Advanced Cellular Biology and biotechnology

CMB: NEUROBIOLOGICAL TRACK [Digitare qui] ACBB PERROTEAU 22/23

ADVANCED CELL BIOLOGY AND BIOTECHNOLOGY (ACBB)

ANCHORING JUNCTIONS 05/10/22

The proteins mentioned in the title of paper 1, i.e. vinculin and talin, are anchoring proteins which

are responsible for the anchoring of the actin cytoskeleton to focal adhesion (FAK).

Examples of cell junctions include:

- TIGHT junctions are a type of cell-to-cell junctions that are primarily found in the epithelial

tissue. They are precisely located at the apical membrane of the lateral side connecting

two neighboring cells. In fact, it is thanks to the tight junctions that it is possible to

distinguish the apical membrane from the basal one in a cell.

- DESMOSOMES are a symmetrical type of cell-to-cell junctions and are involved in the

bearing of forces and tension. They are large transmembrane proteins found on the

lateral side of two neighboring cells. Their constituent protein plakin (specific cadherin

molecule) is connected to the cell cytoskeleton, precisely to the intermediate filaments,

so that they can keep the cytoskeleton in place, whilst tension (force, deformation) can go

from one cell to another. Thus, if one cell is pulled in one direction, the neighboring cell

will follow.

- GAP junctions are a type of cell-to-cell junctions that allows for passage of molecules

between neighboring cells, thus allowing for cell-to-cell communication.

- HEMIDESMOSOMES are a type of cell-to-ECM (ECM= extracellular matrix) junctions and,

because of this, they are found on the basal membrane of epithelial cells. Since on one

side of the hemidesmosome there is the cell and on the other side there is the ECM

(composed of collagen fibers and adhesion molecules), hemidesmosomes are not

symmetrical. However, hemidesmosome structural organization on the cell cytoplasm is

very similar to that of desmosomes.

- FOCAL ADHESIONS are a type of cell-to-ECM adhesion, which is primarily found in

fibroblasts as these are surrounded by ECM.

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PROTEINS FORMING JUNCTIONS

Cadherins and integrins are not junctions, but are transmembrane proteins involved in both

adhesion and junctions. Each of them has their own subfamilies.

Cadherins mediate cell-to-cell contact and mediate homophilic interactions, meaning that the

same type of molecules is found on the lateral side of each cell. This explains the formation of

clumps of cells of the same type in culture: for example, epithelial cells form clumps with each

other, whilst neuronal cells form clumps with each other. This also explains why cells of different

types do not interact, as they each have different types of cadherin molecules (E-cadherins in

epithelial tissue, N-cadherin in nervous tissue). Furthermore, if there is a change in the expression

of the same cadherin type, the cells will reorganize themselves (visible in 3D cell cultures) to

adapt to the new concentration of cadherins.

Integrins mediate cell-to-ECM contact. They are composed of alpha and beta subunits and have

only one transmembrane domain. Their specificity with respect to binding properties is given by

the association of the alpha and beta subtypes, meaning that depending on which subtypes are

present, integrins preferentially bind collagen, or fibronectin or laminins.

Integrins main ligands are the extracellular ones, meaning those in the ECM, thus collagen fibers,

fibronectin, adhesion proteins, laminins, sugars and hyaluronic acid. The fibers and adhesion

proteins found on the basal membrane are produced by the epithelial cells and not by the

connective tissue underneath. Other cell types producing basal membrane proteins and

molecules include endothelial and muscle cells.

Integrins bind to the actin cytoskeleton on the intracellular side.

Note that proteins that are not connected neither to the ECM nor the intracellular side are the

only proteins that are free to move in the bilayer, and thus go from the basal side to the lateral

one.

Occludins are the constitutive molecules of tight junctions. They impede proteins from going

from the lateral or basal side to the apical side of the cell. The only way by which proteins from

the basal side reach the apical one is by exocytosis, precisely by the specificity of the interaction

of the exocytotic vesicles with the apical membrane which is mediated by the proteins v-SNARES

(vesicle SNARES) and t-SNARES (target SNARES on apical membrane).

INTERACTION WITH CYTOSKELETON

Desmosomes (cell-to-cell) and hemidesmosomes (cell-to-ECM) interact with the intermediate

filaments of the cytoskeleton.

Adherens junctions (cell-to-cell), instead, interact with the actin filaments of the cytoskeleton

which are parallel to the plasma membrane.

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Tight junctions (cell-to-cell) are not connected to any part of the cytoskeleton. They only allow

the passage of small ions, nucleotides and secondary messengers like cAMP, single sugars and

amino acids, thus are present in non-skeletal and cardiac muscle cells.

Focal adhesions (cell-to-ECM) interact with the actin filaments of the cytoskeleton.

The integrins constituting focal adhesion interact with actin and form filopodia when actin is in

straight bundles or lamellipodia when actin is in branched networks.

Stress fibers are formed when there are focal adhesions on opposite sides of the cell and they

interact with actin filaments that are in tension and that move with respect to each other (not

with respect to myosin like in muscles), meaning that the actin filaments are interconnected by

specific myosin. This allows for proper tension organization.

Invadopodia and clathrin-containing adhesion complexes are not connected to intermediate

filaments of the cytoskeleton, but they interact with actin instead.

Clathrins interacting with actin are responsible for the formation of vesicles of lysosomes and

endocytosis, as well as playing a role in mechano-transduction as they are sensitive to tension.

Invadopodia have a more circular actin organization than clathrin-containing adhesion complexes

and are responsible for the release of hydrolytic enzymes necessary to cut and destroy the ECM.

Invadopodia are characteristic of osteoclasts (same family of leukocytes, different from

osteoblasts) as these cells mediate bone resorption by dissolving the bone matrix.

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Actin, microfilaments and microtubules are dynamic structures that allow for stretching and

shrinking, whilst intermediate filaments are much more stable and are important in resistance

against pulling forces.

MECHANO-SIGNALING

The signaling processes can be electrical (action potential), chemical (ligand and receptor) and

mechanical (equilibrium and hearing, not vision because it is physical and not mechanical).

Mechano-signaling refers to a process where the signal is triggered by a mechanical force such as

shear stress or a pull/push applied to biomolecules. Usually, application of such force would

induce a conformational change in the protein/receptor, thus exposing functional domains to the

environment.

Mechano-signaling can often trigger cellular signaling processes much faster than purely

chemical means of activation. For example, the mechanical stimulation of focal adhesions in

smooth muscle cells where Src was activated in <0.3s and in 12s as it would under chemical

activation.

Any pressure on cells acts as a mechanical signal.

For example, if there is a healing wound, the cells are in tension during the healing process as

there are not many of them yet and the tension acts as a signal to stimulate cell proliferation.

Note that everything that has to do with adhesion also has to do with signaling. The stress fibers

act as sensors for the organization of the adhesion and spreading.

Therefore, adhesion and cell communication are complementary. Their regulation is altered in

cancer cells, which can be differentiated from normal cells in vitro by means of 3D soft agar gel.

Here, cells are placed in the center of the 3D gel made of pure agar solution, meaning that there is

nothing for the cells to organize adhesion, so that normal cells will be unable to spread and

proliferate and will die by the end of the day, whereas cancer cells will still be able to form

colonies and proliferate.

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MECHANO-TRANSDUCTION

Cells and tissues can sense and react to the modifications of the physio-chemical ECM properties

through integrin-based adhesion sites and adapt their physiological response in the process of

mechano-transduction. Rigidity of the substrate on the ECM plays a role in mechano-signaling,

since the more rigid the substrate the more efficient the organization of the forces.

Molecular signals, on one hand allow for molecular activation because the tension causes a

conformational change in molecules and enzymes in a similar fashion to phosphorylation

changing the charge (making them more negative) of tyrosine-kinase or G-protein coupled

receptors in chemical signaling. On the other hand, tension on the cytoskeleton can also change

organization in other organelles, including the nucleus and thus reflect on chromatin.

Focal adhesion (red dots in the picture below) serve as crucial sites for both outside-in and inside-

out mechano-transduction through the recruitment of transmembrane integrins.

Due to their critical localization at the cell-ECM interface, transmembrane integrins are

mediators of bidirectional signaling, playing a key role in outside-in and inside-out signal

transduction.

Outside-in mechano-transduction mediates the cellular responses that are induced by the ligand

binding to integrins.

Inside-out mechano-transduction activates the ligand-binding function of integrins.

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TRANSDUCTION OF MECHANICAL STIMULI

By changing the adhesion, it is possible to induce different signaling cascades on the cytosolic

side of the cell.

Molecules involved in these signaling cascade include Srcs, which are tyrosine-kinases (together

with serine-kinase, tyrosine-kinases are the most prevalent kinases in the cytosol leaflet) that are

synthesized by free ribosomes and subjected to lipidation (post-translational modification) to be

then associated to the cytosolic leaflet of the plasma membrane.

Src is the first tyrosine-kinase molecule to be discovered as its oncogenic form is present in

viruses and it lacks the C-terminus. It is the C-terminus of Src that allows for the inhibition of the

kinase, since it is the part that contains the tyrosine which, upon phosphorylation, binds the SH2

domain so that the tyrosine-kinase takes its closed conformation and thus becomes inactive.

Therefore, the SH2 domain interacts with the phosphorylated tyrosine present on the C-terminus

of Src and in this conformation the Src kinase is inactive.

In response to physical stimuli, multiple signaling pathways can be activated.

One is the FAK-Src signaling which controls the turnover of focal adhesions, cell adhesion, and

migration. Other pathways involve the activity of the small GTPases RhoA and Rac, which play a

role in the organization of the cytoskeleton, actin polymerization, and actomyosin contractility.

These pathways regulate the activity of YAP/TAZ, which controls gene expression. At cell–cell

adhesions, the RhoA activity is controlled by the guanine nucleotide exchange factor (GEF) Solo,

which promotes the formation of desmosomes and slows down collective cell migration.

Actomyosin-generated tension, for example, can be transmitted and applied to the ECM by focal

adhesion (integrins), whilst it is transmitted and applied to the cell-cell interface by adherens

junctions (cadherins).

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Src AND FAK: NON-RECEPTOR TYROSINE KINASES 07/10/22

As previously mentioned, substrate rigidity plays a crucial role in what happens inside the cell,

meaning that it affects and regulates all the different mechano-signaling pathways.

Cell adhesion has complementary information regarding the soluble factors that stimulate the

cell, so if the cell is not in the right conditions/situation for adhesion, then the cell will not

respond to mitogenic factors.

There are two main regulatory aspects to consider:

1) the regulation of the cytoskeleton by means of small monomeric G-proteins of the Rho

family such as GTPases RhoA and Rac.

2) the regulation of signal transduction operated by integrins interacting with kinase

molecules that are also associated with adhesion. Note that in this instance, these are

molecules with kinase activity, they are not receptors.

In general, tyrosine-kinases are divided into two types:

a) receptors are membrane-spanning proteins with extracellular ligand binding site.

à

b) non-receptors are cytosolic proteins and the majority of them are coupled to

à

transmembrane proteins.

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PROTO-ONCOGENE c-Src

Srcs are synthesized by free ribosomes and subjected to lipidation (post-translational

modification) to be then associated to the cytosolic leaflet of the plasma membrane.

As any other tyrosine-kinase, Src is activated by dephosphorylation, and it is inactivated by

phosphorylation.

The general structure of c-Src includes:

- C-terminus where tyrosine-527 is, which is the first tyrosine to be phosphorylated and

à

has inhibitory activity.

- SH1 domain where tyrosine-416 is, which has stimulatory activity and thus it is the

à

catalytic domain.

- SH2 domain phospho-tyrosine binding domain, it is the domain where Src interacts

à

with the phosphorylated tyrosine-527.

- SH3 domain proline-rich binding domain, it is the domain where Src interacts with

à

proline. allows for protein-protein interaction.

à

- SH4 domain N-terminus where lipidation occurs so that Src can be on the cytosolic

à

leaflet of the plasma membrane. It is unique to each kinase molecule.

c-Src phosphorylates specific tyrosine residues in other tyrosine-kinases. Moreover, it plays a role

in the regulation of embryonic development and cell growth. Elevated activity levels of c-Src are

suggested to be linked to cancer progression by promoting other signals.

The discovery of the cellular homolog c-Src of the viral oncogene v-Src implicated that the

precursor form of the cancer-causing gene (oncogene) is encoded by the human genome,

representing a keystone for modern tumor biology.

Src was the first tyrosine-kinase molecule to be discovered, approx. 100yrs ago, and one of the

few molecules for which 3 different Nobel prizes were given.

The first Nobel prize was given in 1909 to Peyton Rous, who discovered a chicken sarcoma virus,

hence the name RSV (Rous Sarcoma Virus).

He removed the sarcoma from the breast muscle of affected chickens, ground it with sand,

filtered it and injected it into young healthy chicken. These young healthy chickens ended up also

bearing the sarcoma. This was the first evidence that made scientists at that time assume that

tumors are viral diseases, as they saw that sarcoma could be transmitted via virus (structure and

genome of the virus were not known). This conclusion was made because at that time oncogenes

were not known.

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Not all oncogenes are of viral origin, the majority of them are of cellular origin and have

homology with viral ones.

The second Nobel prize was given to Temin and Baltimore who discovered the reverse

transcriptase enzyme, as they found out that the RSV is a retro-virus and as such uses reverse

transcriptase for its replication:

1) As soon as the sarcoma virus infects the cell, its reverse transcriptase enzyme synthesizes

DNA copies of its own genome.

2) These copies enter the nucleus of the cell and randomly insert themselves throughout the

DNA of the host chromosomes.

3) Normal gene transcription within the nucleus now produces an RSV mRNA and this re-

enters the cytoplasm.

4) Some copies of this mRNA are then translated by host ribosomes into protein products.

5) Other copies of this mRNA are incorporated into new viral particles.

Nowadays, reverse transcriptase is widely used in recombinant DNA technologies to produce

cDNA, which enables amplification via PCR, transfection and cloning with vectors.

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The third Nobel prize was given to Bishop and Varmus who discovered the Src oncogene, so they

were able to understand why the RSV is able to cause the sarcoma.

The RSV has extra genetic information (in pink in the picture below) compared to the murine

leukemia virus. In fact, instead of 3 genes, the RSV has 4 genes:

• gag encodes the capsid protein

à

• pol encodes the reverse transcriptase.

à

• env encodes the envelope protein.

à

src

• the “extra” genetic information that encodes a tyrosine kinase, an enzyme that

à

attaches phosphate groups to tyrosine residues on a variety of host cell proteins.

Therefore, Bishop and Varmus discovered the presence of Src, which is not directly useful to the

virus, in the sense that the virus does not use it for any of its own mechanisms, but rather to act

on the host. They discovered that this “extra” gene is an information that is coming from a

cellular gene. The difference is that the cellular gene is regulated, whilst the viral one is no longer

regulated and it is always active.

Nowadays, the regulated cellular gene is defined as protooncogene, whilst the mutated, non-

regulated and always active version of the protooncogene is called oncogene.

Their discovery that the viral Src derives from the cellular one was achieved thanks to

hybridization which evidenced the presence of a match between the cellular Src exons and the

viral Src exons.

The difference is that the oncogene Src (viral Src) lacks the C-terminus, which is needed