this manner it comes into being the primitive
ocean, called the prebiotic soup. Simple organic molecules seem that are produced under such
conditions and this phenomenon was also proved experimentally. Thus, weather a mixture of
gases such as CH 4 , NH 3 , H 2 is warmed together with water into a closed tool and the mixture is
forced to electrical discharges it is observed the forming of small organic molecules as HCN and
HCHO that combine slowly in aqueous solution to form cyanhydrine. In the presence of
ammonia, this forms glycerol, the simplest amino acid. The formed simple organic molecules
polymerized to realize macromolecules that were accumulated in ocean to born organic soups.
The four major classes of small organic molecules formed in cells are aminoacids,
nucleotides, sugars, and fatty acids. Simple organic molecules such as aminoacids and
nucleotides can associate to form large polymers. One aminoacid can join with another by
forming a peptide bond, while two nucleotides can join by a phosphodiester bond. The repetition
of these reactions leads to linear polymers known as polypeptides and polynucleotides. In
present days, in the living organisms, the polypeptides (known as proteins), and polynucleotides
[as both ribonucleic acids (RNA) and deoxyribonucleic acids (DNA)] are commonly viewed as
the most important constituents.
Figure 1. Miller experience, simulating
conditions on primitive earth.
The formation of polynucleotides and proteins requires the presence of specific protein
catalysts or enzymes, which were not present in the “prebiotic soup”. However, less efficient
catalysts as minerals or metal ions would have been present.
The appearance of protein synthesis controlled by nucleic acids was one of the crucial
events leading to the formation of this first cell. Another important step must have been the
development of an external membrane. It was postulated that the first assembled into such
membranous structures, enclosing a self – replicating mixture of RNA and protein molecules. At
some later stages in evolutionary process, DNA replaced RNA as hereditary material.
So, the first living cells probably appeared on Earth 3.5 billions years ago as a result of
spontaneous reactions between the molecules of medium, which is in chemical unstable
equilibrium. The performed recently studies on old sedimentary rocks made evident the
presence of the unicellular organisms similar to blue-green algae on Earth 3.6 billions years ago
that lead to the hypothesis that life appeared during the first thousand billion year of the planet.
The research results done until now lead to the hypothesis that the first living organisms
developed trough autocatalytic mechanisms starting with the RNA molecular family evolution
that can catalyze their owner replication. In the process of time, some of these, probably,
developed their capacity to manage the polypeptide synthesis. As more protein catalysts
accumulated, they became more efficiently allowing the cells evolution, RNA being took by
double helix DNA that has molecules more stable and able to storage the complex genetic
information.
So, the hypothesis would be RNA preceded DNA in the evolution having genetic and
catalytic properties.
It is thought that all organisms living now on earth derive from one simple primordial cell
born several thousand million years ago. This cell, reproducing out its competitors, takes the
lead in the process of cell division and evolution that would eventually cover the earth in green,
changes the composition of the atmosphere, and makes it the human intelligent life.
One important landmark along this evolutionary road occurred about 1.5 billion years ago,
when there was a transition from small cells with a relatively simple internal structure (the
prokaryotes, that include the various types of bacteria and the blue-green alga) to the larger and
radically more complex eukaryotic cells such as those we found in superior animals and plants.
The eukaryotic cells can be unicellular or pluricellular and the prokaryotes are only
unicellular. Between eukaryotes and prokaryotes are the following differences:
Glycolipid
Transmembrane
Glycocalix protein
Lipid
bilayer
CYTOPLASM
Figure 2. Schematic diagram of the cell cover, which is made up of three
components: cell coat (glycocalyx), plasma membrane and membranar
cytoskeleton.
The plasma membrane is the most important constituent of the cell coat. Bowman
in 1840 and Remack in 1852 inferred the existence of the plasma membrane. They
assumed the presence of this structure at the boundary of the cell. Due to its thickness
and to the photonic microscope resolution power limits, the existence of the plasma
membrane was denied for a long period of time.
At the end of the nineteenth century and the beginning of the XXth century,
indirect data were brought about the existence of the plasma membrane.
Since 1950, after the discovery of the electron microscope, there have been
studied ultra-thin sections from animal tissues at a resolution under 50 nm.
The use of KMnO4 as a fixing agent and of epoxidic resins as embedding
materials permitted the study of plasma membrane and gave the proof of their
existence.
The data about the plasma membranes, obtained with the electron microscope, X-
ray diffraction, differential ultracentrifugation, biochemical analysis, freeze-fracture,
were synthesized in a membrane model, with dynamic features.
In 1976, Cifuentes assumed that the plasma membrane is a biochemical complex
with morpho-functional expression.
The degree of saturation and the length of carbon chains are important for the
fluidity of the membrane. The polar head is formed by a phosphoric acid residue,
bound to an alcohol (that can be choline, serine or inositol).
The most important phosphoglycerids are: phosphatidyl-choline, phosphatidyl-
serine, phosphatidyl-inositol, phosphatidyl-ethanolamine.
2. Sphingolipids look like phospholipids, but contain sphingosine, an aminoalcohol
with a long unsaturated hydrocarbonated chain.
The hydrolipidic head is the polar group and an alcohol and a phosphate residue
form it. Two chains form the hydrophobic tails: one of them is always the same
(formed by a part of the sphingosine molecule) and the other can be different, being
linked with the rest of the molecule by an amidic bound. The most common
sphingolipid in the membrane is sphingomyeline, with the same polar group as
lecithin.
Membrane glycolipids.
The membrane glycolipids are asymmetric, being present in the external layer of
the membrane in animal cells. They have the polar group formed by a carbohydrate
residue, located in the external part of the plasma membrane. There are two classes of
glycolipids: cerebrosides and gangliosides.
1. The simplest glycolipids are the galactocerebrosides (neutral glycolipids) that
have coupled to the polar group a galactose residue; they form 40% of Schwann
cell’s membrane.
2. The gangliosides have a more complex structure. At the polar group they have
attached a sialic acid residue, which is negative charged. More than 30
gangliosides are known today, but their functions remains uncertain. They appear
to act as receptors on epithelial cells surface; they can also play a role in Ca++
sequestering at cell surface due to their negative charge. For example, the
ganglioside GM1 acts as a cell-surface receptor for the bacterial toxin that causes
the debilitating diarrhea from cholera; cholera toxin binds to and enters the cells
that have GM1 on their surface, including cells from the intestinal epithelia.
Although binding bacterial toxins cannot be the normal function of gangliosides
such observations suggest that the gangliosides may also serve as receptors for
natural signaling between cells.
Electrochemical
gradient
facilitated
diffusion ENERGY
simple
diffusion ACTIVE
PASSIVE TRANSPORT TRANSPORT
According to the direction followed by the transported molecules we can classify the
transport systems as follows:
Uniport system - a single molecule type is transported from one face of the
membrane to another.
Co-transport system - two molecule types are transported by:
- symport - both substances are transported in the same direction
- antiport - substances are transported in opposite directions (e.g. H+-K+
pump introduces 1 K+ in the cell moving out 1H+ at the same time).
There are two classes of membrane transport proteins that function in passive
transport.
1. Carrier proteins that have the characteristics of the membrane bound enzymes;
the carrier proteins bind the specific molecule to be transported and transfer it across
the membrane, in a process called facilitated diffusion.
2. Proteins that form transmembrane hydrophilic channels through which solutes
of appropriate size and charge can pass by relatively simple diffusion.
Simple Diffusion.
Simple Diffusion through the Lipid Bilayer.
This type of passive transport is realized according to the oil/water partition
coefficient. Overton observed that liposoluble substances passed easier through
membranes than non-liposoluble ones. There is a proportional dependence between
the permeability constant and this oil/water partition coefficient.
The permeability constant is expressed with the formula:
kxD P = permeability constant
P = K = oil/water partition coefficient
t D = diffusion coefficient
t = plasma membrane thickness
The oil/water partition coefficient (k) is measured by the oil/water partition
degree. We mix oil and water and we inject the substance whose k we want to know.
We shake the mixture and after the two phase's separations (oil/water), we measure
the substance concentration in oil and in water. When the coefficient k (that means
liposolubility) is high then the substance can pass easily through the plasma
membrane. Some exceptions of mentioned above are water, urea, and methyl alcohol.
This rule is important to explain the penetrating mechanism of some liposoluble drugs
through the membrane.
The diffusion coefficient (D) represents the substance quantity that passes from a
medium to another, in the time unit. To measure it, we have to know the surface
where changes are made and the substance concentration gradient in both mediums.
The permeability constant (P) represents the speed (cm/s) of the substance that
passes through the membrane.
Ionophores.
Ionophores are small hydrophilic molecules that dissolve in lipid bilayer and
increase the ionic permeability of the bilayer. Most of them are synthesised by
microorganisms and some are used as antibiotics. They have been widely used to
increase membrane permeability to specific ions in studies on synthetic bilayers, cell
organelles, and more recently on intact cells.
There are two classes of ionophores:
• mobile ion carriers
• channel formers
Valinomicin is an example of mobile ion carrier. It is a ring-shaped polymer that
increases the permeability of the membrane to K+. The ring has a hydrophobic
exterior that contacts the carbohydrate core of the lipid bilayer, and a polar interior
where a single K+ can fit. Valinomicin transports K+ down its electrochemical
gradient by picking up a K+ on one side of the membrane diffusing across the bilayer
and releasing K+ on the other side.
The ionophore A23187 is another example for a mobile ion carrier, but it
transports divalent cations such as Ca++ and Mg++. At low concentrations the
ionophore acts as an ion-exchange shuttle carrying two H+ out of the cell for every
cation it carries inside. This ionophore is widely used in cell biology experiments to
increase the concentration of free calcium in the cytosol. If the temperature of the
membrane is lowered below its freezing point, mobile carriers can not longer diffuse
across the lipid bilayer and the transport will stop. This temperature dependence
serves to identify an ionophore as a mobile ion carrier, since by contrast, channel
forming ionophores continue to transport normally the substances when the bilayer is
frozen.
Gramicidin A is an example of a channel forming ionophore. It is a linear peptide
of 15 aminoacids, all with hydrophobic side chains. Two such molecules are though
to come together in the bilayer to form a transmembrane channel that selectively
allows monovalent cations to flow down their electrochemical gradient. Gramicidin A
can transport about 2x107 cations per open channel in 1 second, that is a thousand
times more than it can be transported by a single mobile carrier at the same time.
Pong Ping
Concentration
Lipid bilayer gradient
transport protein
meadiating facilitated
diffusion
lipid
bilayer
SYMPORT ANTIPORT
UNIPORT COTRANSPORT
Sodium
concentration Potassium
gradient concentration
gradient
ATP ADP+Pi
2 K
Figure 10. Schematic drawing of glucose symport, showing how the active
transport of glucose is driven by a Na+ gradient that is generated and
maintained by the Na+-K+-ATPase
The same mechanism is used for the aminoacids transport, the carrier being
always the Na+. In this case, five-transport protein is used. Each protein type binds the
aminoacid with a similar chemical structure. Some bacteria, such Escherichia coli
uses the H+ pump to help glucose to enter into the cell.
Group Translocation.
It is found especially in bacteria. The mechanism involves the binding of a
molecule on the external face of a membrane protein, the molecule entering the cell
with a modified structure.
The protein that facilitates this transport is the phospho-transferase system.
For instance, the sugars will be fixed on the external face of the membrane protein.
The sugars will enter inside the cell, changing their chemical structure as a result of
the phosphorylation. The substrate for this process is the phospho-enolpyruvate,
which will be divided into P and pyruvate.
Many active sugars and aminoacid transport systems in bacteria involve a special
class of water-soluble proteins located in the space between the plasma membrane and
the cell wall - the periplasmic space. These proteins have specific binding sites for the
solute and are therefore called periplasmic binding proteins.
Each of these binding proteins that function in transport also seem to serve as
receptors in chemotaxis; chemotaxis is an important process that enables bacteria to
swim toward an increasing concentration of a specific nutrient (Figure 11).
Biologic receptors.
In 1878, Langley used for the first time the term receptor. In 1910, Erlich
sustained the existence of receptors.
A biologic receptor is a glycoprotein macromolecular structure that
complementary interacts with a ligand from the extracellular environment.
Receptors Classification.
Classification criteria:
- according to the position in the cell
- according to the transduction mechanism
- according to molecules that they are coupling (according to the ligand)
A. According to the position in the cell:
- membrane located receptors (receptors for polypeptide hormons)
- receptors in the cell (recptors for vitamin D, steroid hormons, retinoids,
and thiroid hormons )
- intracytoplasmatic receptors
- intranuclear receptors
Intracellular receptors can be coupled with inhibitor proteins that release the receptor
when the ligand is near.
B. Accoding to the transduction mechanism:
- ionic channel coupled receptors
- G protein coupled receptors (G protein receptors are 7-pass
transmembrane proteins; there are many types of G proteins - Gs,
stimulating, Gi, inhibitor, etc.
- enzyme coupled receptors - the receptors are enzymes that become active
when a ligand is bound or receptor activation leads to other enzymes
activation in cytoplasm (as proteinkinases)
C. According to the ligand, biologic receptors are of two types:
I. Receptors for endogenous substances:
1. Receptors for neurotransmitters located in the post-synaptic external
membrane (e.g. receptors for acetylcholine, histamine, noradrenaline).
2. Receptors for humoral transported substances:
• hormone receptors (receptors for insulin, gastrin, etc. )
• endogenous antigen receptors (on the surface of T lymphocytes), playing a
role in the immune response releasing.
• antibody receptors (receptors for IgE located in the surface of basophils and
mast cells)
• complement receptors (receptors that bind the serum C3 fraction of the
complement system and are specific for the phagocytic mononuclear cells).
• low density lipoprotein receptor - that induces receptor mediated endocytosis.
II. Receptors for exogenous substances:
1. For viruses and bacteria
2. For microbial toxins
3. For non-self exogenous antigens (receptors on B-lymphocyte surface)
4. For drugs
The receptor’s type and density varies with the cell type and the considered area.
The same receptor type can be present in one or more cell populations (e.g. insulin
receptors are present in hepatocytes, muscle cells and adipocytes).
The active catalytic subunits will catalyse the following type of reaction:
Glycocalyx, also called the sweet cell cover (Bennett, 1963) is a glycoprotein
layer located over the membrane outer layer; its integrity is necessary for the normal
development of cell functions and activities.
The Morphological Structure.
In electron microscopy, glycocalyx looks like a fibrillar material of 50 nm thick,
made up of two layers:
• an internal layer or the surface cover, 20 nm thick, less electron-dense
• an external layer or the external lamina, 30 nm thick, denser than the first
Glycocalyx Molecular Structure.
The glycocalyx is a carbohydrate-rich peripheral zone at the surface of most
eukaryotic cells. The carbohydrate consists of the oligosaccharide side chains of
membrane-bound glycoproteins and glycolipids, although it often includes, in
addition, both glycoproteins and proteoglycans that have been secreted and then
absorbed on the cell surface. The proteoglycans (mucoproteins) consist of many
polysaccharide chains linked to a core protein.
Glycocalyx functions.
The most important functions of the glycocalyx are:
1. Protective role - it represents a resistant structure to mucolytic and proteolytic
enzymes, excepting the hyaluronidase and the neuraminidase.
2. Absorptive role - glycocalyx can absorb or fix cytophilic antibodies that
modify the phagocytosis. It has an important role in endocytosis, because in
glycocalyx are found specific receptor zones for different molecules: biological
molecules (hormones), drugs and toxins.
3. Glycocalyx permeability is emphasized by treatment with neuraminidase.
4. Cells adhesion - glycocalyx glycoproteins belonging to adjacent cells
participate to junctions' formation (e.g. desmosomes).
5. Role in cell recognition. Cells belonging to the same type, the same origin and
the same organism have the property to recognize one another. This function also
represents a defense mechanism (phagocytosis).
2.5. Cell membrane specialization.
2.5.1. Microvillia.
Microvillia are cell expansions with a core of crossed-linked actin filaments
covered by membrane; they participate in absorption processes by:
1. Increasing the cell contact surface for substances that will enter the cell.
2. An increased number of receptors on the surface area unit.
Microvillia cover the exposed surfaces of many epithelial cells, especially where
cell functions require a maximum surface area for absorption, such as in the gut or
kidney. These finger-like extensions are about 1 μm length and 0.1 μm diameter.
Microvillia can be grouped in two ways:
1. Isolated microvillia, separated by a certain free space, having variable
dimensions and shape.
2. Grouped microvillia that concentrates a high number of receptors on the
surface unit (an enterocyte has 3000 microvillia per mm2).
In photon microscopy, microvillia look like perpendicular striations on the
membrane surface. Microvillia can be grouped in:
the striated plateau
the brush border
The striated plateau is formed by microvillia with the same shape and
dimensions; it is found at the apical pole of the enterocytes in the gut mucosa.
The brush border is formed by microvillia with unequal dimensions; it is
found at the apical pole.
In electron microscope, a three-layered external membrane, 9-10 nm thick,
similar in structure to common membranes, forms the structure of microvillia.
Inside the membrane, there are a lot of ATP-ases (carrier proteins). Under the
membrane there is an unstructured cytoplasm zone, called ectoplasm. The microvillia
core contains about 40-50 actin filaments that run in a parallel bundle along its length.
At the tip of the microvillia the actin filaments are embedded in an ill-defined cap of
amorphous material that contain myosin, while at their base they extend into a
perpendicular network, composed by many filaments, called terminal web (Figure
15).
Figure 15. Schematic
drawing showing the
polarity of actin filaments
in a microvillus (A) and in
a muscle sarcomere (B).
The actin filament bundle is tied to the inner face of the membrane of the
microvillia free pole by α-actinin. There are also fine lateral bindings between the
actin filament bundle and the entire length of microvillia membrane. Proteins called
fimbrin and villin form these bindings. The interaction of the actin filament bundle
with the lateral membrane of the microvillia is due to arms of myosin and calmodulin
(a Ca++-dependent protein). The microvillia membrane is covered by a rich
glycocalyx.
2.5.2. Stereocilia.
Stereocilia are cytoplasm extensions covered by the membrane. They are not
mobile but their structure is like the microvillia structure. Many of them agglutinate in
the same region and direct a secretion product. They are found in the epididymis and
in the deferent channel cells apical pole.
2.5.3. Cilia.
Cilia are cytoplasm extensions covered by a membrane; they have a high mobility
and are found in the superior airways mucous cells and in some parts of the oviduct.
The tail of the spermatozoon is a flagellum. Cilia are tiny hair-like appendages about
0.25 μm in diameter that contain a core formed by bundle of parallel microtubes.
They extend from the surface of many cell types and are found in most animal species
and some lower plants. They primary function is to move fluid over the surface of a
cell or to propel single cells through a fluid.
The cilia dimensions are under the resolution power of the photonic
microscope.
In electron microscope, cilia appear structured as follows:
1. Cilium stalk that is a free region formed by:
• an external membrane;
• an amorphous zone less electron-dense named ectoplasm;
• the axoneme, a complex structure composed entirely of microtubules and their
associated proteins.
The axoneme is formed by 10 microtubules pairs disposed as follows:
a central pair;
9 peripheral pairs arranged in a ring, around the central pair.
The peripheral pairs are located from 50 to 50 nm. While central pair is formed
by complete wall microtubules, the outer doublets are composed by a complete wall
microtubule and an incomplete wall one; they are called A and B microtubule. They
fuse together to form a pair, A microtubule sharing its wall to the B one. A
microtubule is located closer to the central pair. Its wall is formed by 13 tubulin
protofilaments, each protofilament being formed by αβ tubulin dimmers. B
microtubule wall is incomplete, being formed by 10 protofilaments and it uses 3
protofilaments from A microtubule wall. In A microtubule wall, from 13 to 13 nm,
there are attached two fibrillar arms that project from each doublet and extend toward
an adjacent outer pair in the ring. These arms occur in pairs and are composed by a
protein called dynein. Dynein is an ATP-ase with a 300,000-400,000 MW and it
specifically interacts with tubulin. The interaction between tubulin and dynein plays
essential role in cilia movements, being stimulated by Mg++ and Ca++. By
electrophoresis there were found two types of dynein: dynein 1 and 2. In the cilium
stalk there is more dynein 1. A specific protein called nexin couples the adjacent
doublets in the outer ring; it is thought to be highly elastic and to form “straps” around
the entire axoneme, rather than hoops around a barrel. Projecting inward from each
doublet is a radial spoke, which ends in a globular zone, very near to the inner sheath.
The inner sheath is composed by slender protein arms that project outward from the
central pair of microtubules and curve around them. It apparently enables the central
pair to help regulate the movement of the axoneme. Each of the structures described -
dynein side arms, nexin links, radial spokes, the arms of the inner sheath - can be seen
as series of regular projections, each with its own periodicity. The radial spokes
maintain the geometric conformation of the moving cilia (figure 16).
2. The transient zone is the place where the central doublet microtubules stop in
an electron-dense structure, called basal-plaque. The outer doublet microtubules
continue and pass through this zone, forming the next structure.
3. The basal body (called also kinetosome) has a centriole-like structure. It has a
cylindrical shape, with the wall formed by 9 fused triplet microtubules. Each triplet of
microtubules contains the previous doublet and one more partial microtubule.
4. Actin microfilaments associations form the cilia root that form 70 nm thick
striations, structures that alternate with more clear zones. The cilia root has two
functions:
a. mechanical function for the kinetosome and other cilia structures anchorage;
b. contractile function due to actin microfilaments.
Figure 16. Cross section through the cilium stalk
Cilia movements.
Cilia movements are very difficult to study because its speed (600-1700 beats per
minute). There can be two types of movements:
• bending movements
• undulating movements
It is considered that cilia movements are the result of dynein-tubulin interactions,
guided by ATP, Ca++ and Mg++.
If the cells make connection with the extracellular matrix, actin filaments ends at
the level of specialized regions in the membrane called focal contacts. The
transmembrane linker proteins that mediate the contact between actin filaments and
the extracellular matrix are some cell-surface matrix receptors called integrins. Their
extracellular domain binds protein compounds in the extracellular matrix while the
intracellular domain binds indirectly the actin filaments via a complex formed by
talin, α-actinin, and vinculin.
Septate junctions are found in invertebrate tissues.
2. Desmosomes and hemidesmosomes are connection sites for the intermediate
filaments. They act as rivets and they distribute tensile or shearing forces in specific
tissues like epithelium.
Desmosomes connect intermediate filaments in two adjacent cells. Inside the
cells they are anchoring sites for keratin filaments in epithelial cells and for desmin in
heart muscle cells. Three parts form desmosomes:
1. Extracellular elements represented by the linkers from cadherin family.
Intercellular space is kept intact (15-35 nm).
2. Cell elements:
• an internal plasma membrane thickening in both cells.
• cytoplasmic plate formed by a cytoplasmic condensation located under the
membrane.
• tonofilaments - keratin or desmin (intermediate filaments) - that enter the
cytoplasmic plate and return into cytoplasm. The cytoplasmic plate is separated from
the plasma membrane thickening by an ectoplasm zone, less electron-dense.
3. associated structures, common to both cells - actin filaments.
Hemidesmosomes or half-desmosomes seems to desmosomes except the fact
that they are joining the basal surface of epithelial cells to the underlying basal lamina
(a specialized structure of the extracellular matrix). Keratin filaments (tonofilaments)
are buried in the cytoplasmic plaque. The transmembrane linker proteins are integrins,
similar to focal contacts.
As we have seen cadherins in a plasma membrane anchor it to cadherins in the
membrane of adjacent cells; integrins in a plasma membrane anchor the cell to
extracellular molecules in the matrix.
Tight junctions make possible the transport in two ways. First, they act as
diffusion barriers within the lipid bilayer in the plasma membrane. Thus, they prevent
the transport proteins in the apical membrane from diffusing into the baso-lateral
membrane and vice-versa. Secondly, they tie neighbouring cells together to create a
continuous sheet of cells between which even small molecules are unable to pass. In a
tight junction, the interacting plasma membranes are so closely apposed that there is
no intercellular space: if an electron-dense marker is added to one side of the cell
sheet, it will not pass beyond the tight junction. This junction can be disrupted either
by treatment with proteolytic enzymes or by agents that chelate Ca++ or Mg++, both
special proteins and divalent cations are required for maintaining their integrity. The
morphological structure of tight junctions shows a pentalamelar aspect, formed by
two peripheral electron-dense layers, two layers less electron-dense, situated under the
previous layer, and a central layer electron-dense. This structure is due to the tight
adhesion of the neighbour cell plasma membranes; the intercellular space is missing.
Because tight junctions can be very dynamic structures, they can assembly or
disassembly either partially or completely during many development and
physiological processes. These include the trans-epithelial migration of neutrophils
during inflammation, the migration of maturing spermatocytes across the
seminiferous tubule epithelium during spermatogenesis, changes in cell associations
during morphogenesis and perhaps even hormone regulation of the paracellular
pathway in ion transporting epithelia.
I. Gap junctions are said to be communicating junctions because they allow small
water soluble molecules to pass directly from the cytoplasm of one cell to another; by
chemical synapses the cells communicate only indirectly, even though they are in
physical contact: the pre-synaptic cells secretes a chemical signal (called
neurotransmitter) that diffuses across the synaptic space and signals the post-synaptic
cell. Chemical synapses should not be confused with the less common electrical
synapses, at which electrical impulses pass directly from one nerve cell to another via
gap junctions.
The gap junction is the most common type cell junction, which is widely
distributed in tissues of all animals. This junction is build by proteins that extend out
from the plasma membrane to form structures called connexons, which are believed to
connect the two cells interiors by a continuous aqueous channel. Each of the two
interacting cells contributes enough protein to form one connexon, and these
connexon forms half the length of a channel (Figure 20).
The earliest microscopic observations on living cells revealed that the cytoplasm
is a viscous fluid that can change from a more fluid state to one resembling to a
deformable solid. In the period among 1870 and 1885, it was widely believed
(because the appearance of the cells following fixation and staining) that the
cytoplasm contained a three dimensional network of protein fibers.
The term of cytoskeleton was introduced in 1929 by Koltzoff, who affirmed that
each cell has its own system of liquid compounds and rigid skeletons that gives its
shape. The cytoskeleton represents in fact the muscles and the bones of a cell.
The essential filaments in the cytoskeleton are extensively interconnected by a
three-dimensional network of fine threads, presumably composed by the some
proteins that can removed in detergent-treated cells. This network has been called the
microtrabecular network.
The cytoskeleton is a complex, three-dimensional network, formed by protein
filaments and microtubules, that cross in any direction inside the cell.
It has the following functions:
- it induces the cell’s shape
- it participates in cell motility
- it keeps the spatial internal organization of the cells (it maintain the
position of the organelles in a cell or it associates some metabolic enzymes)
Cytoskeleton is a very dynamic structure due to the continuous actin filaments
and microtubules polymerization and depolymerization. The actin filaments are in
equilibrium with actin monomers and the microtubules are in equilibrium with the
tubulin dimmers. The cytoskeleton contains also some accessory proteins that bind
together its different parts or bind these compounds with other cell structures. The
cytoskeleton can be seen after the treatment of a cell with fluorescent marked
antibodies oriented against the main cytoskeleton proteins. Actin filaments,
microtubules, intermediate filaments and their associated proteins are regulated by
unknown mechanisms in order to produce changes in cell shape and various cell
movements. In addition, the cytoskeleton seems to organize the cytoplasm by binding
various membrane-bounded organelles and soluble proteins. Microtubules emerging
from the cell center (also called MTOC – microtubule-organizing center) induce the
distribution of intermediate filaments and appear to be responsible to determine and
maintain the cell polarity. The organization of a cell’s cytoskeleton can be influenced
by that of its neighbors either through intercellular junctions or by the extracellular
matrix and it can be transmitted to its daughter cells when the cell divides.
In non-muscle cells there were described 5 different types of actin that differ in
molecular weight and aminoacid sequence. In non-muscle cells, actin form
microfilaments while in muscle cells it organizes in thin filaments. In non-muscle
cells, actin makes up 5-15% of the total cell protein mass.
Actin can exist in two forms:
the monomer form (the G-actin);
the polymerized form (the F-actin).
The globular actin is formed by a single polypeptide, with a molecular weight of
45,000 Da, a globular shape, a length of 6 nm and a width of 4 nm. The G-actin is
coupled with a protein called profilin and forms the profilactin complex and thus is
inhibited the polymerization of G-actin. The monomer form of actin has a binding site
for myosin, one for ATP and another one for Ca2+. In instead of Ca2+, Mg2+ is bound,
the profilin can detach from the actin and the latter can polymerize.
The fibrilar actin (the filamentous actin) is formed by the polymerization of G-
actin, in the presence of Ca2+ and energy brought by G-actin bounded ATP
hydrolysis. The polymerization of actin occurs in two steps:
1. At the beginning, in the phase called “lag-phase”, the polymerization
takes place at low-speed; the first 2-3 actin molecules are difficult to bind.
The lag-phase is thought to reflect the fact that the step in filament formation,
in which 2 or 3 actin molecules must come together in a specific geometric
conformation, is particularly difficult.
2. After this step, the ATP hydrolysis accelerates the actin
polymerization. The polymerization process stops when the cytoplasm
monomer concentration is at the “threshold level” (critical concentration).
The actin filament is formed by two strands of globular molecules twisted in a
helix with a length of 500 nm and a diameter of 6.5 nm. G-actin are stringed like
pearls in a necklace in both the F-actin chains.
Actin filaments are polar structures; their two ends differ. At one end prevail
the polymerization while at the other prevails the depolymerization. The polarity of
the actin filament, which is essential to its function in cell motility, is detectable in the
way it interacts with myosin. The actin filament polarity is important to realize the
actin-myosin interaction and to maintain the equilibrium between the monomer form
and the polymerized one. In non-muscle cells, actin filaments can be present
separately or in bundles; many kind of cellular extensions have a core of crossed
linked actin filaments. The actin bundles can be situated in the peripheral cytoplasm,
in the core cytoplasm and under the plasma membrane. Inside these bundles, the actin
filaments are parallel and are cross-linked by a protein called fimbrin. There are
various other actin-binding proteins in the actin bundle, for example villin.
The actin filament bundles forms some contractile rings:
a. The transient ring that appears at the cell division finish, dividing the
two daughters cells.
b. The actin filament belt from the latero-apical cell’s pole.
c. Inside the permanent cell specialization (microvillia and stereocilia).
d. Inside the cell’s temporary prolongation.
In non-muscle cells, the bundles of actin filaments have double functions:
1. Structural role
2. Dynamic role
The actin bundles in non-muscle cells are compared with the muscles. The actin
polymerization produces an increase in the cytoplasm viscosity. The actin
polymerization is influenced by actin binding drugs, cytochalasins (B, D and E) that
are metabolites excreted by molds; these drugs inhibits the addition of actin molecules
to actin filaments, leading to filament depolymerization and inhibit cell movements
based on actin-myosin interaction. The substance called phalloidin, a highly
poisonous alkaloid produced by the mushroom Amanita phalloides, inhibits the actin
depolymerization. By contrast with the cytochalasins, phalloidin stabilizes actin
filaments and inhibits their depolymerization. In muscle cells, the actin filaments form
the thin filament that interacts with myosin thick filaments in order to produce the
muscle contraction.
Myosin filaments were observed for the first time in muscle cells, where they
form the “thick” filaments. In non-muscle cells myosin filaments are difficult to
evidentiate because they are transient structures.
Myosin can be extracted from almost every kind of cell in vertebrates and it is
always present there where actin bundles exist. The skeletal muscle myosin
polymerizes spontaneously in vitro and forms polymers that are much larger than
those formed by other forms of myosin. The myosin filaments are formed by the
addition of myosin molecules, associated by their tails. Myosin can be extracted from
the skeletal muscle by the treatment with concentrated salt solutions, in which myosin
is soluble. Muscle treated in this way loses only its thick filaments that are formed
only by myosin molecules. The latter have a molecular weight of about 500,000 Da
and are seen in electron micrographs as long rod-like molecules, each containing two
globular heads. 6 polypeptidic chains form the myosin molecule: two heavy chains of
200,000 Da each and two pairs of light chains of about 20,000 Da and 16,000 Da
each.
The heavy chain consist of two regions:
- a prolonged region, α-helical;
- a folded region, where they are separated.
This heavy chain segmentation is due to the myosin molecules configuration that
consists of a prolonged part named tail (with 2-coiled α-helices) and two heads which
emerge laterally from the rod-like tail. On each globular head are attached laterally
the two pairs of light chains. The tail is 134 nm length. Between the bicephalic head
and the tail is a flexible zone that allow the myosin head movement when it interacts
with the actin (figure 22).
A protease called papain cuts the myosin subunit at the base of the head region
releasing an almost complete tail, called the myosin rod, and two separates myosin
heads. The myosin rod, as the intact myosin molecule, self-assembles at physiological
salt concentrations into large, ordered aggregates, which, however, lack side
extensions. The two separate heads produced by papain cleavage are each about
120,000 Da and are called myosin subfragment 1 (S1 fragments) (Figure 23).
The heads retain all the ATP-ase activity and the actin filament binding
properties of the intact myosin molecule and can be used to analyze the interaction
between actin and myosin. The two pairs of light chains form the S2 subfragment.
Myosin filaments are formed by the polymerization of myosin molecules
when the myosin tails are associated and the myosin heads project in the external part.
In muscle cells, thick filament have a bare central region where two oppositely S1
subfragments project to the outside (figure 24).
Figure 24. Schematic diagram of a myosin thick filament from skeletal muscle.
Figure 26. Schematic diagram of a muscle thin filament showing the position of
tropomyosin and troponin along the actin filament.
Microtubules are one of the two most important types of filaments of the
cytoskeleton and they participate to organize cilia, flagella, division spindle,
centrioles and the basal body, contributing to the cellular form maintenance and also
to the cellular motility; these functions are due to the tubulin-dynein system.
The microtubules morphology.
Microtubules consist by molecules of tubulin, a globular polypeptide with a
molecular weight of 50,000 Da. Tubulin is a dimmer of about 100,000 Da, each
dimmer being composed by two polypeptides, α-tubulin and β-tubulin, which have
closely related aminoacid sequences. It was described the third type of tubulin, γ-
tubulin, which is not polymerizing in the microtubules but which can play an
important role as nucleation center for the αβ dimmers. When tubulin molecules
assembles into microtubules, they first form protofilaments in which tubulin
polypeptides are aligned in rows, with β-tubulin of one dimmer joined with the α-
tubulin of the next dimmer. Usually 13 such protofilaments are arranged side-by-side
around a central core that appears to be empty on electron micrographs.
Even if a microtubule is usually composed by 13 protofilaments, there are
some variations in different species from 11 to 15 protofilaments. In some cases
protofilaments can associate in structures like doublets (in cilia or flagella) or triplets
(centriols).
The assembly of the tubulin molecules into microtubules resembles the assembly
of actin into filaments in several important features. It occurs spontaneously in vitro
and is normally accompanied by the hydrolysis of one molecule of bound nucleotide;
for tubulin polymerization, the nucleotide is rather GTP than ATP. The nucleotide
hydrolysis plays a crucial role in the kinetics of polymerization. But, in addition to the
exchangeable GTP that is hydrolysed to GDP during polymerization, each tubulin
dimmer contains one firmly bound molecule of GTP that does not participate to the
polymerization reaction and whose significance is unknown.
The resulting microtubule consequently contains one molecule of GTP and one
molecule of GDP for each tubulin dimmer.
The microtubules polymerize at high temperature (37°C) and depolymerize at
low temperature (4°C). Polymers of tubulin have a polarity due to the arrangement of
their asymmetrical subunits in a specific orientation in the polymer. The structural
polarity of the polymer is essential in cilia to induce the organized movements. It also
makes the two ends of the polymer different in a way that is important for the control
of polymer growth. It has recently been found that under certain unusual experimental
conditions, free tubulin molecules will add to the outside of the existing microtubules
to form curved protofilament sheets that in cross-sections resemble hooks. Depending
on the polarity of the microtubules, the hooks appear to be pointing either clockwise
or counterclockwise. Microtubules have been shown to extend with uniform and
identical polarity in nerve cell axons, in cilia, and from the centriole-containing region
in mitotic and interphase cells.
In microtubules, the two opposite ends grow and depolymerize at very different
rates. The growth asymmetry of the microtubules can be observed by allowing tubulin
molecules to polymerize for a short time on small fragments of ciliary axoneme and
then examining them in electron microscopy. In this way, one end of the microtubule
can be seen to elongate three times faster than the other end. The fast growing end
(defined as the (+)-end) has been shown to be the end that points away from the basal
body in a cilium and also the end to which tubulin molecules add in a growing cilium
in a cell.
The growth rate for microtubules differs according to the concentration of free
tubulin dimmers. They can assemble and disassemble rapidly in a cell, being always
in equilibrium with the microtubules. At equilibrium, tubulin molecules (as αβ
heterodimmers) are continuously adding to and are lost from both ends of the
microtubule. In the presence of GTP, however, there is a marked addition at one end
(+) and a marked loss at the other (-). Consequently, there is a continuous movement
of tubulin from one end of the microtubule to another.
The presence of (+) and (-) ends having different critical concentrations (the
critical concentration is defined as the concentration at which the rate of addition
balances the rate of dissociation) for polymerization also has important implications
for the spatial organization of filaments in cell.
Regarding the tubulin, here is evidence that the concentration of free molecules in
the cytoplasm is so low that an unattached microtubule will loose dimmers faster from
its depolymerizing (-) end than it would add the to it’s (+) end and would,
consequently disappear.
Many of the microtubule arrays in the cell are labile, and there are some whose
function depends on its lability. During interphase, microtubules radiate from the cell
center throughout the cytoplasm, but at the mitosis onset these cytoplasmic
microtubules disassemble as the microtubules in the mitotic spindle begin to form.
When mitosis finishes the process is reversed. The stability of different microtubular
structures varies considerably, the mitotic spindle being one of the most labile. This
lability can be seen in the extreme sensitivity of the spindle to various drugs that bind
to tubulin and prevent from polymerizing. From these, colchicine, an alkaloid
extracted from the meaow saffron, has the longest history, having been used
medicinally since ancient Egyptian as a treatment for gout. One molecule of
colchicine will bind tightly to one tubulin dimmer and there it prevents its
polymerization. The addition of colchicine (or of a related compound, colcemid) to a
dividing cell causes the disappearance of the mitotic spindle and blocks cells in
mitosis. For this reason, such compounds are known as antimitotic drugs.
Vinblastine and Vincristine, which also inhibit microtubule formation, have been
widely used as anticancer drugs; they are useful because the disruption of mitotic
spindle microtubules preferentially kills fast dividing cells.
The addition of a different type of drug, taxol, increases (rather than decreases)
the tubulin polymerization of tubulin: when added to the cells, it causes much of the
free tubulin to assemble into microtubules. Similarly, D2O (heavy water) causes a
shift toward tubulin polymerization and thus, for example, increases the number of
microtubules in a mitotic spindle. The additional stabilization of the microtubules in
the cell caused by either taxol or D2O is a deleterious to the function of microtubules
as their depolymerization: a cell with microtubules stabilized by either taxol or D2O is
unable to progress through its growth cycle and it stops at mitosis.
Intermediate filaments (IF) forms the third compound of cell cytoskeleton. Their
name derives from their diameter (about 10 nm) that is intermediate between that of
actin microfilaments and that of microtubules. IF are found in almost all the human
cells, especially in epithelial cells and neurons. They are organized in a network that
crosses the cytoplasm and that insures the intracellular organization and the
disposition of the cells in tissue. In the following table we can see the classification
and the tissue distribution of the IF.
Intermediate filaments Tissue type
TYPE I
Acid keratins Epithelia
TYPE II
Basic keratins Epithelia
TYPE III
Desmin Muscles
Acid fibrilar glial protein Glial cells and astrocytes
Vimentin Mezenchimal cells
Peripherin Neurons
TYPE IV
NF-L Mature neurons in central and
NF-M peripheral nervous system
NF-H
Internexin
Nestin
TYPE V
A laminin In all cells
B laminin
C laminin
NF = neurofilaments
L,M,H = low, medium, high refers to the molecular weights
IF Type I and II:
Keratins are formed by complementary acid and basic intermediate filaments
that form hetero-dimmers. They are frequently found in epithelial cells, associated
with the desmosomes or to hemidesmosomes, and they form flexible networks; in this
way they play a supportive role.
IF type III contains 4 proteins: vimentin, desmin, acid glial fibrillar protein, and
peripherin.
Vimentin appears in endothelial cells, in some of the epithelial cells and in
mezenchimal cells as fibroblasts, forming fibers that end in the nucleus membrane.
They can maintain the localization of the nucleus and of other organelles.
Desmin is found especially in muscle cells, bundling myofibrils. In striated
muscle fibers, desmin filaments are circling the Z disks and connect it to the neighbor
Z disk or to the membrane aligning the disks.
Acid glial fibrilar protein forms the IF in glial cells in central nervous system
and in astrocytes.
Peripherin is less known and is found in neurons in peripheral nervous system.
IF of IV type are called neurofilaments and they fill the axons core in neurons,
where they are usually associated to the microtubules. Another protein called nestin,
found in neurons and miocytes, and is included in this class because of genic
similarity.
Neurofilaments function is to support radial growth of the axons in neurons.
IF of type V are found exclusively in the nucleus, playing a supportive role for
nuclear membrane. These IFs are composed by laminins A, B and C. Nuclear
laminins form the nuclear lamina, a filamentous network situated on the ineer part of
the nuclear membrane. Laminins play an important role in nucleus assembly.
Cellular movements are classified according to the proteic system involved in the
movement:
1. Mobility due to muscle contraction, realized through the actin-myosin
system activation.
2. Cellular mobility that modifies the relation between the cell and the
environment realized by:
a. Ameboidal movement realized by the actin-myosin system: import of
substances during the phagocytosis.
b. Flagellary movement based on the interaction between dynein and
tubulin: the spermatozoon movement.
c. Ciliary movement.
3. Cell mobility that do not modify the relation between the cell and the
environment, realized by:
- exocytosis;
- endocytosis;
- boiling movements, chromosomal movements during the cell
division.
CHAPTER IV
A. The first type is called endocytosis; there are two types of endocytosis,
distinguished according to vesicle size:
I. Phagocytosis (“cell eating”), that involves the large particle ingestion such
as micro-organisms or cell debris, via large vesicles (often called vacuoles).
II. Pinocytosis (“cell drinking”), that involves fluid and/or solutes ingestion
via small vesicles.
B. The second transport type is called exocytosis.
C. The third transport type is called transcytosis. By transcytosis macromolecules are
transported through the capillary endothelium cells. Transcytosis can be done by:
independent vesicles that pass through endothelial cell, from the luminal
face to the interstitial one.
through a channel formed by fused vesicles.
Transcytosis realises the changes between plasma and interstitial fluid.
Macromolecules transport depends on particles dimension, their electric charge
and their chemical properties.
A. Endocytosis is the process where cells ingest macromolecules and liquid or solid
particles from extracellular medium. It is a kind of nutrition for cells or a defence
modality against external aggressors (intercellular space is cleaned by specialised
cells);
I. Pinocytosis (“cell drinking”) is a process that involves the ingestion of
liquid particles. Virtually all eukaryotic cells are continuously ingesting bits of their
plasma membrane as small endocytic (pinocytic) vesicles. The extracellular fluid (and
every substance that can dissolve in it) becomes trapped in the vesicles and is ingested
as well. There are two types of pinocytosis:
Fluid phase endocytosis (receptor independent pinocytosis) is visualised with the
aid of a tracer, such as an enzyme peroxidase, introduced into the extracellular fluid.
A liquid molecule binds on the external face of plasma membrane produces
interactions between plasma membrane and membrane cytoskeleton; plasma
membrane invaginates and the liquid drops are introduced in the cytoplasm, covered
by a plasma membrane fragment. The structure formed is called pinosome and has a
diameter of 50-250 nm. Once formed, the pinosome can evolve in three ways:
a. It binds a primary lysosome and forms a secondary lysosome where the
ingested molecule is disrupted in simple elements, because of lysosomal
enzymes; by this process, from greases there are obtained fatty acids, from
polypeptides result simple peptides). Simple substances pass through
secondary lysosome membrane, enter the cytoplasm and are used to synthesise
new cell structures or are used in cell metabolism.
b. The pinosome remain in cytoplasm as stock substances.
c. The pinosome content is released to the exterior by exocytosis.
The vesicle substance concentration is the same as in the extracellular fluid
concentration (it is a non-selective type of endocytosis). Cell surface area and volume
remain unchanged during this process because the membrane is added to cell surface
by exocytosis as fast as it is removed by endocytosis.
Receptor-mediated endocytosis (adsorptive endocytosis) is called also
concentrative or selective pinocytosis. It takes place in some plasma membrane
depressions that concentrate a large number of receptors for different molecules.
These depressions are coated on the inner face by specific molecules as clathrin and
adaptin that plays an important role in receptors recognition. Receptors are
recognized by these proteins and are concentrated at plasma membrane depressions
level. The depressions act as molecular filters, being permissive only for specific
molecules.
After ligand recognition and binding by the receptor, the depression (also called
caveolae) goes deep inside and then closes. As a result, it appears a clathrin-coated
vesicle. Clathrin coat has a specific structure, being an assembly of three-legged
protein complexes (or triskelion). Three clathrin molecules and three small
polypeptides compose each triskelion. Triskelions are arranged as a basket-like
network of hexagons and pentagons at coated-vesicles surface. The second major
protein of coated vesicles is adaptin. Adaptin plays an important role in clathrin-coat
binding to the membrane and in receptor concentration in caveolae. Adaptin is
recognizing a signal sequence formed by 4 aminoacid residues from the cytoplasmic
receptor tail.
Immediately after forming, the vesicle loses its clathrin coat; clathrin elements
are directed by cytoskeleton components to the inner face of the plasma membrane,
where they will coat another caveolae.
Once formed, the vesicle can follow three ways:
1. It stops in early endosomes. Early endosomes represent a membranous cavities
network with an acid interior (pH=5). This is a dynamic compartment that appears
and disappears according to cell needs. They act as a sorting compartment on the
endocytic way. Because of acid medium, receptors that are bringing ligand
molecules are changing their conformation, delivering the ligand. Endocyted
molecules (ligands) will group in a specific region of the endosome. Here are
formed some vesicles by budding processes. These vesicles contain endocyted
molecules. At the same time, receptors are accumulating in another region of the
endosome. Another budding processes take place in this region including the
receptors in vesicles; these receptor-containing vesicles will be sent back to the
membrane to fit in new caveolae – receptor-recycling way.
2. The second way is similar with the first but receptors reach the baso-lateral pole
of the cell instead the apical pole, serving for transcytosis.
3. The third way seems also the first one before the vesicles enter the early
endosome. Receptors are included in vesicles together with the ligand molecules.
In both Ist and IIIrd ways, vesicles reach the late endosome. From here, part of vesicles
is directed toward lysosomes or to Golgi complex. In lysosomes these structures will
be digested while in Golgi complex they will be used.
Early and late endosomes differs by forming time: 1 minute for early endosome
and 5-15 minutes for late endosomes. Both types interior is very acid, with a pH
around 6, maintained by a proton pump located in their membrane.
An important example is cholesterol uptake by animal cells; this process is
realised by receptor mediated pinocytosis, and it is important in order to prevent
cholesterol accumulation in the blood, with aterosclerotic plaque formation. Most of
the cholesterol is transported in the blood compacted (bounded) in low-density
lipoproteins (LDLs). These LDLs are large spherical particles (22 nm diameter) that
contain a cholesterol core of about 1500 molecules, surrounded by a lipid bilayer
containing a single protein species. When a cell needs cholesterol, it expresses LDL-
receptors on its surface.
Most receptors for LDL associates spontaneously to coated pits but there are also
receptors that bind the coated pits only after LDL-binding. All particles that bind to
LDL receptors are internalised very fast. The coated vesicles lose their coat and fuse
with early endosomes, than with late endosomes, and than with lysosomes. Thus,
within 10-15 minutes of binding to cell-surface receptors, the LDL is delivered to
lysosomes where the cholesterol esters are hydrolysed to free cholesterol and
therefore become available for new membrane synthesis in the cell. If too much free
cholesterol accumulates in the cell, the latter stops cholesterol and LDL-receptor
synthesis (Figure 28).
Concluding, LDL-receptors are following the Ist way. They dissociate by LDL in
endosomes and they are recycled while LDL passes into lysosomes.
A more complex recycle undergoes transferin receptors after transferin
endocytosis. Transferin is a plasmatic carrier-protein for Fe. It can be found as
apotransferin if it lacks Fe. Surface cell receptors bind transferin and deliver Fe to
early endosomes. Low pH in early endosomes induces transferin to deliver Fe and
apotransferin. Apotransferin remains bounded to its receptor, being recirculated to the
apical pole and then delivered at neutral pH in order to bind another Fe molecules. By
this way transferin delivers Fe to the cell, without entering the lysososmes. Transferin
receptors follow the same Ist recycling way.
The second recycling way is followed by EGF-receptors (EGF – epidermal
growth factor, a small protein that induces epidermal cell growth). EGF-receptors
accumulate in the coated pits only after EGF binding. Most of the receptors are not
recycled, being degraded into the lysosomes, together with EGF. Concluding, EGF
binding to EGF surface receptors induces a decrease in receptor number so a negative
receptor feedback.
II. Phagocytosis (“cell eating”) is the process that involves solid particle
ingestion. On membrane surface there are receptors that recognise, bind the ligand
and start the ingestion and the bacteria digestion of bacteria, parasites,
microorganisms, senescent and damaged cells and cell debris. Metchinikov described
it in 1905 in protozoa, where it represents a special type of nutrition. In metazoan,
phagocytosis is a type of feeding and of defence against the infection by
microorganisms.
According to the size of ingested material, the phagocytes are:
mobile microphages (polymorphonuclear leukocytes).
mobile macrophages (big circulant monocytes).
fixed macrophages (hystiocytes from lax connective tissue, adventitial cells in
vessels walls, some cell in the lung, Kupffer cells, in liver).
Figure 28. Receptor mediated pinocytosis for LDL (up) and the relation late
endosome - Golgi complex.
The cells are compartmented membrane structures. A eukaryotic cell has two
compartments:
- the first one is the nucleus which hosts the genetic memory of the cell and the
process of DNA replication and RNA synthesis by DNA transcription;
- the second one is the cytoplasm that contains the translation and transport
mechanism of RNA and the machinery that helps the cell biosynthesis.
The prokaryotes have no individual nucleus, even they have in their structure
genetic material condensed in nuclear equivalents.
The eukaryotes have in interphase an individual nucleus that is the control center
of all the cellular activities.
Eukaryotic DNA is tightly complexed with specialized proteins-histones.
The nucleus of all the cells, except nervous cells, has two functional states:
- the metabolic state (interphasic nucleus);
- the mitotic state.
Usually, the nucleus occupies inside the cell a central position that can be
modified by numerous factors:
- the accumulation of reserve substances (for example, in fat cells the nucleus
is pushed at cell periphery-eccentric nucleus);
- the accumulation of secretion granules (for example, in the cells with serous
exocrine secretion-parotid gland, exocrine pancreas, etc. – the nucleus
occupies a medio-basal position).
In the cells with mucous secretion (for example, the goblet cells from the
intestinal epithelium) the nucleus occupies a basal position.
The nucleus size and volume vary with the cellular type. It also depends on the
phase of the cellular cycle and its functional activity, age, etc.
The usually dimensions of the nuclei are between 3 and 25 μm. In the granular
cells from cerebellums cortex nucleus diameter doesn’t exceed 2-3 μm;
spermatozoon’s nucleus has 4 μm and that of the ovule 20-25 μm. Very big nuclei
are also found in the basophilic megakaryocytes from haematogene bone marrow.
Nucleus’ size and volume depend on cell dimensions; there is a nucleoplasmatic ratio
(NPR) which is constant for each cellular type.
Nv 1 1
NPR = = ÷
Cv - Nv 3 20
Where:
Nv – nucleus’ volume
Cv – cell’s volume
Cv-Nv – cytoplasm’ volume
When cell’s volume grows more than nucleus’ volume, the nucleoplasmatic ratio
will be remarked by a cellular division or by a growing in nucleus’ volume. In
pathological conditions, for example in neoplasic cells, NPR can be complete
modified.
Usually, the cells have a single nucleus they are mononucleated cells. There are
some exceptions: adult red cells that have lost the nucleus in order to adapted to the
function of gas transport (oxygen and carbon dioxide).
They are non-nucleated cells. In some cellular types with more intense metabolic
activity there are two nuclei double-nucleated cells (for example, hepatocytes and
condrocytes). There also are cells with more nuclei multi-nucleated cells (e.g., rough
muscular fiber that contains hundreds of nuclei).
According to their forming process, multinucleated cells are classified in:
- Plasmodium cells that are formed from a mononuclear cell where take place
successive nuclear divisions (cariokinesis) without cytoplasm division
(cytodierhesis). An example is the rough muscular fibers’ osteoclasts.
- Sincitium cells are formed by the fusion of more mononucleated cells with the
disappearance of intercellular plasma membrane (e.g., the trophoblastic
sincitium on the surface of placenta villosities).
Multinucleated cells also appear in pathological cases: in tuberculosis appear
giant Langhans cells.
Figure 30
The exam in light microscopy of the nuclei treated with basic staining shows the
existence inside the nuclei of an intense basophilic substance named chromatin. The
spaces less stained between the chromatin masses are named inter-chromatinic spaces.
The chromatin and the chromosomes are two aspects of the same genetic material
(DNA). In interphase the genetic material is represented by chromatin and during the
cellular division it is organized in the shape of chromosomes.
Messenger RNA
It takes the genetic information from DNA and transits it into the cytoplasm, to
the organelles implied in protein synthesis. It is used as mould for aminoacids
assembling in a polypeptide chain. mRNA is very quickly synthesized and destroyed.
It has a single chain, complementary to one of the DNA chains where it was
synthesized. mRNA molecules are responsible for ribosomes association in
polyribosomes functional elements in protein synthesis. The fact that the smallest
proteins have a polypeptide chain formed by 100 aminoacids means that the minimal
length of mRNA chain is of 100 x 3 ribonucleotides (a codon is formed by 3
nucleotides). The molecular weight of mRNA is of 5 x 105 ÷ 40 x 105 Da.
Ribosomal RNA
It is synthesized inside the nucleus and forms two subunits:
- the small subunit (40S)leaves the nucleus together with the molecules of
mRNA; it contains rRNA 18S and 34 ribosomal proteins.
- The large subunit (60S) will combine in the cytoplasm with the small subunit
in the moment of protein biosynthesis and both the subunits will form the
ribosome; the large subunit contains rRNA 28S, rRNA 5, 8S, rRNA 5S and
45 ribosomal proteins.
5.7.3. The Basic Proteins
Generally, basic proteins have a small molecule and a basic character because of
their content in aminoacids (arginine, histidine, and lysine).
They are classified in:
1) Protamines lie especially in the nucleoproteins from fish’s spermatic cells.
They are also found in small amounts in the acids from mammalian cells.
2) Histones are basic proteins tighly bound with the DNA molecule. By
biochemical researches it was found that eukaryotic cells’ nuclei contain 5
types of histones:
- H1 very rich in lysine
- H2A, H2B moderate rich in lysine
- H3, H4 rich in arginine
These kinds of histones, especially the last 4 are found in equal amounts and have
a small molecular weight (10 ÷ 15,000 Da). The molecular weight of H1 is double. In
almost all cellular types the DNA/histone ratio is equal to the unit.
The nuclear zones between the chromatinic bundles are called interchromatinic
spaces. The ultrastructural autoradiography and cytochemical studies showed that
these spaces are occupied by heterodispersed nuclear RNA. It can have the following
aspects:
1) Perichromatic fibrils are represented by fibrillar elements with a diameter of
30-50 Å, disposed at chromatin’s periphery. From chemical point of view
RNA molecules form them.
2) Perichromatinic granules are found in all the animal cell types and appear in
the shape of granules with a diameter of 250-450 Å. They are formed by
particles of ribonucleoproteins. It is supposed that they are evacuated in the
cytoplasm through the nuclear pores in the shape of mRNA with the small
subunits of the ribosomes atatched.
3) The interchromatic granules are groups of particles with a diameter of 200
Å, formed by mRNA molecules covered with proteins.
4) The spiral bodies are spherical aggregates of 0.3-0.5 μm formed by spiral
fibrils of ribonucleoproteins whose role is unknown.
-
Nucleolus ultrastructure
- Pars cromosoma (chromosomal component) contains fibrils with a diameter
of 100 Å disposed both at nucleolus’ periphery (perinucleolar chromatin) and
in its interior (intranucleolar chromatin-DNA). This pars cromosoma contains
DNA from the nucleolar organiser region of a chromosome.
- Pars amorpha (unstructured component) is homogenous, less
electronodense.it is disposed among the first 3 components and is formed by
proteins.
One of the most important phenomena that happen in the interphase is the synthesis and
cell secretion, the central place being occupied by the protein synthesis. The process takes
place in cytoplasm. The organelles involved in this function are the ribosomes, endoplasmic
reticulum, and Golgi complex.
Ribosomes are non-membrane organelles found in the cytoplasm of all eukaryotic cells.
They play an important role in the protein biosynthesis, by placing the aminoacids according
to some fragments from messenger RNA.
The ribosomes lies generally in cell cytoplasm, free or attached to the rough endoplasmic
reticulum membrane or to outer nuclear membrane.
The number of ribosomes in a cell depends on cell type; in the neurons or secretor gland
cells, their number is higher than in other cell types. Their number is variable, according to
the functional state of the cell. In the active mammary gland secreting cells, the number of
ribosomes is higher than in repose period.
6.1.3. Polyribosomes.
They are the functional structures of ribosomes that are tied at the rRNA. The number of
ribosomes involved in the protein synthesis varies according to protein dimensions that must
be synthesized. The mRNA molecule includes the molecule-organizing plan. When the
mRNA is long, the protein is complex. According to mRNA length, the number of ribosomes
organized in polyribosomes varies a lot. The mRNA molecules that code for globine synthesis
have a length of 2600 nm.
The number of ribosomes attached to mRNA is about 4-6. The number of ribosomes that
codes for collagen molecule is around 100.
The polysomes can be free in the cytoplasm, attached to the mRNA where they are
involved in protein synthesis mechanisms, or attached to the RER (rough endoplasmic
reticulum) membrane, involved in the export of synthesized proteins. In the mammary
secretor cells, there are many ribosomes attached to the RER membrane.
The ribosomes are tied to the RER membrane by two glycoproteins, called
glycophorines. There are two types of glycophorines, type I (MW = 65,000 Da) and II (MW =
63,000 Da).
ENZYME
AA + ATP AA-AMP + P-P
++ +
Mg , K
Stage II. The stage consists in some reactions that have like purpose the first aminoacid
insertion into the polypeptidic chain and the protein biosynthesis release. It is formed an
initiation complex, mRNA-ribosome 80S-aminoacyl-tRNA. In these reactions participate
some proteins, individualized by saline extraction from the ribosomes, called initiation
factors. The initiation factors are: IF1, IF2, IF3, IF4A,B,C,D, IF5. The IF1, IF2, IF3 factors plays an
important role in polypeptidic chain initiation. IF1 participates to the binding process between
R60S and the mRNA-aminoacyl-tRNA-R40S complex. IF2 participates to the binding process
between R60S and the mRNA-R40S and aminoacyl-tRNA complex. IF3 binds R40S to
mRNA. This stage has 3 steps and uses as energy source the GTP.
1. In the nucleus are formed mRNA molecules, with their codons. Here are
formed ribosomal particles with two subunits. This step takes place in the nucleus and
R40S binds to mRNA, at a distance of 10 nucleotides from the 5' extremity, there where it
is an AUG codon (the initiation codon). The binding is realised with the intervention of
IF3.
2. The second step is the insertion of the first AA-tRNA initiation complex,
which has always the same aminoacid type named methionine. The initiation complex that
brings the first aminoacid in the polypeptidic chain is formil-methionil-tRNA. It
recognises by the azotate bases of the anticodon the azotate bases of the initiation codon
AUG. The IF2 factor participates to complex initiation and brings a GTP molecule to
supply energy. When the complex is ready, IF3 is released in the cytoplasm.
3. Finally, the large ribosomal subunit joins the complex. The IF1 factor is
essential for this process, bringing another GTP molecule for hydrolysis. Now, ribosomes
presents two sites: P-site (from peptidyl) - occupied by formil-metionil-tRNA and the
neighbour place A (from aminoacyl) - it is free and placed where it is the following
codon of mRNA molecule.
Figure 33. Initiation step in protein biosynthesis.
Stage III. Now takes place some cycle-repeated reactions, for a time. The number of
cycles depends on the number of codons on mRNA that codifies the polypeptidic chain. This
stage has also 3 steps:
1. The insertion - it consists in occupying the A place with the second AA-tRNA
complex, according to the second codon configuration. New complexes aminoacyl-tRNA
are formed (the aminoacid can be lysine) by the intervention of two free cytosolic proteins
named elongation factors (EF-Tu = transfer unstable and EF-Ts = transfer stable). They
participate to the insertion of the second aminoacyl-tRNA complex in the A place,
bringing the second aminoacid in the polypeptidic chain. The reaction needs energy
delivered by a GTP molecule hydrolysis; both places, P and A, are occupied by an
aminoacyl-tRNA complex.
2. The elongation. In this process participates an enzyme called peptidyl-
transferase that cuts the bind between methionine and the tRNA in the P place and makes
the first peptide bound between the first and the second aminoacid. The tRNA in the place
P become free and it will be delivered into cytoplasm. Now, A place is occupied by a
dipeptide (a chain of two aminoacids). EF-Tu, EF-Ts and GDP+P are also delivered from
the complex into cytoplasm.
3. The translocation. First, the tRNA molecule is moved off from the P place into
cytoplasm and the P place is free. The tRNA will be able to bind another aminoacid that
will be always methionine (because of the accepting specificity). Then, the ribosome
moves off along the mRNA molecule toward the 3' end. The translocation needs energy,
delivered by a third GTP molecule hydrolysis. The P place is occupied by a dipeptidyl and
the A place is free. After this, the reaction repeats cyclically (figure 34).
Introduction.
A single continuous sheet enclosing a single closed sac forms the endoplasmic reticulum.
It is highly convoluted and covered by a lipoprotein membrane.
In 1879, G. Garnier studying the gland cells, observes in their cytoplasm the presence
of an intense basophilic organelle, that he named ergastoplasm.
In 1945, Porter observes the presence of a canalicle network, delimited by a
membrane that he named endoplasmis reticulum. Electron microscope complex studies
performed by Porter (1953) and G.E.Palade (1954) show the ultrastructure and the functions
of this organelle.
The endoplasmic reticulum is present in cell types, being largely represented in
secretor cells. It has two functionally distinct regions that differ by structure and function;
they have the same membrane with a thickness of 5-6 nm. These regions are named rough
endoplasmic reticulum and smooth endoplasmic reticulum. The rough endoplasmic reticulum
is studded with ribosomes on the cytoplasmic side of the membrane; the smooth endoplasmic
reticulum lacks of ribosomes. These two regions differ considerably in shape. Rough
endoplasmic reticulum (RER) is organised in stacks or flattened sacs, called cisterns; smooth
endoplasmic reticulum (SER) consists of a meshwork of fine tubules (figure 35).
A. J. Dalton and M. D. Felix showed the real existence of the Golgi complex using the
electron microscope. They showed that the organelle is formed by a fine lamellar structure
and named it “Golgi complex”. It is a cellular organelle, present in all cellular types,
excepting the adult red cells and is well represented in the glandular and nervous cells.
Lysosomes’ biogenesis
The lysosomal enzymes are maturated and transformed in Golgi sacs, recognized by a
receptor from the Golgi complex membrane due to their manose-6-phosphate residue. Thus,
they are segregated, packed in chlatrin coated vesicles and they come in cytoplasm and form
primary lysosomes.
THE MITOCHONDRION
Mitochondria are organelles present in all plant and animal cells and it occupies a
substantial fraction of the cytoplasm of virtually all eukaryotic cells. Although they
are large enough to be seen in the light microscope and were first identified in the
XIX-th century, but the real process in elucidating their function had to wait until
1948, when a procedure was developed for isolating intact mitochondria.
Mitochondria are usually depicted as stiff, elongated cylinders with a diameter of
0,5-1μm. The size of mitochondria is different upon the kind of the cell: in
hepatocytes and nephrocytes they have a length of 3 μm and a diameter of 0,5 – 1 μm;
in muscular cells their length may be until 8 μm and in cells from exocrine pancreas
10 – 12 μm. The number of mitochondria varies with the energetic requires of the cell
– for example in liver, each cell contains 1.000 to 2.000 mitochondria and a fifth of
the total cell’s volume is occupied by this organelles; in fat cells, lymphocytes, etc.,
their number is reduced.
The observation of the living cells reveals a mobility and plasticity of the
mitochondria – rapid changes of shape and that belie their static image in electron
micrographs. As they move about in the cytoplasm, mitochondria often appear to be
associated with the microtubules of the cytoskeleton. This association may determine
the unique orientation and distribution of mitochondria in different types of cells: the
mitochondria of some cells form long moving filaments or chains, while in other
types of cells they are fixed in position near a site of unusually high ATP
consumption.
In the nephrocytes from distal contort tubes they occupy the basal labyrinth, the
delivered energy being used for the active transport of substances; in ciliary cells’
mitochondria are found near the basal corpuscle (the kinetic center of the cilium).
1) The outer membrane contains many copies of a transport protein that forms
large aqueous channels through the lipid bilayer; it is permeable to all molecules of
10.000 Da or less, including small proteins. Such molecules can enter the
intermembranespace, but most of them can not pass the impermeable inner
membrane. This means that, while the intermembrane space is chemical equivalent to
the cytosol with respect to the small molecules it contains, the composition of the
matrix space is much more highly specialized. The outer membrane contains 60%
proteins and 40% lipids (the highest percentage is occupied by cholesterol and
phospholipids). It contains two enzymes such as: monoamine-oxidase (marker
enzyme), NADH2-c-reductase.
2) The inner membrane contains a higher fraction of proteins (80% of the total
membrane weight) and 20% lipids. Cardiolipin, the lipid concentrated in the inner
membrane, is believed to reduce the permeability of the phospholipid bilayer to
protons.
Freeze-fracture studies on the inner membrane indicate that it contains many
protein-rich intramembrane particles that are laterally mobile in the membrane plane.
Some of these particles are permeases that allow otherwise impermeable molecules,
such as ADP and other phosphorylated compounds, to pass from the cytosol to the
matrix. The marker enzyme for the inner membrane is cytochrom-C-oxidase.
It contains 3 major types of proteins:
- the enzymes of the respiratory chain;
- an enzyme complex called ATP synthetase that makes ATP in the matrix;
- the Krebs cycle enzymes (succinate-dehidrogenase-marker enzyme) and
specific transport proteins that regulate the passage of metabolites inside and outside
the matrix.
Since an electrochemical gradient that drives the ATP synthetase is established
across this membrane by the respiratory chain, it is important that the membrane be
impermeable to most small ions.
3) The intermembrane space contains several enzymes like adenilakinase that
maintains the equilibrium ATP/ADP/AMP; it catalyses the reaction: ATP+ AMP=
2ADP. Other enzymes are nucleoside mono and diphosphokinase, etc.
4) The matrix contains a highly concentrated mixture of hundreds of different
enzymes, including those required for the oxidation of pyruvate and fatty acids and
for the citric acid acid cycle. It also contains several identical copies of the
mitochondria DNA genome, special mitochondria’s ribosomes, tRNAs, and various
enzymes that are required for the expression of the mitochondria’s genes (Figure 38a).
Figure 38a
This enzyme is a large protein complex through which proton flows back down
by the electrochemical gradient into the matrix. Like a turbine, this protein complex
converts one form of energy to another, synthesizing ATP from ADP and Pi in the
mitochondrial matrix in a reaction that is coupled to the inward flow of protons.
ATP synthetase is a large complex, with a molecular weight of about a million,
composed of at least nine different polypeptide chains. The entire complex is also
known as the F0F1ATP-ase. Five of its polypeptide chains make up the spherical head
of the complex, known as the F1ATP-ase. ATP synthetase serves as a reversible-
coupling device that interconverts electrochemical-proton-gradient and chemical bond
energies. Its direction of operation depends on the net free-energy change for the
coupled processes of proton translocation across the membrane and the synthesis of
ATP from ADP and Pi. In fact, three parts form ATP synthetase:
1) A hydrophobic proteic complex named the base, situated inside the lipidic
bilayer, that has the H+ translocation system, from the matriceal side to the
cytoplasmic one.
2) A spherical complex, with 9 nm diameter, named the head. This one extends
through the mitochondrial matrix. The complex is named also F1 subunit and has
catalytic role in the reaction of ATP synthesis from ADP+Pi.
3) A proteic stalk that binds the other two parts and contains a protein that gives
olygomycine sensitivity and another protein that binds the F1 portion to the
membrane. When the H+ are transported from the matriceal face to the cytoplasmic
one, it results a pH difference and an electrochemical potential accumulation that
gives the motrice force and determines the H+ back entering the matrix. The internal
mitochondrial membrane is not permeable for H+. These ions enter through the
protonic tunnel from the ATP-synthetase. When the H+ ions pass through this tunnel
and enter inside the matrix, at the F1 level is realized the ATP synthesis from ADP+P.
When is synthesized more ATP than the cell needs, the synthesis stops. When the cell
needs again ATP, the F1 subunit begins again the ATP synthesis from ADP+P.
CHAPTER VIII
Cellular digestion is the phenomena that lead to the digestion of the molecules
penetrated inside the cytoplasm through the endocytosis of some old organelles, some
cell fragments or other structures.
In the digestion phenomena are implied two kinds of cellular organelles:
- lysosomes
- peroxisomes
The word lysosome was introduced by Champy in 1913 and it names some
intense osmiophillic granules. Christian de Duve discovered them in 1957 by
biochemical researches. He ultracentrifugated mitochondria's suspension and he
isolated a subfraction rich in acid phosphatase, an enzyme that is found in great
amount into this “ lytic particles”. In the same period Novikoff, morphogically
identified these organelles on electronomicrographs.
The lysosomes have a spherical or oval shape, with a diameter of 0,25- 8 μm and
they are found into the cytoplasm of all eukaryote cells. They have a rich content of
hydrolazic enzymes.
All beings have a life cycle where take place growing and differentiation
processes, having as final aim the life conserving.
The cells that represent structural and functional units of the organism have their
own cycle called cell cycle.
Cell cycle means the assembly of biochemical, morphological and functional
changes that take place in a cell from its appearance by mother-cell division to its own
division into daughter cells.
Cell cycle has two steps: the interphase and the cell division (Figure 44).
As it is known, the cell cycle can be divided into four stages: G1 (extracellular
environment control, cellular growth before DNA replication), S (DNA synthesis), G2
(the cells verifies if the DNA replication is completed and prepares for division)
(Figure 44 a).
In fact, all the signals that regulates the cell cycle converge to the nucleus-placed
control horologe which is based on a group of interacting proteins capable to trigger
or stop cell division, thus co-ordinating the mechanisms leading to cell division as the
"cell executive manager " (R.Weinberg-1996).
The control horologe of the cell cycle uses a large number of proteic molecules in
order to insure this succession of stages: e.g. cyclins (first D, then E, A and B1),
cyclin-dependent kinases (CDK) playing a major role in controlling the shift from one
stage of cell cycle to the next .
There are several molecular systems that control the activity of different CDK:
the one for synthesis, control and cyclins regulating subunits destruction (R.
King,J.Petors, M.Kirschner-1994), post-translational changes of the kinase subunit
alone by highly specific kinases and phosphatases (T.Coleman,W.D.-1994) as well as
binding/ unbinding to a large variety of inhibitory proteins capable of inhibiting most
of CDK types; among these types there are: p21 (active during all stages of cell cycle;
its expression it is adjusted by p53 protein that is encoded by a tumour suppresser
gene; p53 controls cell 's health. DNA integrity and the proper succession of cell cycle
stages; in the cells do not posses p53, p21 is very reduced and is not able to associate
with CDK),p27 (also called IcK, kip- 1 and Pic- 2 -K.Polyak, M.Lee, A.Koff,
J.Roberts et al.- 1994) and p57; other inhibitory proteins are specific to CDK- 4 and
CDK- 6 e.g. p16, p15, p18 and p19/p20 (also named InK 4D and InK 6B- H.Hirai,
M.Roussel, J.Cato et al.- 1995) .
These inhibitors regulate the cell cycle by inhibiting CDK4- cyclin D1, CDK2-
cyclin E and CDH 2- cyclin A complexes. The importance of these molecules in cell
cycle control and carcinogenesis is proved by the fact that, directly or indirectly, they
are the targets for mutations as it happens in a large number of human malignant
tumors and tumoral cell lines.
Figure 44. The cell cycle.
In the last years, studies linking oncogenesis and the cell cycle found proves
indicating that a misfunction of the cell cycle control horologe induces both
uncontrolled cellular proliferation and genetic instability, which are characteristics of
the tumor cells (T, Hunter and J. Pines - 1994).
The end of stage G1 is crucial for the normal progress of a cell cycle since this is
the moment when the cell decides whether the cell cycle continues or not. Also, the
misfunction of the control horologe of the cell cycle during stage G1 is one of the
most frequent molecular anomalies seen in malignant tumors. When this phase is
concluded and the cell enters the stage S, a “molecular switch” must be turned from
“off” to “on” (Figure 44b).
The way this switch functions is: as the D and E cyclins concentration increases,
they associates with CDK and, as a result they become active. Thus activated, the
cyclin-kinases complexes initiate Rb protein phosphorylation, the main inhibitor of
cell cycle control horologe, and it remains hyperphosphorylated until mithosis is
initiated when it is dephosphorylated. During the first phases of stage G1, Rb protein
is associated to the transcription factors from E2F family, thus inhibiting the
transcription of the genes implicated in DNA synthesis. After phosphorylation, the Rb
protein releases E2F factors, allowing the cell to enter the stage S. If Rb protein is not
phosphorylated at the end of stage G1, the cell cycle is blocked by maintaining the
"switch" in the position "off ".
CELL DIFFERENTIATION
10.1. Introduction.
Cell differentiation is the process that obtains cells with a high degree of
structural and functional organisation, leaving for a primitive, not-differentiated, but
multipotent cell. The multipotent cell contains the genetic information necessary for
the evolution.
Cell differentiation begins in the pre-embryo period when in the gonads of parent
organisms, the primitive germ cells (ovogonia and spermatogonia) pass through
differentiation mitosis that leads to gametes realisation. It is a necessary stage for
fecundating and for keeping the morpho-functional characters of the species. Through
the differentiation process, there are achieved:
species perpetuity
organism growing and development
species evolution and organism adaptation to the medium
The ideal material for cell differentiation study is the embryo, although such
processes take place during whole life, toward the death of the individual.
During the fecundation process, the two pronuclei (male and female) with a
haploid number of chromosomes get near one to another without fusing, achieving the
cariogamy or amphimixy. This stage marks the end of the fecundating phenomena
where the zygote or fecundated egg is obtained. Before approaching, the DNA
replication takes place inside the two pronucleoli, and the DNA amount doubles. In
the fecundating process are achieved:
• the activation of the egg recovers the division capacity (the unfecundated ovule
degenerates);
• the remake of the diploid chromosomal equipment;
• sex determination: X or Y chromosomes;
• it is formed the hereditary dowry of the new individual through the fusion in the
same cell of the paternal and maternal genes. After the zygote forming, it begins a
new stage of the differentiation. This stage is called segmentation. Immediately
after amphimixy, the maternal and paternal chromosomes (46 chromosomes in the
fecundated egg) are longitudinal cleaved and then separated, forming 92
chromosomes. Half of their number is directed toward a pole of the cell and the
other half migrates toward the opposite pole. During the migration, it begins the
cytodierhesis. Finally there will be separated two cells (blastomeres) from the
zygote. Each blastomere has a diploid number of chromosomes: 44+XX or
44+XY.
The fecundated egg and the first four blastomeres born from the first two
segmentation mitoses can form through the differentiation process all cell types of an
adult organism. That is why they are called pluripotent cells. In these blastomeres that
have the same size and shape, all the genes from the nucleus are depressed (are
active) so that every message received by them is accepted. In this way, the cells have
a very different evolution (figure 46).
Figure 46. Cell differentiation in
In the stage of morula appear differences in size and position: small blastomeres
appear. They are named micromeres and occupy the animal pole. Large blastomeres
also appear and they occupy the vegetative pole. The difference in size and position is
due to some extrinsic factors.
In the second week of embryonic life, the differences become more evident: it is
formed the blastula, the embryonic button appears. Its cell will form the first two
embryonic layers: the endoblast formed by small cells and the ectoblast formed by
large cells. The cells in these two layers are obliged to follow a certain evolution and
are named determinate cells. This state is determined by the action of some repressor
agents that inactivate a certain number of genes from cell’s genetic dowry. During the
evolution are repressed a variable number of genes, always others, depending on the
specialized cell type that has to evolve.
The factors that direct the differentiation process toward a certain specialized cell
type are called inductors. The target cells must be permissive (permeable to the
inductor) so that the inductors can act. This property appears as a result of the
appearance of receptor zones located in cell membrane that bind the inductors,
because the activity of some genes. During the differentiation process, the permisivity
for a certain type of inductor is limited in time. The periods of action for the first
inductor and the moment of the first determination are essential for realizing a normal
individual.
In the developing process of the human being (in ontogenesis) the number of the
successive determinations is equal to the number of inductors. These elements finally
create stem cells. From these cells will form different types of specialized cells. For
example, from stem cells of hematogenous bone marrow, there will be differentiated
the leukocytes, red blood cells and platelets.
In an adult organism, there are not pluripotent cells, but each tissue has stores of
stem cells except the heart muscle and the nervous tissue. The differentiation process
is characterised by the appearance of some biochemical, morphological and functional
changes. From biochemical point of view, cells differentiation is connected with
specific protein synthesis during the embryo development. From functional point of
view, the differentiation process is characterised by the appearance of some
characteristic function for each type of new formed cells. For example, muscle cells
are able to contract, because they are synthesising contractile proteins - actin and
myosin.
The nature of the inducting factors, of the mechanisms that control cell
differentiation represents problems of modern biology. Egg fecundation is quickly
followed by more segmentation mitosis. The blastomeres become smaller and smaller
and will form the morula, which dimensions are equal with that of the fecundated egg.
Between the central blastomeres of the morula that is formed by 64 cells, there appear
spaces. These spaces will grow and will form a cavity: central blastomeres are
localised at one of the poles of this cavity. A single layer of blastomeres forming the
embryonic button delimits the cavity. It is the blastula stage.
In this stage, the cytoplasm role is more important than the nucleus’s.
In amphibian eggs, there are two different peripheral zones:
• the animal pole that is dark-brown and
• the vegetative pole that is white.
Immediately after fecundation, on zygote surface appears a zone of intermediate
pigmentation, localised in the subequatorial region called gris semiluna. From this
structure will develop the dorsal part of the frog. If the superficial layer of this zone is
destroyed (dorsal cortex) the development of axial organs does not take place
anymore. The cortex of gris semiluna that appears at a short time after fecundation is
pure cytoplasmic.
The germinative plasma is another cytoplasmic territory localised at the
vegetative pole of the amphibian fecundated egg. It contains big granules of
ribonucleoproteins. Destroying this territory with UV rays gives the appearance of the
individual without gonads. If it is injected in such an irradiated egg, cytoplasm from
the vegetative pole of an non-irradiated egg, the obtained individual is normal. It
results that in the first development stages, all blastomeres nuclei are equipotents, but
the cytoplasm plays an important role in the differentiation process.
In the gastrula stage, the interaction between nucleus and cytoplasm has an
important role in differentiation. In this stage, the cell continues to multiply but two
new events appear: the embryo growth and cell grouping in distinct assemblies. T.H.
MORGAN gave the nucleo-cytoplasmic interaction theory in 1934. He showed that in
this stage, under the cytoplasm influence, in some nuclei, one or more genes would be
derepressed, modifying the biochemical cytoplasm structure that will become more
heterogeneous.
The nucleus transplant techniques in non-nucleated cells with different
differentiation degrees were used to demonstrate an important phenomenon in the
differentiation process. In non-nucleated Xenopus eggs were transplanted nuclei from
different adult cell types, perfect differentiated. There were obtained perfect
developed tadpoles. This experiment demonstrates that the nuclei of adult cells are
identical with that of fecundated cells. They have conserved all the genetic
information contained in ovules and spermatozoon nuclei. The genes from the nuclei
of adult cells are identical with that of the nuclei of fecundated eggs. This
demonstrates that nuclei are not irreversible differentiated during the individual
evolution and that their genes are made active or repressed because of the nucleus-
cytoplasm interaction.
In the gastrulation period, from embryo button are formed three layers:
• the endoblast formed by small cells, localised between the segmentation cavity and
the embryo button;
• the ectoblast formed by big cells, localised between the endoblast and
cytotrofoblast;
• the cordomesoblast is differentiated from the posterior zone of the primary
ectoblast and lies between the endoblast and cytotrofoblast.
The gastrulation marks the beginning of the derepression or of the repression of
some genes through:
• cytoplasm-nucleus interaction
• interaction between cells (induction)
• the control messengers from the surrounding medium.
From the three embryo layers will be formed all organs of an adult individual.
Cells of these layers are “obliged” to follow a certain evolution before to be obliged
or determined, they are competent, in the sense that their development depends on
their answer to different stimuli. When they answer to a stimulus, they become
determined cells.
In the developing embryo, the induction term names the effect of a surrounding
influence that determines the competent cells to undergo a certain differentiation
degree through which they arrive to a certain determination degree. The induction has
a very important role during the development of an embryo and on it depends the
tissue development in comparison with the surrounding tissues.
In mammalian adult organisms there are no pluripotent cell, but only stem cells
both for somatic and sex cells. In adult these differentiation processes are called
regenerative processes and take place in the same conditions as in embryo
(hematogeneous bone marrow, gonads etc.).
CHAPTER XI
APOPTOSIS
11.1. Introduction
Necrosis means pathologic cell death and is induced when major cell alterations are
present. Abrupt cell confrontation with extreme non-physiologic conditions guides to the loss
of control on ionic flux, fast water penetration in the cell, followed by swelling and membrane
rupture. Organelles volume increasing, of mitochondria especially, is characteristic for this
type of cell death. At the same time necrosis is characterised by lysosome destruction and
hydrolytic enzyme overflowing in cytoplasm. Necrosis is a passive process that does not need
active cell participation, cell that finally dies.
Apoptosis or programmed cell death is a regulating physiologic phenomenon that
equilibrates cells number in a normal tissue. It is a part of the normal cell life, like
differentiation or cell division; it is also an active process that engages cell metabolism. The
latter observation leaded to the term of programmed cell death or cell suicide. The cell
participates actively to a self-destruction program similar to a suicide. Apoptosis is
irreversible, and it is not toxic for neighbour cells. It may appear in physiological conditions
(embryo development, normal turnover in the tissues) and also in pathological ones
(malignacy).
The inducing mechanism disturbances of apoptosis may cause various pathological
processes (table 2).
Glucksmann described cell death during normal organism development in 1951 then by
Kerr in 1965 and Sannders in 1966.
Their experiments demonstrate that human embryonic cell or from insects during
metamorphosis carry the information necessary for time delayed self-destruction.
Consequently, it appeared the idea that cells contain a suicidal program controlled by a
biological watch.
11.2.1. Tanatogenes
First of all, cell death programming involves the existence of specific genes, the
tanatogenes, that coding for proteins with lethal effect on a specified cell. Molecular analyses
lead to the discovery of two genes Ced-3 and Ced-4 that become functional in cells that enter
the apoptosis.
If these genes are inactivated, apoptosis do not occur. Both genes act autonomous to
induce programmed cell death. Ced-3 codes for a new protein with phosphorylation sites
while Ced-4 codes for a new protein with two Ca+-binding sites; however,it is not well known
the way of interaction with other proteins or the way by which other proteins are inducing
apoptosis. At the same time, it was shown that other genes are essential to induce apoptosis in
mammalian cells: proto-oncogenes, c-myc - that normally stimulates cell proliferation is
involved in apoptosis induction, P53 - tumor gene suppressor, is another gene associated to
apoptosis; high levels of P53 are associated to programmed cell death.
Physiologic cell death starts when a cell in the organism dies by a mechanism controlled
by proteins coded by the host genome. The purpose of this process is to eliminate undesired
cells; it becomes active in three situations:
• development and homeostasis;
• apoptosis mechanism;
• aging.
A large variety of stimuli are able to induce apoptosis for a specific cell type; for
example: hormones, growth factors, monoclonal antibodies directed over a surface molecule,
etc.
At the same time, stimulus missing - physical agent, metal, biological agent, xenobiotic
agent, antitumoral agent, may have the same consequence. These agents act on extremely
various cell types (Table 4).
As we have seen, apoptosis can be induced by all factors that induce necrosis, but at a
lower concentration or dose. Kerr demonstrates that a reduced degree of hypoxia induces
apoptosis while severe hypoxia provokes infarcts and necrosis; hyperthermia induces
apoptosis or necrosis depending on quantity of heat absorbed, depending also on cell type.
INDUCING SIGNAL
TANATOGENE ACTIVATION
MACROMOLECULAR SYNTHESIS
NUCLEAR FRAGMENTATION
CELL FRAGMENTATION
During aging we can observe the diminishing of cell number in most organs, considering
that apoptosis is inducing agent. Apoptosis induction in senescent cells is due to the lack in
growth factors, anomalies in intercellular and intracellular signal transmission, and
modifications of the cell cycle. Apoptosis acts probably to eliminate altered senescent cells
and its perturbation may involve elimination of healthy cells with the exception of abnormal
one.
Depriving of growth factors and/or surviving factors represents a possibility to induce
apoptosis: a cell can activate the self-destruction program because it did not received
surviving signal. So it is explained fast regression by apoptosis of experimental hyperplasia in
various organs (kidney, liver, pancreas). During aging we can see similar phenomena of
homeostatic mechanism disregulation in order to maintain cell number in organism. Growth
factor and/or surviving factor synthesis decreases with age inducing apoptosis mechanism
activation. This phenomenon was revealed during some organs aging: mammary gland and
uterus involution during menopause happens by apoptotic mechanisms and reported to
deprivation of sexual hormones; cutaneo-mucous modifications are reported to estrogens
deficit and are based on apoptosis mechanisms activation; diminishing of androgen synthesis
with age is partially responsible for some organic atrophy, especially in muscle mass melting
in older man. Muscle and cutaneous atrophy observed during senescence could by of
apoptotic origin and linked to diminished production of growth and surviving factors, like
growth hormone and insulin-like growth hormone (IGF-1). The observation that a hormone
treatment for 6 months with growth hormones leads to a significant increase in muscle mass,
of epidermal thickness and of vertebral bones density in persons over 60 years of age with
IGF-1 deficit, pleads for apoptotic origin or organic atrophy during senescence. Apoptosis
induction by lacking of surviving factors is well demonstrated in nervous system, where
neurones die when neuronal growth factor produced by target cell lacks.
Defects in transmembrane signalling.
Death by apoptosis can occur while signalling ways that lead to cell division are
interrupted or disturbed. C-protein-kinase (PKC) plays an important role for cell option for
death or proliferation. PKC activation inhibits apoptosis while PKC inhibitors have the
reverse effect. Tirosin-kinases are involved in apoptotic processes and their dysfunction can
provoke the arrest of cell cycle and death by apoptosis. During ageing, membrane receptor
bound tyrosin-kinase activity diminishes, leading to alterations in the transmission of
proliferative signals.
Cell cycle alterations.
After ontogenesis, a multicellular organism must realise the equilibrium between
proliferation and cell death for maintaining a constant volume. No doubt that these genes are
involved in the control of cell cycle and also in apoptosis control. During aging, it is possible
that the modification of gene expression that controls proliferation mechanisms to move the
equilibrium death-proliferation over apoptosis. For example, p53 is a cell cycle regulator but,
in some special conditions it can induce apoptosis. Its expression is induced especially by
some events susceptible to alter DNA but also by deprivation of growth factors.
Concluding, apoptosis mechanism that acts during organogenesis and then during adult
life, can be activated during aging processes. During aging, apoptosis eliminates non-
functional cells but it could represent an abusive phenomenon; in other situations apoptosis
control mechanisms are affected by aging and their activation leads to elimination of normal
cells - aberrant apoptosis.
CHAPTER XII
CELL AGING
12.1. Introduction.
12.2.1. Generalities.
The life of the majority of cell population from human organisms can be divided
in four phases: normal functioning, aging (senescence) agony and cellular death. The
most studied model of cellular aging is represented by cultured human fibroblasts.
More than 25 years ago, Hayflick demonstrated that normal diploid fibroblasts have a
limited life span in culture.
The normal human fibroblasts can be multiplied (by duplication) up to 60 times
after what the cells stop dividing.
The proliferate potential can be slightly modified by adding some compounds al
glucocorticoids and growth factors. It was observed a limited time span too for other
cell types as: keratinocytes, chondrocytes, glial cells, smooth muscle cells, granulosa
cells, adrenal cortical cells, vascular endothelial cells and mammary epithelial cells.
Then it became clear that the most important parameter of cellular aging is not the
survival chronological time in culture but the number of complete cell divisions,
because it is known that one of the essential events linked to cellular aging is the
diminishing cell proliferate potential followed by a decreasing cell number in most
organs.
From the morphological and biochemical changes that characterizes cellular
aging in senescent cultured fibroblasts and in other cells in senescent tissues we are
mentioning: the increased number and dimensions of the lysosomes, chromosomal
abnormalities, accumulation of altered forms of structural proteins that drives to the
perturbation of cell functionality (e.g. the accumulation of α-DNA-polymerize is
partial responsible for restraining the proliferate capacity of senescent fibroblasts),
reducing the transcription and translation rate, reducing the protein degradation,
decreasing response to a wide variety of growth factors and hormones, due to the
decreased number of receptors according to aging or to the altered ways of signal
transmission in target cells.
During aging, in the vascular system appears the “aging vasculopathy” – a result
of the adaptation mechanisms in senescent cells – and that represents a distinct
phenomenon from atherosclerosis but it is close to it by the susceptibility of aged
vessels to develop atherosclerotic lesions.
This susceptibility is due to morphological, architectural and molecular
modifications that appears in vessels, especially in senescent endothelial cells; vessels
dimensions are increasing, also increases the thickness of medium and internal layer,
of the extracellular matrix that is more rich in glycosaminoglycans and collagen, the
cell volume, elastin fibers are progressively disorganized and become fragmented,
transendothelial traansport is altered favoring the entrance of plasmatic molecules in
subendothelial space, retaining substances as LDL or AGEs that accelerates
atherosclerosis processes.
The endothelium permeability is increasing by age while the ability to produce
vasoactive substances is decreasing.
At the same time, aging is associated with the accumulation of some AGEs
(high end-glycated products) that produces particular cellular events like: oxidative
stress, leukocyte adhesion molecules expression, transendothelial migration of
monocytes, all being considerated important prelesional events in atherogenesis
process.
Aging affects the ability of endothelial cells to proliferate and to repopulate
nude zones resulted by vascular injuries. Chang and Harley observed that the length
of telomeres in endothelial cells taken from donors of different ages decreases with
aging.
Aging modifies the properties of smooth muscle cells from blood vessels walls
(migration and proliferation), the structure and the composition of extracellular matrix
that increases the susceptibility of the vessels to atherosclerosis. So, at aorta’s level it
was observed a decreasing in the concentration of heparan-sulphate associated to the
endothelial basement membrane while the the quantity of condroitin and dermatan-
sulphate in coronaries increases with aging. These changes in the percent of
glycosaminoglycans are similar to those observed in atherosclerotic coronaries and
are close linked with the accumulation of esterificated cholesterol.
Recent studies show the involvement in this complex phenomenon of two kind of
factors:
⊇ extrinsic factors (influence of medium and of life style)
⊄ intrinsic factors (primary aging processes genetically induced)
According to these two categories of factors, it appeared some theories regarding
the causes of cellular aging, classified as follows. (table 6)
There are two major types of hypotheses to explain cellular aging.
The first proposes that aging is provoked by the passive accumulation of errors in
cellular constituents as DNA, RNA, protein and lipid due to a variety of
environmental insults coupled with imperfect repair mechanisms.
The second considers aging to be an active, genetically programmed event.
Theory Description
Error theories
Rate of living Fixed metabolic units
Waste product Build up of a toxic substance
Somatic mutation Mutations in somatic cells
Cross linking Cross-linked macromolecules
Free radical Hyperactive oxygen radicals
Error catastrophe Exponential errors in protein synthesis
mistakes
Altered proteolysis Changes in protein breakdown
Program theories
Codon restriction Switching codon preferences in early
development restricts codons available
later in life
Repetitive DNA loss Multiple gene copies sequentially
inactivated or lost
Telomere shortening Altered telomerase activity
Terminal diferentiation Growth simulators not expressed or
growth inhibitors overexpressed.
Table 6. Cellular theories of aging
A. Error theories.
The different error theories of aging focus on somatic mutations, faulty proteins,
damaged lipids or a combination of damaged molecules.
1. Rate of living.
2. Waste product accumulation.
3. Somatic mutations.
4. Cross-linking.
5. Free radicals.
6. Error catastrophe
7. Altered protein degradation
B. Program theories.
Preponents of the idea that aging is actively programmed often cite examples of
programmed cell death in early development.
1. Codon restriction
2. Inactivation of multicopy deoxyribonucleic acid sequences
3. Shortening of telomeres
4. Terminal differentiation
In fact, two events are always linked to senescence: diminishing of cells
proliferate potential and diminishing of cell mass.
This progressive cell rarefaction affects the most organs, being an evidence
especially in skin, muscles, kidney, brain and blood; at the same time is associated
with an increased DNA concentration in plasma blood in aged persons. We consider
today that this decreasing in cell mass in senescent organs is probably of apoptotic
reason. Recent progresses in the understanding of biological basics of cellular
senescence drive to the hypothesis that the mechanism of apoptosis induction (cell
programmed death) are activated by physic-pathological modifications characteristics
for cellular aging. The connection between apoptosis and cellular aging is somehow
still intuitive and incomplete explored.
Perspectives.
Numerous deficits underlie age changes. Replacement of these deficient factors,
such as estrogen and growth hormone, is being actively investigated in an effort to
thwart age-related changes in bone and muscle, body composition and other
functions.
With a better defined causes of aging and its molecular bases there is a real
opportunity to develop approaches for reducing age-associated deficits and thus
reduce the incidence of age associated diseases. Notable in this regard is the increase
in longevity via long-term diet restriction in animals, an increase associated with a
reduction in AGE glycation products, reduced oxidative damage and a reduction in
degenerative diseases.
CHAPTER XIII
EXTRACELLULAR MATRIX
The tissues do not contain only the cells in their structure but they have also an
extracellular space filled up with specific substances called extracellular matrix. The
extracellular matrix is a complex network of proteins and polysaccharides between the
intercells space where we find many types of molecules secreted by cells. In addition to
serving as universal biological glue, they also form highly specialized structures such as
cartilage, tendons, basal laminae, and bone and teeth with some forms of calcium phosphate
crystals. The quantitative variations of the different types of the matrix macromolecules
determine the diversity of the matrix shapes, very enclose with the tissue functions. Thus, in
the epithelial tissues the extracellular matrix is limited in volume, but in the connective
tissues its volume is bigger than the cells.
The matrix has an active and a complex role: influencing the cells in their development,
migration, and proliferation and stabilizes the shapes and the metabolic functions. In the
differentiation process the cells need specific components. The cells morphogenesis is very
close dependent of the matrix fibers. The matrix components can bind growth factors and
hormones giving signal abundance for present cells.
Since the different cells secrete varied components of extracellular matrix we can group
them in two types:
1. Polysacharride chains of glycosaminoglycans, which have a covalent bind at a
protein forming proteoglycans.
2. Fibrilar proteins, which could be:
- structural such as colagenes and elastins
- adhesive such as fibronectin and laminin
The glycosaminoglycans and proteoglycans molecules from connective tissues form a
highly hydrated gel-like essential substance in which collagen fibers are embedded. These
collagen fibers strengthen and help to organize the matrix and the polysaccharide gel offer
resistance to the pressure. Also, the aqueous phase of the polysaccharide gel permits a rapid
diffusion of nutrients, metabolites, and hormones between the blood and the tissue cells. In
the same time the elastin fibers give to the matrix the specific elasticity, and some proteins as
fibronectin are responsible with the attaching of the fibroblasts and other cells to
extracellular matrix in the connective tissues. For the epithelial cells laminin realizes them
connecting to basal membrane.
There is a diversity of the presented components due to the multiple gene sets that are
responsible of the collagen IV coding, and the laminin A and B1 chains. Many of these
molecules and their isoforms have a limited tissue distribution; their presence can be
described in specific stages of tissue growth.
13.1.3. Proteoglycans
Proteoglycans are macromolecules find in the all connective tissues. They are formed
from a proteinic core covalently linked with one or more glycosaminoglycans’ molecule.
The polypeptidic chain or proteinic chain of the proteoglycans is synthesized on the
ribosomes of rough endoplasmic reticulum membranes and introduced into their lumen. The
polysaccharide chains are usually assembled in the Golgi complex.
Proteoglycans are classified depending on the repeating disaccharide structure unit of
their glycosaminoglycan component. Sometimes, the proteoglycan molecules are not
exclusively components of the extracellular matrix.
Proteoglycans from cartilages are the best known molecules. It size can be more than 4
μm and a volume bigger than bacterian cell. These proteoglycans confer to the cartilage its
characteristic properties: resistance on the strength and consistence like a gel. A long
hyaluronic acid molecule forms the central component of proteoglycans. At it is linked the
core proteins of proteoglycans to chondroitin sulfate. A linker protein assures the proteins’
linkage on the hyaluronic acid. On each proteinic core is attached multiple chondroitin
sulfate and keratan sulfate chains.
Schematic drawing of the various intracellular and extracellular events involved in the
formation of a collagen fibril. While the extension peptide cleavage and fibril formation
are shown occurring after secretion, there is some evidence that cleavage of the amino-
terminal peptides and some aggregation of collagen molecules occurs just prior to
secretion from the cell. Because the larger extracellular aggregates of collagen
molecules are stabilized by covalent cross-links as an example of how the collagen
fibrils can form ordered arrays in the extracellular space, they are shown further
assembling into large collagen fibers that are visible in the light microscope.
The structural properties of each collagen fibril assure their specific functions.
The structural unit of the collagen is long protein, with the length about 300nm and the
thickness around 1.5 nm, formed by three polypeptidic chains, triple-stranded: two chains of
α1, and one chain α2. Each chain is formed by 1050 aminoacids, and a α chain contains 3
aminoacid per each complete helix. Characteristic for collagen is its great contents of glycine
and proline as well as hydroxyproline and hydroxylysine. Glycine is the single small
aminoacid to occupy the space from the interior of the triple-stranded. Hydroxyproline and
hydroxylysine realize the hydrogen bindings between the α chains stabilizing the molecule.
The collagen synthesis has place in the ribosomes attached to the endoplasmic reticulum as
pro-α-chains, then being hydroxylated and glycosylated. Of them, three associates to form
triple-stranded procollagen molecules in the endoplasmic reticulum and the Golgi complex.
After the secretion off the cell, some peptides from the end of the molecules will be cut
forming the microfibrils through the covalent bindings. The microfibrils are grouped in
bundles forming the collagen fibers observed on the light microscope.
This structure has a great mechanical resistance especially on traction.
13.2.2. Elastin
Elastin is other protein rich also in proline and glycine, but un-hydroxylated. Secreted
in the extracellular space, elongated elastin molecules are linked into a network. Each
molecule being stranded as random-coil in the network, as fibers or layers, has the rubber
elasticity because the “eyes” of the network modifies its shape according with the force
direction.
Elastic fibers are not composed solely of elastin, however; they also contain a
glycoprotein that is usually distributed as microfibrils on the surface of the elastic fibers. In
developing elastic tissues, these glycoprotein microfibrils often appear before elastin does
and may serve to organize the secreted elastin molecules into the fibers and sheets which
they later form.
It would be found in skin, lung and blood vessels.
Therefore, elastin is a cross-linked, random-coil protein that gives tissues their
elasticity.
13.3.1. Laminin
Laminin is a molecule with a specific shape having 70nm the length. The molecule is
formed from three polypeptides and contains sites with a good linkage affinity for other
components of basal lamina. Basal laminae are thin layers of specialized extracellular matrix
that underlie all epithelial cell sheets and tubes. They also surround individual muscle cells,
and fat cells. The basal lamina thus separates these cells and cell sheets from underlying or
surrounding connective tissue.
The basal lamina is synthesized by the cells that rest on it (see the figure below).
Although the precise composition varies from tissue to tissue and even from the region
within the same lamina, a major component of basal laminae is type IV collagen. Type IV
pro-α-chains is unusual in having extra-long extension peptides that are probably not cleaved
after secretion.
In addition to proteoglycans and fibronectin, which are important constituents of basal
laminae, the large glycoprotein laminin has been shown to be a major component of all basal
laminae studied so far, together with entactin, perlecan and type IV collagen. It consists of at
least two subunits that are disulfide-bonded to each other. Basal laminae undoubtedly
contain many other proteins yet to be identified. The detailed molecular organization of basal
laminae is unknown, although there is some evidence that laminin and proteoglycan
molecules are concentrated along the inner and outer surfaces of the basal lamina, with
collagen molecules sandwiched in the middle.
13.3.2. Fibronectin
The fibronectin form a major class of glycoproteins from the matrix. Fibronectin
molecules assure the cells attachment on different extracellular matrix types. The presence
on the surface of un-transformed culture cells and the absence on transformed tumoral cells
of fibronectin confirm us the fibronectin implication in cellular adhesive. In this way,
fibronectin regulates the cell shape and the cytoskeleton organization. In embryogenesis, in
the cellular migration and the differentiation process fibronectin is found.
The fibronectin is a dimmer molecule formed from 2 similar peptides, which are
bounded with disulfuric links. Each chain is embedded in many globular domains, separated
by linker polypeptide segments. The individual domains are specialized for specific binding
on other different molecules from matrix. Thus, fibronectin has binding sites on the surface
receptors, collagen and sulfate proteoglycans.
13.3.3. Integrin
Between cells and matrix the complex relations are established. Due to multiple
interactions achieved on the level is very difficult to precisely establish where the membrane
components are finished and where the matrix begins. Sometimes, the cellular matrix
components are linked on the cells’ surface through the specific glycoproteic receptors.
The most known receptor for matrix is fibronectin receptor found on the surface of
mammalian cells. This receptor has 2 polypeptides called α and β, it acts as transmembranar
linker which mediates the interaction between intracellular skeleton (formed through actin)
and fibronectin (found in extracellular matrix).
There fore integrins are the mainly cell receptors to link the extracellular matrix, but
integrins are also connectors between matrix and cytoskeleton.