Limited proteolysis of Hansenula polymorpha yeast amine oxidase: isolation of a C-terminal fragment containing both a copper and quino-cofactor

AbstractLimited proteolysis of recombinant Hansenula polymorpha yeast amino oxidase produces a 48 kDa fragment which corresponds to the C-terminal two-thirds of the protein. The fragment contains both TOPA (2,4,5-trihydroxyphenylalanine) and copper, as well as the histidine ligands implicated in copper binding. The fragment is proposed to be the domain responsible for cofactor production in yeast amine oxidase

Relaxing the actin cytoskeleton for adhesion and movement with Ena/VASP

At cell–cell contacts, as well as at the leading edge of motile cells, the plasticity of actin structures is maintained, in part, through labile connections to the plasma membrane. Here we explain how and why Drosophila enabled/vasodilator stimulated phosphoprotein (Ena/VASP) proteins are candidates for driving this cytoskeleton modulation under the membrane

Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking

International audienceCells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an 'outside geometry'. The actin shell is either grown from an activator of actin polymeriz-ation immobilized at the membrane by a biotin–streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications

Etude du rôle de la protéine VASP dans la dynamique et la mécanique des réseaux d'actine avec un système biomimétique de la motilité cellulaire

Nous étudions dans cette thèse l influence de la protéine VASP sur la dynamique et les propriétés mécaniques des réseaux d actine à l origine de la motilité cellulaire. Dans une première partie, nous utilisons un système biomimétique du mouvement constitué de billes. Incubées dans un mélange minimal de protéines soutenant la polymérisation, ces billes forment un réseau d actine appelé comète les propulsant vers l avant, semblable à celui qui sous-tend la membrane du front de migration des cellules en mouvement. Grace à des formes mutantes de VASP, nous explorons la contribution à son activité des domaines qui la composent. Dans nos conditions, nous montrons également que VASP agit au travers d un mécanisme de coopération avec le complexe Arp2/3 qui est à l origine de la formation des réseaux d actine. Enfin, nous étudions les modalités de recrutement de VASP par les activateurs de la famille WASP/WAVE en mutant les sites potentiels de recrutement de VASP. Dans une seconde partie, nous nous intéressons, en collaboration avec deux équipes de recherche, au rôle de VASP dans la mécanique des réseaux d actine. Nous caractérisons en rhéologie couplée à des observations en microcopie confocale les mécanismes moléculaires de VASP dans la réorganisation de l architecture des réseaux d actine, en lien avec leurs propriétés mécaniques, puis, nous étudions en AFM par nano indentation l augmentation de rigidité des réseaux d actine formés en présence de VASPWe study the influence of the VASP protein on dynamics and mechanics of the actin networks that drive cell motility. Our experimental setup is an in vitro bead system that mimics cell movement. The beads are induced to form a network of actin on their surface that resembles a comet and propels the bead forward. The actin comet reproduces in many ways the actin network that pushes forward the front of a moving cell. Using mutant forms of VASP, we define which functional domains of VASP are necessary for its function in enhancement of motility. We show that VASP exercises its effect on actin polymerization via synergy with the Arp2/3 complex, an actin polymerization nucleator and an important component of branched actin networks in the cell. We study how this synergy is brought about by co-recruitment of VASP and the Arp2/3 complex by actin polymerization activators of the WASP/WAVE family. In collaboration, we also study how VASP affects the mechanical properties of actin networks. Via AFM nano-indentation of actin comets, we show that VASP increases actin network rigidity, and we corroborate this result by rheological measurements of the elasticity of pure actin networks in the presence of VASP, where we also gain insights into how VASP affects actin network architecture. Our results define the mechanism of action of VASP for enhancement of actin dynamics and cell motility, and shed light on why VASP is implicated in various pathologies, including cancer cell metastasisPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Adaptive Actin Networks

International audienceDespite their fundamental importance in the regulation of cell physiology, the mechanisms that confer cell adaptability to changes in the microenvironment are poorly understood. A recent study in Cell (Mueller et al., 2017) examines the capability of branched actin networks to respond and adapt to mechanical load in vivo

Dynamic stability of the actin ecosystem

International audienceIn cells, actin filaments continuously assemble and disassemble while maintaining an apparently constant network structure. This suggests a perfect balance between dynamic processes. Such behavior, operating far out of equilibrium by the hydrolysis of ATP, is called a dynamic steady state. This dynamic steady state confers a high degree of plasticity to cytoskeleton networks that allows them to adapt and optimize their architecture in response to external changes on short time-scales, thus permitting cells to adjust to their environment. In this Review, we summarize what is known about the cellular actin steady state, and what gaps remain in our understanding of this fundamental dynamic process that balances the different forms of actin organization in a cell. We focus on the minimal steps to achieve a steady state, discuss the potential feedback mechanisms at play to balance this steady state and conclude with an outlook on what is needed to fully understand its molecular nature

Roles of Actin in the Morphogenesis of the Early Caenorhabditis elegans Embryo

The cell shape changes that ensure asymmetric cell divisions are crucial for correct development, as asymmetric divisions allow for the formation of different cell types and therefore different tissues. The first division of the Caenorhabditis elegans embryo has emerged as a powerful model for understanding asymmetric cell division. The dynamics of microtubules, polarity proteins, and the actin cytoskeleton are all key for this process. In this review, we highlight studies from the last five years revealing new insights about the role of actin dynamics in the first asymmetric cell division of the early C. elegans embryo. Recent results concerning the roles of actin and actin binding proteins in symmetry breaking, cortical flows, cortical integrity, and cleavage furrow formation are described