HAY DIÁLOGO

221

Entrapment of Intracytosolic Bacteria by Septin Cage-Like Structures.

S. Mostowy1, P. Cossart1; 1Bacteria Cell Interactions, Institut Pasteur, Paris, France

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However their role during interphase has remained elusive. We have discovered that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin-tail formation and cell-to-cell spread. Finally we show that septin cages entrap bacteria targeted to autophagy. Together these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens, and highlight the septins as a novel and unconventional component of the cytoskeleton.

222

The Shs1p C-terminus is required for septin ring geometry and cell morphology. R. A. Meseroll1, A. S. Gladfelter1; 1Department of Biological Sciences, Dartmouth College, Hanover, NH

Septins are a class of conserved GTP-binding proteins that function in diverse cellular processes. Individual septin proteins assemble into hetero-oligomeric complexes, which can further polymerize into nonpolar filaments. These filaments complex to form higher-order structures, including rings and bundled filaments, that function in cytokinesis and act as membrane barriers. Although in vitro analyses have unveiled the composition and arrangement of individual septin filaments, the mechanisms of septin assembly into the higher-order structures seen in vivo are not well understood. The filamentous fungus Ashbya gossypii expresses five septins (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7/Shs1p) that localize to hyphal tips and form cortically-attached rings throughout the hyphae. These rings maintain a persistent but dynamic association with the plasma membrane over many hours of growth. We have shown that the kinases Elm1p and Gin4p regulate the stable association of septin structures with the cell cortex. A possible kinase target is the C-terminus of Shs1p, a region containing nine phosphorylation sites that we have identified by mass spectrometry. Additionally, the Shs1p C-terminus harbors a predicted coiled-coil domain, which may play a role in septin-septin interactions. Expression of Shs1p lacking the C-terminus as the only Shs1p in Ashbya results in aberrant formation of a specific subset of septin rings that arise at hyphal branch points. Branch rings containing Shs1ΔC-GFP assemble extra cortical filamentous extensions adjacent to otherwise normal-appearing rings. Shs1ΔC-GFP immunoprecipitates with the other four septins, indicating that a loss of septin complex association is not the cause of the aberrant branch ring phenotype. Time-lapse microscopy reveals that the extensions appear concurrently with the septin ring formation, suggesting that loss of the C-terminus of Shs1p leads to misregulation of the septin assembly process, specifically an inability to scale the ring properly at assembly. In addition to septin abnormalities, these cells display increased hyphal branching frequency, suggesting that either the C-terminus of Shs1p is required for normal cell morphology and/or that aberrantly scaled septin rings can alter sites of polarity. We hypothesize that the phosphorylation sites and/or the coiled-coil domain of Shs1p may be

responsible for the inability of Shs1ΔC-GFP to grow and form septin rings properly and are currently analyzing alleles containing point mutations in the phosphorylation sites. This work illuminates the role of specific septin domains in higher-order septin structure regulation.

223

Self-Organization of Cytoplasmic Actin-Myosin-Formin Network.

W. Luo1, A. Mogilner2, A. Bershadsky3, M. Sheetz4; 1Mechanobiology Institute, National University of Singapore, Singapore, Singapore, Singapore, 2Department of Neurobiology, Physiology and Behavior and Department of Mathematics, University of California at Davis, USA, 3Mechanobiology Institute, National University of Singapore, Singapore and Weizmann Institute of Science, Rehovot, Israel, Singapore, Singapore, 4Mechanobiology Institute, National University of Singapore, Singapore and Columbia University, New York, USA, Singapore, Singapore

In eukaryotic cells, specialized actomyosin structures such as stress fibers and cytokinetic contractile ring are well known, but there is very limited understanding of the cytoplasmic actomyosin network. To elucidate this actomyosin network organization, we perturb it using Latrunculin A (LA). This treatment led to emergence of asters with diameter of 0.5-1.2µm, visualized by fluorescently tagged Lifeact or beta-actin in regions of cytoplasm not associated with remaining stress fiber or focal adhesions. These asters were very dynamic moving vigorously, often fusing with each other. Using dual color TIRF microscopy live imaging, myosin clusters were found either between or co-localized with actin aster centers, while filamin A and formin protein DAAM1 co-localized with the centers. Notably, PALM microscopy revealed that even in non-treated cells DAAM1 was localized in patches spaced similarly to the asters that emerged after the LA treatment. Actin aster movements induced by LA can be stopped by adding blebbistatin, a myosin II inhibitor, or largely reduced by treatment with formin inhibitor SMIFH2. On the other hand, in filamin A-/- cells, LA treatment resulted in formation of asters moving more rapidly and fusing and splitting more frequently than in control cells. Re- introduction of filamin A into the knockout cells decreased the asters’ dynamics significantly. Increasing the level of filamin A in the control cells caused the reduced velocity or complete block of the aster motility. We developed a computational model which, in agreement with these data, suggests that myosin motors slide actin filaments into the multiple asters with centers stabilized by cross-linkers and reinforced by formin-mediated nucleation, while myosin is stabilized at the edges between the asters where its contractile action causes aster movements. Modeling suggests that the dynamic-aster mode of self-organization is optimal for mechanical connectedness of the cytoplasm in perturbed actin networks.

224

Actin Assembly Kinetics Determine the Architecture of α-Actinin Crosslinked F-actin Networks.

T. Falzone1, M. Lenz2, D. Kovar3, M. Gardel4; 1Biophysics Graduate Program, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, 2James Franck Institute and Department of Physics, University of Chicago, 3Department of Molecular Genetics & Cell Biology, Department of Biochemistry & Molecular Biology, University of Chicago, 4Institute for Biophysical Dynamics, James Franck Institute and Department of Physics, University of Chicago

The spatial and temporal regulation of the actin cytoskeleton is required for numerous aspects of eukaryotic cell physiology including adhesion, polarity, migration, division, endocytosis, and intracellular trafficking. In concert with actin regulatory proteins, actin filaments (F-actin) are organized into bundles of axially aligned filaments or meshworks cross-linked at high angles.

Understanding the biochemical and physical processes regulating the assembly of actin filaments into meshworks or bundles is central to developing a quantitative and predictive understanding of cytoskeletal organization. Here we show that the kinetics of actin polymerization play a crucial role in controlling the morphology of in vitro filament networks assembled with α-actinin by a combination of quantitative imaging, microrheology and computational modeling approaches. Cross-link mediated bundle formation only occurs in dilute solutions of actin filaments with a predominately viscous microenvironment facilitating rotational and translational diffusion of actin filaments. During the time period of high filament mobility, α- actinin concentration and filament density controls the rate of bundle formation. Furthermore, we show that the network morphology can be modulated from a highly bundled network to an isotropic meshwork that by altering only nucleation kinetics. Thus, the morphology of cross- linked F-actin networks reflects a metastable state determined by the kinetics of filament nucleation, elongation and α-actinin mediated aggregation. These results underscore the importance of physical constraints in regulation of actin cytoskeletal organization into higher ordered structures in a dense and crowded cytoplasm.

225

In-vivo measurement of stress fiber contraction dynamics with high resolution and dynamics range.

B. Axelrod1, A. Tadmor2, M. Roukes3; 1Physics, California Inst Technol, Pasadena, CA,

2_{Biochemistry and Molecular Biophysics, California Inst Technol,} 3_{Physics, Applied Physics and}

Bioengineering, California Inst Technol

Force generation is critical to the regulation of many fundamental cellular processes including stem cell differentiation, tissue development, and tumor progression. Stress fibers, the principle generators of contractile forces, are linear chains of bundles of actin filaments of opposing polarity that are pulled together by bipolar myosin filaments in a manner similar, though less ordered, to muscle myofibrils. Though force was not directly measured, recent experiments suggest a surprising richness in how stress fiber dynamics regulate force and mechanotransduction.

We present a high resolution, high dynamic range Nano-Electro-Mechanical Systems (NEMS) based force measurement tool with wide applicability to measure forces generated by individual adherent cells. Which we demonstrate here with measurement of the force from a single stress fiber within a cell as the cell is perturbed by Cytochalasin D (CD) and recovers after CD removal. We measure a contraction upon initial exposure to CD of ~4nN, force steps during both CD induced force collapse and post-CD force recovery of ~1nN, and small force oscillations, ~400pN peak-peak, with frequency linearly dependent upon the cell generated force throughout.

The data validate a two part model – steady state and kinetic – of stress fiber force generation. The steady state model, adapted from recent work that features actin treadmilling and polymerization forces in addition to actomyosin contraction, describes the initial contraction and force steps. The kinetic model is based upon stochastic, abrupt failure and activation of sarcomere like units within the stress fiber and describes the overall force-time response. This work demonstrates the ability of high performance NEMS to connect quantitative biophysical modeling with specific cell level function. With these capabilities we confirm the importance of actin polymerization dynamics in understanding stress fiber response and present a detailed mechanism by which cytochalasins reversibly disrupt cellular force generation.

226

In silico myosin motors and contractile networks.

J. Alberts1, E. Munro2, M. Gardel3; 1Biology, University of Washington, Friday Harbor, WA, 2Cell and Molecular Biology, University of Chicago, Chicago, IL, 3Physics, University of Chicago, Chicago, IL

Motor proteins are critical to cells in processes such as cellular contractility, motility, cell division, and intracellular transportation and compartmentalization. Thus detailed simulations of these processes require motor representations that can be “benchmarked” to encapsulate key features of the biochemical and mechanical motor characteristics.

A biological myosin molecule is continuously flexible (with distinct regions of differing rigidity) and mechanical models might vary from molecular dynamics approaches (i.e. representation of each molecule and the various force fields of interaction) to multiple rigid bodies connected by springs, to simple elastic representations. Ever more detailed and physically realistic models of a motor protein have the potential to capture finer facets of motor behavior, but at a computationally cost.

Our eventual goal with this model is to simulate very large networks of actin filaments, myosin motors, and crosslinkers. Thus a very simple and computationally inexpensive motor representation is appropriate. We propose a two-spring myosin motor for 2-dimensional simulations. One spring is co-linear with the actin filament —this spring has the stiffness measured for a myosin crossbridge and a bi-modal rest length to enact a powerstroke— while an orthogonal spring keeps the filament and motor from drifting apart.

Each single motor is encoded through Monte Carlo methods to exhibit the measured transition rates between biochemical states (i.e. the state of the bound nucleotide). The ADP-release rate is modulated by force-state, as indicated by experiment. The model prescribes a 5.5nm step- size and a measured value for crossbridge stiffness, which guarantees appropriate force interaction between filament and myosin with each powerstroke.

We show that groups of motors exhibit the expected average duty-ratios. In in silico gliding filament assays filaments demonstrate appropriate speed dependence and eventual saturation with increasing myosin density. And under resisting loads these model motors can reproduce the expected concave force-speed curve.

As an element in networks with filaments and crosslinkers we demonstrate how these motors can lead to filament buckling and different contractile topologies, dependent on motor heterogeneity and density.

227

Actin pattern formation in reconstituted actomyosin cortices.

S. K. Vogel1, P. Schwille1; 1Institute of Biophysics, BIOTEC/TU Dresden, Dresden, Germany In eukaryotic systems the actomyosin cortex is involved in fundamental processes such as cytokinesis, cell migration and cell polarization. Actomyosin contractility is involved in the generation of cortical flows helping cells to polarize, which is essential for asymmetric cell division and hence for the development of multicellular organisms. In Caenorhabditis elegans zygotes cortical flows are accompanied by the formation of dynamic actomyosin networks. However, the physical relation between actomyosin contractility, network formation and cortical flows are not well understood.

We therefore set out to build minimal in vitro systems, namely consisting of membrane bound actin filaments and myosin (filaments) motors in order to reconstitute and mimic general features of contractile actomyosin cortices present in model organisms. In particular, we aim to understand the behavior of motor filaments and the role of the membrane during actomyosin network formation.

By imaging fluorescently labeled actin and myosin filaments with TIRF microscopy we show that the addition of myosin filaments to the membrane bound actin layer leads to the formation of interconnected actin clusters. By varying the density of the membrane bound actin layer we obtain different morphologies of the actin pattern after addition of the motors. We demonstrate that similar to active gels in bulk solution, the process of actin pattern formation depends on the concentration of ATP. At saturating ATP concentration actin pattern formation is inhibited, while lowering the ATP concentration to a critical level leads to the formation of actin clusters. This process is reversible by the addition of ATP to actomyosin clusters thereby resuming the original morphology of the actin meshwork. However, addition of the crosslinker Fascin at ATP saturating conditions leads to pattern formation, although more slowly. This indicates that crosslinking is necessary for the contraction of the actin filament layer. Visualization and tracking the motion of individual fluorescently labeled myosin filaments revealed that the processivity of the motor filaments increases when the ATP concentration is lowered. We propose that upon ATP depletion some myosin filaments start to function as crosslinkers inducing the actin meshwork contraction. We hypothesize that the concomitant overall increase of the processivity of myosin filaments further accelerates the contraction of the actin filaments into actomyosin clusters.

228

Thickness and dynamics of the actomyosin cortex.

A. G. Clark1, K. Dierkes2, S. Simmert1, E. Paluch1; 1Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 2Max Planck Institute for the Physics of Complex Systems, Dresden, Germany

Cellular shape changes are, in essence, mechanical processes that are governed by the physical properties of the cell. One physical property in particular that has been implicated in cell shape change is tension, which is controlled by the actomyosin cortex, a thin layer of proteins that directly underlies the plasma membrane. Cell tension arises from two major properties of the cortex: (1) the amount of stress generated by myosin motors within the cortex and (2) cortex thickness. In order to further understand the regulation of cortical tension, we have developed an assay to measure cortex thickness in living cells that relies on a precise, quantitative analysis of fluorescence images of the cortex and the plasma membrane. In this assay, we measure cortex thickness by determining the spatial separation between the cortex and the plasma membrane, and we have used this technique to track changes in thickness during dynamic cell shape changes, including bleb retraction and cell division. Furthermore, we have used a similar image analysis approach, combined with the use of photoactivatable probes, to investigate the dynamics and spatial regulation of actin assembly in the steady-state cortical network. By interpreting these photoactivation data through a simple geometrical description of the cortex, we are not only able to more precisely extract information about the spatial dynamics of actin assembly, but we also attain an independent measurement of cortex thickness that is consistent with our thickness measurements using cortex and membrane peak separation. Comparing the results of these experiments with photobleaching data, we are further trying to understand the regulation of actin dynamics and thickness at different stages of the cell cycle, both in order to uncover the underlying mechanisms involved in previously observed changes in cells’ physical properties and shape throughout the cell cycle and also to more generally determine the relationship between cortical actin dynamics and cortex thickness.

The understanding of such relationships and of the basic principles of cortical actin dynamics are essential in understanding how the coordinated actions of these proteins contribute to larger-scale physical properties.

229

Actin network architecture determines myosin motor activity.

A-C. Reymann1, R. Boujemaa-Paterski1, J-L. Martiel1, W. Cao2, H. Chin2, E. De La Cruz2, M. Théry1, L. Blanchoin1; 1Institut de Recherches en Technologies et Sciences pour le Vivant,

CNRS/CEA/INRA/UJF, Grenoble, France, 2Department of Molecular Biophysics and

Biochemistry,Yale University, New Haven, CT

The organization of actin filaments into higher-ordered networks governs overall eukaryotic cell shape, mechanical integrity and directed movement. The global architecture of the actin cytoskeleton is determined by coordinated actions of a large number of actin regulatory proteins that modulate filament assembly and disassembly dynamics. Myosin motors also plays a critical role in these processes and reorganize filament structures through sliding (e.g. contractility) and/ or depolymerization. Understanding the molecular mechanism of such complex spatiotemporal orchestration is extremely challenging in cells, where hundreds of different proteins act simultaneously on overlapping actin sub-structures. Here, we use geometrically controlled and polarized in vitro actin networks to evaluate how myosin motors influences filament architecture. Direct visualization of filaments indicates that myosins selectively disassemble randomly-oriented and anti-parallel actin filament structures while parallel actin filament bundles are unaffected by myosin contractility. This “orientation selection” reveals how the overall organization and dynamics of the actin cytoskeleton is controlled by actomyosin contractility. General principles governing the spatial organization of actin filaments in cells emerge from this work.

230

Spatial structure of actin cytoskeletons associated with nuclear membrane.

J. Usukura1, S. Minakata2; 1EcoTopia Science Institute, Nagoya University, Nagoya, Japan,

2_{School of Engineering, Nagoya University, Nagoya, Japan}

Current study aims to elucidate the spatial organization of cytoskeletal actin filaments in the cytoplasm with special reference to the association with nuclear membrane and its alternations during cell cycle. Cytoskeletal actin filaments in living cell have been investigated so far exclusively with fluorescent light microscopy in conjunction with GFP tag method. However, fine structural changes with time were not detected well. Observation was restricted on the stress fiber with relatively strong fluorescence and at ventral side of the cell. We compared live cell images with electron microscopic images in a strict sense. In order to detect real spatial structure of cytoskeleton, high voltage TEM (1000 KV), high resolution SEM and immune-freeze etching technique were applied to unroofed whole cells in addition to light microscopic live cell