Geometrical and mechanical properties control actin filament organization.

The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model

Reversal of contractility as a signature of self-organization in cytoskeletal bundles.

Funder: FP7 People: Marie-Curie Actions; FundRef: http://dx.doi.org/10.13039/100011264; Grant(s): PCIG12-GA-2012-334053Bundles of cytoskeletal filaments and molecular motors generate motion in living cells, and have internal structures ranging from very organized to apparently disordered. The mechanisms powering the disordered structures are debated, and existing models predominantly predict that they are contractile. We reexamine this prediction through a theoretical treatment of the interplay between three well-characterized internal dynamical processes in cytoskeletal bundles: filament assembly and disassembly, the attachement-detachment dynamics of motors and that of crosslinking proteins. The resulting self-organization is easily understood in terms of motor and crosslink localization, and allows for an extensive control of the active bundle mechanics, including reversals of the filaments' apparent velocities and the possibility of generating extension instead of contraction. This reversal mirrors some recent experimental observations, and provides a robust criterion to experimentally elucidate the underpinnings of both actomyosin activity and the dynamics of microtubule/motor assemblies in vitro as well as in diverse intracellular structures ranging from contractile bundles to the mitotic spindle

Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in eLife 6 (2017): e30867, doi:10.7554/eLife.30867.The actomyosin ring generates force to ingress the cytokinetic cleavage furrow in animal cells, yet its filament organization and the mechanism of contractility is not well understood. We quantified actin filament order in human cells using fluorescence polarization microscopy and found that cleavage furrow ingression initiates by contraction of an equatorial actin network with randomly oriented filaments. The network subsequently gradually reoriented actin filaments along the cell equator. This strictly depended on myosin II activity, suggesting local network reorganization by mechanical forces. Cortical laser microsurgery revealed that during cytokinesis progression, mechanical tension increased substantially along the direction of the cell equator, while the network contracted laterally along the pole-to-pole axis without a detectable increase in tension. Our data suggest that an asymmetric increase in cortical tension promotes filament reorientation along the cytokinetic cleavage furrow, which might have implications for diverse other biological processes involving actomyosin rings.DWG has received funding from the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 241548 (MitoSys) and no. 258068 (Systems Microscopy), an ERC Starting Grant under agreement no. 281198 (DIVIMAGE), and from the Austrian Science Fund (FWF) project no. SFB F34-06 (Chromosome Dynamics). FS has received funding from an EMBO long-term fellowship (ALTF 1447–2012). SM has received funding from Human Frontier Science Program cross-disciplinary fellowship (LT000096/2011)

Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction

Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially

Plastin increases cortical connectivity to facilitate robust polarization and timely cytokinesis.

The cell cortex is essential to maintain animal cell shape, and contractile forces generated within it by nonmuscle myosin II (NMY-2) drive cellular morphogenetic processes such as cytokinesis. The role of actin cross-linking proteins in cortical dynamics is still incompletely understood. Here, we show that the evolutionarily conserved actin bundling/cross-linking protein plastin is instrumental for the generation of potent cortical actomyosin contractility in the Caenorhabditis elegans zygote. PLST-1 was enriched in contractile structures and was required for effective coalescence of NMY-2 filaments into large contractile foci and for long-range coordinated contractility in the cortex. In the absence of PLST-1, polarization was compromised, cytokinesis was delayed or failed, and 50% of embryos died during development. Moreover, mathematical modeling showed that an optimal amount of bundling agents enhanced the ability of a network to contract. We propose that by increasing the connectivity of the F-actin meshwork, plastin enables the cortex to generate stronger and more coordinated forces to accomplish cellular morphogenesis

Cytoskeletal control of B cell responses to antigens.

The actin cytoskeleton is essential for cell mechanics and has increasingly been implicated in the regulation of cell signalling. In B cells, the actin cytoskeleton is extensively coupled to B cell receptor (BCR) signalling pathways, and defects of the actin cytoskeleton can either promote or suppress B cell activation. Recent insights from studies using single-cell imaging and biophysical techniques suggest that actin orchestrates BCR signalling at the plasma membrane through effects on protein diffusion and that it regulates antigen discrimination through the biomechanics of immune synapses. These mechanical functions also have a role in the adaptation of B cell subsets to specialized tasks during antibody responses

The mechanics of mitotic cell rounding

When animal cells enter mitosis, they round up to become spherical. This shape change is accompanied by changes in mechanical properties. Multiple studies using different measurement methods have revealed that cell surface tension, intracellular pressure and cortical stiffness increase upon entry into mitosis. These cell-scale, biophysical changes are driven by alterations in the composition and architecture of the contractile acto-myosin cortex together with osmotic swelling and enable a mitotic cell to exert force against the environment. When the ability of cells to round is limited, for example by physical confinement, cells suffer severe defects in spindle assembly and cell division. The requirement to push against the environment to create space for spindle formation is especially important for cells dividing in tissues. Here we summarize the evidence and the tools used to show that cells exert rounding forces in mitosis in vitro and in vivo, review the molecular basis for this force generation and discuss its function for ensuring successful cell division in single cells and for cells dividing in normal or diseased tissues

Contractile response of biomimetic actomyosin systems

Cellular contractility – the internal generation of force by a cell orchestrated by theactomyosin machinery – is a critical regulator of a wide range of cellular processes includingthe establishment of cell polarity, cell migration, tissue integrity or morphogenesis duringdevelopment. Disruptions of the force generation and of mechanical properties of living cellsaffect their physiological functions and consequently can lead to pathological defectsincluding cancer. However, the parameters or mechanisms that drive force production by theactin-myosin system and their mode of regulation in cells are not fully understood. During myPhD, I used biomimetic system made of a minimum set of proteins to study the properties ofactomyosin contractile systems. The goal was to understand how/if the actin architecture canmediate the contractile response. For this purpose, I was first interested in building a varietyof actin organization that will serve next as substrate for myosin during contraction. Tounderstand the general principles that dictate geometrically-controlled actin assembly, wedeveloped a model that allowed us to identify key parameters including filaments/filamentsinteraction, filament mechanical property and contact activation between actin filamentsgrowing from the adjacent pattern and the nucleation area. These actin templates were usedthen to evaluate the response of oriented actin structures to myosin-induced contractility. Idemonstrated that crosslinking level modulates the myosin-induced deformation of actinnetworks according to their architecture. I showed also that crosslinkers are necessary tosustain myosin-driven deformation and force production of dynamic actin networks. Inaddition, we developed numerical simulation in order to relate the observed myosin-drivenactin deformation with the underlying microscopic mechanism. This work revealed howdiverse cellular actin networks contract differently to a define set of biochemical conditionsand hence how dynamic rearrangements can modulate network contractilityLa contractilité cellulaire, un phénomène orchestrée par le système d'actomyosine, est un régulateur critique d'une large gamme de processus cellulaires, y compris l'établissement de la polarité cellulaire, la migration cellulaire, l'intégrité des tissus au cours de la morphogenèse ou du développement. Une simple perturbation de la génération de la force et des propriétés mécaniques des cellules peut affecter leurs fonctions physiologiques et par conséquent peut conduire à des défauts pathologiques y compris le cancer.Cependant, les mécanismes qui contrôlent la production de la force par le système acto-myosine et leurs modes de régulation dans les cellules ne sont pas pleinement compris. Au cours de ma thèse, j'ai utilisé un système biomimétique fait d'un ensemble minimal de protéines purifiées pour étudier les propriétés contractiles du système actomyosin.L'objectif était de comprendre comment l'architecture des filaments d'actine peut modifier la réponse contractile. A cet effet, j'étais d'abord intéressée par la construction d'une variété d'organisation de l'actine qui servira après comme substrat pour les moteurs moléculaires (la myosine) lors de la contraction.Afin de comprendre les principes généraux qui dictent l'assemblage de l'actine, nous avons développé un modèle numérique qui nous a permis d'identifier les paramètres clés, y compris l'interaction entre les filaments d'actine, les propriétés mécaniques de ces filaments et l'activation par contact entre une région de nucléation et les filaments d'actine qui poussent à partir d'un motif adjacent. Ce modèle a été utilisé en premier lieu pour implémenter les propriétés reliées à l'actine et en second lieu pour évaluer la réponse contractile des structures d'actine induite par la myosine.Durant ma thèse, j'ai pu démontrer que le niveau de connectivité module la déformation du réseau d'actine induite par la myosine, selon leur architecture. J'ai montré aussi que les protéines de pontages des filaments d'actine sont nécessaires pour effectuer une déformation et générer des forces au niveau des réseaux d'actine dynamiques en présence de la myosine. De plus, nous avons développé les simulations numériques dans le but de relier la déformation macroscopique des structures d'actines due à la myosine avec le mécanisme microscopique sous-jacent.Ce travail a révélé comment la variété des réseaux d'actine contracte d'une façon différente même en respectant les mêmes conditions biochimiques et a démontré l'importance de l'effet du réarrangement dynamique des structures d'actine sur la modulation de sa contractilité

Réponse contractile des systèmes actomyosines biomimétique

La contractilité cellulaire, un phénomène orchestrée par le système d'actomyosine, est un régulateur critique d'une large gamme de processus cellulaires, y compris l'établissement de la polarité cellulaire, la migration cellulaire, l'intégrité des tissus au cours de la morphogenèse ou du développement. Une simple perturbation de la génération de la force et des propriétés mécaniques des cellules peut affecter leurs fonctions physiologiques et par conséquent peut conduire à des défauts pathologiques y compris le cancer.Cependant, les mécanismes qui contrôlent la production de la force par le système acto-myosine et leurs modes de régulation dans les cellules ne sont pas pleinement compris. Au cours de ma thèse, j'ai utilisé un système biomimétique fait d'un ensemble minimal de protéines purifiées pour étudier les propriétés contractiles du système actomyosin.L'objectif était de comprendre comment l'architecture des filaments d'actine peut modifier la réponse contractile. A cet effet, j'étais d'abord intéressée par la construction d'une variété d'organisation de l'actine qui servira après comme substrat pour les moteurs moléculaires (la myosine) lors de la contraction.Afin de comprendre les principes généraux qui dictent l'assemblage de l'actine, nous avons développé un modèle numérique qui nous a permis d'identifier les paramètres clés, y compris l'interaction entre les filaments d'actine, les propriétés mécaniques de ces filaments et l'activation par contact entre une région de nucléation et les filaments d'actine qui poussent à partir d'un motif adjacent. Ce modèle a été utilisé en premier lieu pour implémenter les propriétés reliées à l'actine et en second lieu pour évaluer la réponse contractile des structures d'actine induite par la myosine.Durant ma thèse, j'ai pu démontrer que le niveau de connectivité module la déformation du réseau d'actine induite par la myosine, selon leur architecture. J'ai montré aussi que les protéines de pontages des filaments d'actine sont nécessaires pour effectuer une déformation et générer des forces au niveau des réseaux d'actine dynamiques en présence de la myosine. De plus, nous avons développé les simulations numériques dans le but de relier la déformation macroscopique des structures d'actines due à la myosine avec le mécanisme microscopique sous-jacent.Ce travail a révélé comment la variété des réseaux d'actine contracte d'une façon différente même en respectant les mêmes conditions biochimiques et a démontré l'importance de l'effet du réarrangement dynamique des structures d'actine sur la modulation de sa contractilité.Cellular contractility – the internal generation of force by a cell orchestrated by theactomyosin machinery – is a critical regulator of a wide range of cellular processes includingthe establishment of cell polarity, cell migration, tissue integrity or morphogenesis duringdevelopment. Disruptions of the force generation and of mechanical properties of living cellsaffect their physiological functions and consequently can lead to pathological defectsincluding cancer. However, the parameters or mechanisms that drive force production by theactin-myosin system and their mode of regulation in cells are not fully understood. During myPhD, I used biomimetic system made of a minimum set of proteins to study the properties ofactomyosin contractile systems. The goal was to understand how/if the actin architecture canmediate the contractile response. For this purpose, I was first interested in building a varietyof actin organization that will serve next as substrate for myosin during contraction. Tounderstand the general principles that dictate geometrically-controlled actin assembly, wedeveloped a model that allowed us to identify key parameters including filaments/filamentsinteraction, filament mechanical property and contact activation between actin filamentsgrowing from the adjacent pattern and the nucleation area. These actin templates were usedthen to evaluate the response of oriented actin structures to myosin-induced contractility. Idemonstrated that crosslinking level modulates the myosin-induced deformation of actinnetworks according to their architecture. I showed also that crosslinkers are necessary tosustain myosin-driven deformation and force production of dynamic actin networks. Inaddition, we developed numerical simulation in order to relate the observed myosin-drivenactin deformation with the underlying microscopic mechanism. This work revealed howdiverse cellular actin networks contract differently to a define set of biochemical conditionsand hence how dynamic rearrangements can modulate network contractilit