FUNCTIONAL IMPLICATIONS OF VINCULIN SUPRA-ACTIVATION ON CELL ADHESION 

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Actin sequestering proteins

In the intracellular milieu, actin binds to profilin. This complex represents the main source of monomers for polymerization. Profilin catalyzes the ADP/ATP exchange in actin monomers, while inhibiting the spontaneous filament nucleation property of actin monomers (Dominguez and Holmes 2011). Profilin-actin also binds to other proteins involved in actin polymerization, such as formins and Ena/VASP proteins (Kovar et al. 2006) (Figure 7).
Thymosin-β4 is a peptide of 43 residues. In its N-terminus it has short helix that binds the actin barbed end and the rest of the helix, forms an extended region that binds the front surface of actin and a second helix that caps actin pointed end (Xue et al. 2014). This results in the blockage of engagement of actin monomers into polymerization. However, profilin and thymosin-β4 are constantly exchanging actin monomers, allowing elongation of actin filaments (Pantaloni and Carlier 1993).

Severing proteins

Depolymerization in cells is enhanced by proteins of the ADF/Cofilin family, which includes Actin Depolymerization Factor (ADF), cofilin-1, cofilin-2 and twinfilin. This family of proteins have a higher affinity for ADP-actin than for ATP-actin, and therefore bind to ADP-actin fiber stretches and promote the severing of these filaments (Barbara W. Bernstein 2010).

Daniel Isui AGUILAR SALVADOR – Thèse de doctorat – 2019

Gelsolin proteins also sever actin filaments and cap their barbed ends. Most of its family members’ activity is regulated by calcium binding. Mutants of this protein have defects in cellular motility and decreased blood clotting.
Figure 7. Dynamics of actin filaments. A. Actin polymerization. B. Actin regulation.
(1) Arp2/3 recruitment (2) Actin branching (3) Formin elongation of actin fibers (4) Ena/VASP elongation of actin fibers (5) Filament capping (6) Depolimerization of actin filaments. C. Formin and Ena/VASP nucleation and elongation activity.

Actin nucleators

Actin nucleators initiate the de novo polymerization of actin filaments. The Arp2/3 complex is one of the best characterized actin nucleators. It is composed of a core of 7 proteins. Upon the interaction of Nucleation Promoting Factors (members of the Wiskott-Aldrich Syndrome Protein family, WAVE, among others), actin filaments and actin monomers, Arp2/3 is recruited and activated by Arf1 and Rac1 (Koronakis et al. 2011). Through the VCA domains of the Nucleation Promoting Factors, Arp2/3 is connected to the mother actin filament and nucleates the polymerization of a new branched actin filament (Pollard 2007). Given the Arp2/3 structure, the daughter filament elongates at an angle of 70° from the mother filament (Amann and Pollard 2001). At a large scale, polymerization of new branched filaments upon existing filaments give rise to branched actin networks, which are enriched in lamellipodia (Figure 7B).
In opposition to Arp2/3, formins nucleate and elongate unbranched actin filaments in filopodia, stress fibers and actin cables. They are characterized by the presence of the Formin Homology Domains 1 and 2, which mediate their interaction with profilin and their nucleation activity, respectively. Their ATP-dependent activity results in a highly processive actin polymerization (Romero et al. 2004). In a similar fashion, Ena/VASP proteins also promote the nucleation and elongation of actin filaments (Figure 7C), and works in neurons show that they are particularly important for filopodial formation (Lebrand et al. 2004).

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Capping proteins

Capping protein and CapZ bind to the barbed end of actin filaments, blocking both the addition and loss of actin subunits (Isenberg, Aebi, and Pollard 1980; J. Xu, Casella, and Pollard 1999), therefore preventing the assembly/disassembly of filaments. In conjunction with the branching activity of Arp2/3, a network of short and highly branched actin filaments can be formed (Figure 7B) (M. Edwards et al. 2014).

Cross-linking proteins

Individual actin filaments can be bundled together through the action of actin cross-linkers, such as α-actinin isoforms 1 and 4 or fimbrin. These proteins contain Actin-Binding Domains (ABD) that connects two separate actin fibers. α-actinin has also the ability to associate with other cytoskeletal, signaling and membrane molecules (Sjöblom, Salmazo, and Djinović-Carugo 2008).
The organization into bundles by filament bundling facilitates the mechanical contraction exerted by myosins molecular motors. In particular, Myosin II transforms the chemical energy released by ATP hydrolysis into mechanical work that results in pulling of actin filaments (Figure 8). The pulling activity is the result of the myosin II polymerization into bipolar filaments, with the motor domains walking in opposing cross-linked actin filaments (Figure 8) (Sweeney and Holzbaur 2018).
Figure 8. Myosin II and α-actinin in actin filament contraction. Myosin II polymerizes into bipolar filaments, with the motor domains walking in opposing actin filaments cross-linked by α-actinin. Modified from (Svitkina 2018).
The intracellular actomyosin cytoskeleton dynamically interacts with the extracellular environment through linker molecules anchoring transmembrane receptors that connect with the extracellular environment. Integrins and cadherins are two of the main receptors that connect the actomyosin cytoskeleton with the outer environment and to respond to mechanical and biochemical cues.

Table of contents :

1 INTRODUCTION 
1.1 BACILLARY DYSENTERY
1.1.1 CLINICAL MANIFESTATIONS, COMPLICATIONS AND TRANSMISSION
1.1.2 EPIDEMIOLOGY
1.2 SHIGELLA SPP
1.2.1 SHIGELLA CLASSIFICATION
1.2.2 EVOLUTION OF SHIGELLA
1.2.3 SHIGELLA VIRULENCE PLASMID
1.3 OVERVIEW OF SHIGELLA INVASION
1.3.1 ANATOMY OF THE INTESTINAL EPITHELIUM
1.3.2 SHIGELLA INVASION ROUTES OF THE INTESTINAL EPITHELIUM
1.4 THE SHIGELLA TYPE III SECRETION SYSTEM
1.4.1 THE SHIGELLA T3SS STRUCTURE
1.4.2 SHIGELLA T3SS EFFECTOR PROTEINS
1.5 MAIN PLAYERS OF THE CYTOSKELETON
1.5.1 ACTIN MONOMERS
1.5.2 ACTIN SEQUESTERING PROTEINS
1.5.3 SEVERING PROTEINS
1.5.4 ACTIN NUCLEATORS
1.5.5 CAPPING PROTEINS
1.5.6 CROSS-LINKING PROTEINS
1.6 ADHESION STRUCTURES
1.6.1 INTEGRINS
1.6.2 ADHESION FORMATION AND MATURATION
1.6.3 TALIN AND VINCULIN MOLECULAR CLUTCH
1.6.4 FORCE GENERATION AND MATURATION
1.7 ACTIN CYTOSKELETON REORGANIZATION BY BACTERIAL PATHOGENS
1.7.1 BACTERIAL ADHESION AND INTERNALIZATION STRATEGIES
1.7.2 E. COLI FIMH CATCH-BOND ADHESION
1.7.3 ZIPPERING: LISTERIA MONOCYTOGENES INVASION MECHANISM
1.7.4 ATTACHING AND EFFACING LESION PATHOGENS
1.7.5 SALMONELLA “TRIGGERED” INTERNALIZATION
1.7.6 SHIGELLA INVASION AND CYTOSKELETON REMODELLING
1.8 IPAA
1.8.1 IPAA VINCULIN BINDING SITES
2 RATIONALE OF THE PHD PROJECT 
3 ARTICLE 1
3.1 OVERVIEW
4 ARTICLE 2 
4.1 OVERVIEW
5 ARTICLE 3
5.1 OVERVIEW
6 DISCUSSION 
6.1 IPAA VBS3 BINDING TO TALIN
6.2 VINCULIN SUPRA-ACTIVATION: A NOVEL INTERACTION MODE
6.3 FUNCTIONAL IMPLICATIONS OF VINCULIN SUPRA-ACTIVATION ON CELL ADHESION
6.4 IPAA VBSS ROLE DURING SHIGELLA INVASION
6.5 GENERAL CONCLUSION
7 REFERENCES

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