Bulletin of the American Physical Society
APS March Meeting 2019
Volume 64, Number 2
Monday–Friday, March 4–8, 2019; Boston, Massachusetts
Session H64: Physics of the Cytoskeleton Across Scales IIIFocus
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Sponsoring Units: DBIO GSOFT Chair: Olivia Du Roure, ESPCI ParisTech Room: BCEC 259B |
Tuesday, March 5, 2019 2:30PM - 3:06PM |
H64.00001: Nonlinear microscale mechanics and macromolecular mobility of tunable cytoskeleton composites Invited Speaker: Rae Robertson-Anderson Actin and microtubules are two key protein filaments that comprise the cytoskeleton, enabling cells to exhibit multifunctional nonlinear mechanics. However, it remains an open question as to how the structure, interactions, and dynamics of these proteins map to the nonlinear and non-equilibrium mechanics that the cytoskeleton exhibits. We address this open problem by using a robust approach that combines: tunable in vitro cytoskeleton networks, rheology that spans from molecular to mesoscopic scales, and single-molecule transport and mobility measurements. Specifically, we use optical tweezers microrheology to characterize the nonlinear mechanics of cytoskeleton networks while simultaneously using fluorescence microscopy and particle-tracking to determine macromolecular mobility and network stress propagation. To directly map network properties to stress response, we perform measurements using custom-designed in vitro networks of actin and microtubules with structural properties and interactions that can be precisely tuned. I will describe these methods as well as our recent intriguing results that demonstrate the elegant couplings that can emerge between network structure, stress response, and macromolecular mobility in cytoskeleton networks. |
Tuesday, March 5, 2019 3:06PM - 3:42PM |
H64.00002: Reconstitution of basic mitotic spindles in cell-like confinement Invited Speaker: Marileen Dogterom Bipolar organization of the mitotic spindle is the result of forces generated by dynamic microtubules and associated proteins in interaction with chromosomes and the cell boundary. Biophysical experiments on isolated spindle components have provided important insights into the force-generating properties of different components, but a quantitative understanding of the force balance that results from their concerted action is lacking. Here we present an experimental platform based on water-in-oil emulsion droplets that allows for the bottom-up reconstitution of basic spindles. We find a typical metaphase organization, where two microtubule asters position symmetrically at moderate distancefrom the mid-zone, is readily obtained even in the absence of chromosomes. Consistent with simulations, we observe an intrinsic repulsive force between two asters that can be counterbalanced alternatively by cortical pulling forces, anti-parallel microtubule crosslinking, or adjustment of microtubule dynamics, emphasizing the robustness of thesystem. Adding motor proteins that slide anti-parallel microtubules apart drives the asters to maximum separation, as observed in cells during anaphase. Our platform offers a valuable complementary approach to in vivo experiments where essential mitotic components are typically removed, instead of added, one by one. |
Tuesday, March 5, 2019 3:42PM - 3:54PM |
H64.00003: Revealing Cytoskeletal Dynamics and Avalanches via Active Micropost Arrays Yu Shi, Katherine Xiang, Shankar Sivarajan, Christopher L Porter, Daniel H Reich, John Crocker The cytoskeleton is critical for a wide range of cellular behavior, including motility, morphology, and mechanotransduction. However, understanding of the connections between molecular-scale processes and cell-scale dynamics is not complete. Here we present results using poly(dimethylsiloxane) active micropost array detectors (AMPADs) with embedded magnetic actuators to measure the fluctuations and local rheology of cells’ actomyosin stress fiber network and cortex in detail. We find that both structures display consistent power law rheology, along with highly heterogeneous and intermittent fluctuations. Notably, the fluctuating motion is dominated by large step-like displacements, resembling the dynamics observed in avalanches and earthquakes. The effects of substrate stiffness and geometry will also be discussed. Our results imply that actomyosin contractile units act in a highly collective manner and that cellular actomyosin networks self-organize into marginally stable plastic networks whose properties influence the biomechanical behavior of cells. |
Tuesday, March 5, 2019 3:54PM - 4:06PM |
H64.00004: A model of sliding and stalling in microtubule bundles Shane Fiorenza, Matt Glaser, M. Betterton Microtubules, motor proteins, and crosslinkers self-assemble a variety of cytoskeletal networks within the cell. Minimal systems of two antiparallel microtubules, kinesin-4 motors, and PRC1 crosslinkers reconstitute controlled sliding and stalling, leading to stable antiparallel overlaps like those seen in the mitotic spindle. Experiments show that the final overlap length and initial sliding velocity are both linearly proportional to the two microtubules’ initial overlap length. However, the mechanisms behind this length-sensing are not fully understood. We develop a model to show how crosslinker-motor interactions produce these regulated microtubule overlaps. We observe sliding even when motors exert no direct forces on neighboring microtubules due to purely steric interactions. Direct binding interactions between crosslinkers and motors can significantly increase the lifetime of the final overlap. |
Tuesday, March 5, 2019 4:06PM - 4:18PM |
H64.00005: How actin dynamics affect membrane nanotube mechanics Antoine Allard, Flavien Brette, Alexandre Deslys, Guillaume Lamour, Fabrice Valentino, Timo Betz, Clément Campillo, Cécile Sykes The living cell is an out-of equilibrium system that constantly remodels its architecture to ensure biological functions such as division or intracellular transport. The latter involves the formation of intermediate cylindrical membrane nanotubes. These nanotubes are then split into membrane compartments transported in other areas of the cell. Whereas mechanics of pure membrane nanotubes are now well understood, the role of the actin cytoskeleton on tube stability remains unclear. |
Tuesday, March 5, 2019 4:18PM - 4:30PM |
H64.00006: Tuning Migratory and Cytoskeletal Response of Cells to Texture with Collagen-IV Coating Matt J. Hourwitz, John T Fourkas, Wolfgang Losert Cell migration is vital for many physiological processes, both beneficial and detrimental. Actin is a cytoskeletal protein scaffold that assembles and disassembles to accomplish cell motion in a specific region and direction. A stimulus must be provided to influence guided cellular motion. In addition to biochemical signals, physical cues such as the texture of the environment also provide a guiding stimulus. To better understand how cytoskeletal and migratory processes integrate biochemical information with texture information, we studied actin dynamics and migration of MCF10A cells, an immortalized human breast epithelial cell line, on 3D nanotopographical surfaces. We systematically varied the coating with collagen IV, a basement membrane protein representing key biochemistry of the local ECM. Preliminary studies show that the response of actin to surface texture significantly depends on collagen coating concentration. |
Tuesday, March 5, 2019 4:30PM - 4:42PM |
H64.00007: Microrheology of Microtubule-Actin-Vimentin Composite Cytoskeletal Networks Yinan Shen, Marjan Shayegan, Arturo Moncho, Hui Li, Huayin Wu, Weichao Shi, Songlei Liu, Jing Xia, Dianzhuo Wang, Liheng Cai, Meng Zhang, Ruihua Ding, Frederick MacKintosh, David A Weitz Mechanics of the cytoskeleton is known to be responsible for maintaining cell mechanical integrity and determining cellular functions. We develop a method that enables us to reconstruct a three-component in-vitro network composed of intermediate filaments (vimentin filaments), microtubules and F-actin filaments, which are three fundamental cytoskeletal components. This composition is more physiologically relevant compared with any of the previously reconstituted cytoskeletal networks, which are composed of one or two components only. We investigate the structure and mechanical properties of this multicomponent cytoskeletal network using a combination of several microscopies and microrheology. We show that vimentin filaments couple the other cytoskeletal filaments together by introducing steric constraints between cytoskeletal polymers; these inter-network interactions extend the composite network elastic behavior to a longer time scale, prolong the network relaxation time, and facilitate the stress propagation within the network. These findings are helpful to deepen our understanding of the mechanical role vimentin plays in regulating cellular activities. |
Tuesday, March 5, 2019 4:42PM - 4:54PM |
H64.00008: Maximal entropy production rates in non-contractile actomyosin Daniel S. Seara, Vikrant Yadav, Ian Linsmeier, Pasha Tabatabai, Patrick W. Oakes, Ali Tabei, Shiladitya Banerjee, Michael Murrell The actin cytoskeleton is an active semi-flexible polymer network whose non-equilibrium properties coordinate both stable and contractile behaviors to maintain or change cell shape. While myosin motors drive the actin cytoskeleton out-of-equilibrium, the role of myosin-driven active stresses in stable states of actomyosin is unclear. To investigate this, we synthesize an actomyosin material in vitro whose active stress content can tune the network from stable to contractile and analyze the resulting filament dynamics using the framework of stochastic thermodynamics. We find that the entropy production rate does not increase monotonically with myosin content, but instead is maximized in a non-contractile, stable state. Our study provides evidence that the origins of system entropy production and activity-dependent dissipation relate to disorder in the molecular interactions between actin and myosin. |
Tuesday, March 5, 2019 4:54PM - 5:06PM |
H64.00009: Actively crosslinked microtubule networks: mechanics, dynamics and filament sliding Sebastian Fuerthauer, Bezia Lemma, Peter Foster, Stephanie C Ems-McClung, Claire E Walczak, Zvonimir Dogic, Daniel Needleman, Michael John Shelley Cellular components such as cytoskeletal filaments and motors are the essential |
Tuesday, March 5, 2019 5:06PM - 5:18PM |
H64.00010: Particle delivery using bead-loading gives insight into heterogeneity of the cellular cytoplasm Wenlong Xu, Ashok Prasad Microrheology is an important technique for probing the properties of the cytoplasm, but requires particle delivery inside cells. Here we report a particle delivery method based on a method of protein delivery called bead-loading, requiring no specialized instruments. Bead loading is able to deliver 100nm fluorescent particles that are widely dispersed into the cellular cytoplasm. Particle tracking reveals that the fluorescent particles probe two very different regions of the cytoplasm. One set of beads are significantly confined, and treatment by drugs indicate that the confinement is due to the actin cytoskeleton. The second set of beads seemingly display free diffusion, but ATP depletion experiments show that the motion has an actively driven component. Most strikingly, the main effect of most drug treatments is through changing the relative distribution of these two populations. Our analysis suggests that the specific mode of particle delivery may be strongly affecting the measured properties of cells, underlining the importance of intracellular heterogeneity. The general applicability of our particle delivery technique and the distribution of intracellular movement were confirmed in three different cell lines. |
Tuesday, March 5, 2019 5:18PM - 5:30PM |
H64.00011: Onset and Arrest of Catastrophic Depolymerization in Microtubules Controlled by Tubulin Subunit Shape Mark Stevens, Jonathan Bollinger Microtubules are biopolymers critical for cellular function that display rich dynamic and mechanical behaviors. While microtubules are one of the stiffest known polymers, they possess a distinct dynamic instability: tubules self-assemble via the addition of GTP-tubulin dimers (tubulin bound to GTP), but hydrolysis of GTP- to GDP-tubulin within the tubules eventually destabilizes them toward catastrophically-fast depolymerization, if the leading cap of GTP-tubulin is lost. The molecular mechanisms and features of the individual tubulin proteins that drive such apparently contradictory behavior are still not well-understood. Using molecular dynamics simulations of whole microtubules built from a new coarse-grained model of tubulin, we demonstrate that conformational changes in subunits that frustrate tubulin binding, which have long been hypothesized as a part of microtubule dynamics, drive depolymerization via the unpeeling ``ram's horns'' consistent with experiments. We also show how depolymerization can be prevented or even arrested in-progress (the latter allowing rescue and regrowth) by the presence of even very few GTP-tubulin dimers, and explore the ranges of binding interaction strengths and degrees of shape frustration for which these behaviors are possible. |
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