The Multicellular Effects of VDAC1 N-Terminal-Derived Peptide

The mitochondrial voltage-dependent anion channel-1 (VDAC1) protein functions in a variety of mitochondria-linked physiological and pathological processes, including metabolism and cell signaling, as well as in mitochondria-mediated apoptosis. VDAC1 interacts with about 150 proteins to regulate the integration of mitochondrial functions with other cellular activities. Recently, we developed VDAC1-based peptides that have multiple effects on cancer cells and tumors including apoptosis induction. Here, we designed several cell-penetrating VDAC1 N-terminal-derived peptides with the goal of identifying the shortest peptide with improved cellular stability and activity. We identified the D-Δ(1-18)N-Ter-Antp comprising the VDAC1 N-terminal region (19-26 amino acids) fused to the Antp, a cell-penetrating peptide. We demonstrated that this peptide induced apoptosis, autophagy, senescence, cell volume enlargement, and the refusion of divided daughter cells into a single cell, it was responsible for reorganization of actin and tubulin filaments, and increased cell adhesion. In addition, the peptide induced alterations in the expression of proteins associated with cell metabolism, signaling, and division, such as enhancing the expression of nuclear factor kappa B and decreasing the expression of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha. These cellular effects may result from the peptide interfering with VDAC1 interaction with its interacting proteins, thereby blocking multiple mitochondrial/VDAC1 pathways associated with cell functions. The results of this study further support the role of VDAC1 as a mitochondrial gatekeeper protein in controlling a variety of cell functions via interaction with associated proteins

Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries.

International audienceActin-based motility demands the spatial and temporal coordination of numerous regulatory actin-binding proteins (ABPs) [1], many of which bind with affinities that depend on the nucleotide state of actin filament. Cofilin, one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro [2], binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments [3]. Deficiencies in methodologies to track in real time the nucleotide state of actin filaments, as well as cofilin severing, limit the molecular understanding of coupling between actin filament chemical and mechanical states and severing. We engineered a fluorescently labeled cofilin that retains actin filament binding and severing activities. Because cofilin binding depends strongly on the actin-bound nucleotide, direct visualization of fluorescent cofilin binding serves as a marker of the actin filament nucleotide state during assembly. Bound cofilin allosterically accelerates P(i) release from unoccupied filament subunits, which shortens the filament ATP/ADP-P(i) cap length by nearly an order of magnitude. Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments, thereby controlling the overall filament length, depending on cofilin binding density

Allosteric activation of vinculin by talin

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s$^{-1}$. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere

Inhibitors target actin nucleators.

International audienceIn this issue of Chemistry and Biology, Rizvi and colleagues indentify a small molecule that inhibits formin-mediated actin assembly. Together with recently characterized inhibitors of the Arp2/3 complex (Nolen et al., 2009) and formins (Gauvin et al., 2009), these small molecules provide useful laboratory tools to dissect the link between actin nucleators and actin-based structures in living cells

Dynamique des réseaux d'actine d'architecture contrôlée

Mon travail fut de développer différents projets en vue de mieux comprendre la dynamique et l'organisation des réseaux d'actine et les mécanismes moléculaires à l'origine de la production de force, cela en systèmes reconstitués bio-mimétiques. Dans un premier temps je me suis intéressée à l'étude de l'organisation spatio-temporelle des réseaux d'actine et de ses protéines associées durant la motilité de particules recouverte de promoteurs de nucléation (Achard et al, Current Biology, 2010 et Reymann et al, sous presse à MBOC). J'ai suivi en temps réel l'incorporation de deux régulateurs de l'actine (capping protein et ADF/cofiline) et montré que leur contrôle biochimique sur l'actine gouverne également ces propriétés mécaniques. Afin de mieux caractériser les propriétés mécaniques de ces réseaux d'actine en expension, j'ai ensuite développé un système biomimétique novateur utilisant un set-up de micro-patterning permettant un contrôle spatial reproductible des sites de nucléation d'actine. Cela m'a permis de montrer comment des barrières géométriques, semblables à celles trouvées dans les cellules, peuvent influencer la formation dynamique de réseaux organisés d'actine et ainsi contrôler la localisation de la production de forces. (Reymann et al, Nature Materials, 2010). De plus l'addition de moteurs moléculaires sur ce système versatile nous a permis d'étudier la contraction induite par des myosines. En particulier les myosines VI-HMM interagissent de manière sélective sur différentes architectures d'actine (organisation parallèle ou antiparallèle, réseau enchevêtré), aboutissant à un processus en trois phase : tension puis déformation des réseaux d'actine fortement couplé à un désassemblage massif des filaments. Ce phénomène est intimement dépendant de l'architecture du réseau d'actine et pourrait donc jouer un rôle essentiel dans la régulation spatiale des zones d'expansion et de contraction du cytosquelette in vivo. (Travail en cours d'écriture).I have developed different projects in order to tackle the problem of actin network dynamics and organization as well as the molecular mechanism at the origin of force production in biomimetic reconstituted systems. My first interest concerned the spatiotemporal organization of actin networks and actin-binding proteins during actin based motility of nucleation promoting factor-coated particles (Achard et al, Current Biology, 2010 and Reymann et al, in press at MBOC). I tracked in real time the incorporation of two actin regulators and showed that their biochemical control of actin dynamics also governs its mechanical properties. To further characterize mechanical properties of expanding actin networks, I used an innovative micro-patterning set-up allowing a reproducible spatial control of actin nucleation sites. It allowed me to show that geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures and hence control the location of force production (Reymann et al, Nature Materials, 2010). Finally the addition of molecular motors on this tunable system allowed me to study implications for myosin-induced contractility. In particular, HMM-MyosinVI selectively interact with the different actin network architectures (parallel, anti-parallel organization or entangled networks) and leads to a selective three-phase process of tension, deformation of actin networks tightly coupled to massive filament disassembly. This phenomenon being highly dependent on actin network architecture could therefore play an essential role in the spatial regulation of expanding and contracting regions of actin cytoskeleton in cells. (Work in writing process).SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Tiopronin-Protected Gold Nanoparticles as a Potential Marker for Cryo-EM and Tomography

Gold nanoparticles (AuNPs) and their conjugation to biological samples have numerous potential applications. When combined with cryo-electron microscopy and tomography analysis, AuNPs may provide a versatile and powerful tool to identify and precisely localize proteins even when attached to cellular components. Here, we describe a general and facile approach for the synthesis of homogeneous and stable AuNPs, which can readily be conjugated to a molecule of interest and imaged by cryo-electron tomography (cryo-ET). We demonstrate the synthesis of 2.2 ± 0.45-nm tiopronin-protected AuNPs, followed by their conjugation with recombinant proteins and peptides. Visualization of the ∼2.2-nm gold-tagged peptides by cryo-ET reveals the potential use of this strategy to label and localize accessible proteins in a cellular environment with nanometric resolution

Talin-activated vinculin interacts with branched actin networks to initiate bundles

Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions

Force Production by a Bundle of Growing Actin Filaments Is Limited by Its Mechanical Properties

International audienceBundles of actin filaments are central to a large variety of cellular structures such as filopodia, stress fibers, cytokinetic rings, and focal adhesions. The mechanical properties of these bundles are critical for proper force transmission and force bearing. Previous mathematical modeling efforts have focused on bundles' rigidity and shape. However, it remains unknown how bundle length and buckling are controlled by external physical factors. In this work, we present a biophysical model for dynamic bundles of actin filaments submitted to an external load. In combination with in vitro motility assays of beads coated with formins, our model allowed us to characterize conditions for bead movement and bundle buckling. From the deformation profiles, we determined key biophysical properties of tethered actin bundles such as their rigidity and filament density

Geometrical control of actin assembly and contractility

International audienceThe actin cytoskeleton is a fundamental player in many cellular processes. Ultrastructural studies have revealed its extremely complex organization, where actin filaments self-organize into defined and specialized structures of distinct functions and, yet, are able to selectively recruit biochemical regulators that are available in the entire cell volume. To overcome this extraordinary complexity, simplified reconstituted systems significantly improve our understanding of actin dynamics and self-organization. However, little is known regarding physical rules governing actin networks organization and to which extent network structure may direct and regulate selective interactions with specific regulators. Here, we describe the first method to direct actin filament assembly to specific 2D motifs with a finely tuned geometry and relative distribution. This method enables the study of how geometrical confinement governs actin network structural organization and how, in return, structural cues can control selective contraction by myosin motor. The protocol relies on the use of surface micropatterning and functionalization procedures in order to selectively direct actin filament assembly to specific sites of nucleation