Advanced Cellular Biology and biotechnology

Bi-directional Signaling and Receptor Activation

This is similar to the way different receptors activate different signaling cascades. Usually, the ligand is outside the cell, it binds the membrane receptor and this binding activates the signaling cascade inside the cell. Different receptors might have different affinity for the ligand. If the ligand is transmembrane, then the signaling can occur in the two cells that are directly interacting. This type of signaling is defined as bi-directional.

Bi-directional signaling referred to integrins is different from the bi-directional signaling that occurs in two cells that are directly interacting. When integrins are not active, they are in a closed conformation on the extracellular side and it is the interaction with the substrate on the extracellular side that allows for the opening of the integrins. This binding of the substrate to the extracellular side of the integrin dictates a change in the

intracellular side of the molecules. Again, this process is defined as outside-in signaling.

OUTSIDE-IN signaling: Ligand binding to the external domain causes conformational changes that increase ligand affinity, modify protein-interaction sites in the cytoplasmic domains and thus the resulting signals.

The opposite process can also take place. Modifications inside the cell can induce a change in the structure of the integrin that will increase the affinity for the ligand on the extracellular side of the molecule. This process is, instead, defined as INSIDE-OUT signaling, where what happens inside modifies what happens outside. This type of signaling changes the adhesion properties of the cell (e.g. diapedesis during inflammation, or PSA N-cams, adhesion molecules of the nervous system that are modified in the already present post-translation modification so that they negative change increases and the two N-cams can no longer interact).

INSIDE-OUT signaling: Signals received by other receptors

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In order to modify the adhesivity of a cell, charges of interacting molecules can be changed so that the adhesion decreases – as in the case of negative charges on both interacting molecules -, or increases – if interacting molecules have opposite charges.

Thus, by changing the expression of the adhesion receptors, or by modifying the structure of the interacting molecules by means of covalent changes, it is possible to change the adhesivity of cells.

Moreover, by modifying the intracellular regulator proteins that bind to the cytoplasmic C-terminal of the integrin β subunit, as well as that intracellular signaling, it is possible to also change cell adhesivity.

This concept is at the basis of cell migration, and it also applies to walking. In fact, when we

haveto take a step forward

we increase the adhesion of one leg, the one making the step forward, whilst we decrease the adhesion of the other leg. If the adhesion stays the same, movement cannot take place.

This same process occurs at the lamellipodia of cells, where the adhesion of the front lamellipodium increases, whilst in the back the adhesion is removed. The direction of this movement is dictated by the actin filaments in the ventral stress fibers.

This explains why in ventral stress fibers there are 2 focal adhesions (one on each side of the stress fiber) and myosin. The focal adhesion in the back is temporarily removed to move the cell forwards.

This change is not dictated by the ECM nor from other outside signals, but rather by structural changes inside the cell, possibly by integrins taking their closed conformational shape and thus decreasing their affinity for the substrate.

This means that the migrating cell is highly polarized, i.e. the regulation of integrins in the front part of

The cell is different from the regulation of integrins found in the back of the cell. In the front part of the cell, integrins are regulated in such a way that adhesion is increased, whilst in the back of the cell, integrins are regulated in such a way that adhesion is decreased.

Note that once adhesion in the front has stabilized, the adhesion in the back is removed so that all the fibers that are in tension can allow for the organelles to be moved inside the cell and towards the direction of the movement.

When cells change their adhesion, they also change their shape. In order to have more membrane in the front of the lamellipodium exocytosis takes place, whilst endocytosis takes place in the back of the cell to remove the membrane.

Intracellularly activation of integrins begins with the activation of a GTPase (GDP is exchanged into GTP by GEF) such as Rac1 (in lamellipoidia), Rho (in stress fibers),

or even Rap1, which interacts with proteins such as RIAM to bind to and activate talin, so that the GTPase-RIAM-talin complex recruits kindilin to the plasma membrane, promoting the binding between talin and the cytoplasmic end of the integrin β subunit through vinculin. This latter binding leads to the opening of the extracellular domain of the integrins.

The extracellular interaction between the N-terminus of integrins and the ligand/substrate can also promote the interaction of the cytoplasmic end of the integrin β subunit with talin which then allows for the opening of integrins.

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PAPER 1: FORCE-DEPENDENT VINCULIN BINDING TO TALIN 10/10/22

The topic of this paper is the interaction between talin and vinculin, as talin plays a key role in adhesion by acting as an adaptor molecule between integrins and actin.

This study is performed in vitro on live cells, and it analyzes whether the

control.Chamber slides are used for immunohistochemistry because simple Petri dishes are not suited for this approach. Chamber slides have sealed glass-slip at the bottom, on top of which are located different wells so that the sample slices can be placed in there along with the culture medium. Said medium can be then removed, cells washed with PBS or buffer, methanol added on the slides and this will be enough for permeabilization. The wells are finally removed from the glass-slip and sample slices on there are treated for immunohistochemistry.

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After permeabilization, the samples are incubated with a solution that lacks the primary antibody to remove a-specific bindings and background effects. Said solution (dry milk) has a high protein concentration so that it can saturate all a-specific bindings.

Sometimes some antigens need to be revealed, thus samples are microwaved for a short period of time.

phalloidin can color actin filaments. To recognize the full-length vinculin, they used the monoclonal antibody anti-vinculin, so that it would not recognize the dominant negative construct they used.

In the immunohistochemistry results the 5 integrin (α subunit of the integrin receptor) is used as control in both upper and lower panel.

In the upper panel, talin is detected in the middle, and its merging with 5 integrin is detected on the right.

In the lower panel, vinculin is detected in the middle and its merging with 5 integrin is detected on the right.

In cells, the expression of talin and/or vinculin depends on cell adhesion.

For the pharmacological approach, they used inhibitors of the Rho activation pathway, which is the G-protein pathway responsible for the organization of stress fibers. Stress fibers are connected to focal adhesion points on both sides and rich in myosin.

When Rho is

bound to GTP it is active, and it will activate the ROCK kinase that phosphorylates the myosin light chain (MLC). This phosphorylation activates the contractility of the stress fiber.

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Another activity of ROCK kinase, once activated by Rho, is that of phosphorylating LIMK which in turn inhibits the activation of cofilin by adding a phosphate onto it (= phosphorylates it), so that actin filaments can be stabilized.

Thus, when Rho is inhibited, so is the activity of ROCK, i.e. Its phosphorylation and activation of myosin light chain, reducing contractility, as well as its phosphorylation and inactivation of cofilin, reducing actin filament stabilization.

Using blebbistatin, specifically inhibits actomyosin contractility within the stress fibers. Once blebbistatin (myosin inhibitor) was administered, they investigated if there would be a relationship between the actin tension due to its interaction.

with myosin differe