Bulletin of the American Physical Society
APS March Meeting 2020
Volume 65, Number 1
Monday–Friday, March 2–6, 2020; Denver, Colorado
Session L20: Physics of the Cytoskeleton Across Scales II: Enzymes to NetworksFocus
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Sponsoring Units: DBIO Chair: Jennifer Ross Room: 301 |
Wednesday, March 4, 2020 8:00AM - 8:12AM |
L20.00001: Tubulin shape controls the kinetics and mechanism of microtubule depolymerization Jonathan Bollinger, Zachary Imam, Mark Stevens, George Bachand Microtubules exhibit alternating phases of growth and shrinkage thought to be controlled by the conformation of tubulin dimers. Specifically, compression of tubulin due to the hydrolysis of GTP has been suggested to generate stress and drive catastrophic depolymerization. We use molecular dynamics simulations and ex vivo experiments to investigate how depolymerization is affected by the presence of uncompressed (unhydrolysed) dimers in the microtubule lattice. Both methods reveal exponential decay in the kinetics of depolymerization corresponding to the relative number of uncompressed dimers. This slowdown is accompanied by a morphological change from ram's horns to blunt-ended dissociation. Collectively these data show that uncompressed dimers can alter depolymerization consistent with promoting rescue events. |
Wednesday, March 4, 2020 8:12AM - 8:48AM |
L20.00002: Stutter: a Transient Microtubule Dynamic Instability Phase that is Strongly Associated with Catastrophe Invited Speaker: Holly Goodson Microtubules (MTs) are dynamic polymers with critical roles in processes ranging from membrane transport to chromosome separation. Central to MT function is dynamic instability (DI), a behavior typically assumed to consist of growth and shortening, with sharp transitions in between. However, this two-state assumption disregards details in MT behavior that are evident in high-resolution data. For example, MTs exhibit growth rate variability, and pinpointing where transitions begin can be difficult when viewed at high spatiotemporal resolution. These observations suggest that MT behavior is more complicated than implied by standard quantification methods. To address these problems, we developed STADIA (Statistical Tool for Automated Dynamic Instability Analysis). STADIA’s methods are rooted in machine learning to objectively analyze and quantify macro-level DI behaviors exhibited by MTs. Applying STADIA to MT length-history data revealed a transient, intermediate phase that we term ‘stutter’, during which the rate of MT length change is smaller in magnitude than growth or shortening phases. Significantly, most catastrophe events in both simulations and experiments are preceded by stutters, suggesting that this newly recognized phase is mechanistically involved in catastrophes. Consistent with this idea, a MT anti-catastrophe factor (CLASP2γ) increases the likelihood of growth following a stutter phase in experiments. We conclude that STADIA enables unbiased identification of DI phases including stutters, producing more complete and accurate DI measurements than possible with classical analysis methods. Identifying stutters as a distinct and quantifiable phase provides a new target for mechanistic studies regarding DI phase transitions and their regulation by MT binding proteins. |
Wednesday, March 4, 2020 8:48AM - 9:00AM |
L20.00003: Molecular dynamics study of Katanin oligomeres: A MT-severing enzyme Mangesh Damre, Rohith Anand Varikoti, Ruxandra I Dima Microtubule (MT) severing enzymes, such as katanin, belong to the AAA ATPases family and play an important role in meiosis and mitosis through their cutting action on MTs. The severing action is driven by ATP hydrolysis, which induces transitions between distinct conformations of the oligomeric state of the severing enzyme. Two of these oligomeric structures, one in a spiral and the other in a closed ring arrangement, have recently been solved using cryo-EM. ATP is present in all the chains from the spiral conformation, while there is only partial occupancy of ATP in the ring conformation. At the same time, reports from the literature suggest that such high-order oligomeric states of severing enzymes are stable only in the presence of nucleotides and the substrate. We will describe our all-atom molecular dynamics simulations performed on the apo, the ATP bound, and minimal substrate-bound states of katanin. Our results help identify the factors that account for the stability of the oligomeric states and characterize the allosteric transitions that underlie the action of severing enzymes. |
Wednesday, March 4, 2020 9:00AM - 9:12AM |
L20.00004: Role of substrate cooperativity and motor concentration in microtubule severing Rohith Anand Varikoti, Jennifer L Ross, Ruxandra I Dima Microtubules (MTs) associated proteins (MAPs) regulate the dynamic behavior of MTs during cellular processes. Severing enzymes are MAPs which destabilize MTs by removing subunits from the filament. Because severing enzymes belong to the AAA+ unfoldases family, we probed the severing mechanism characterized by the enzymes applying pulling forces on the C-terminal regions of MT subunits. Due to the large size of the system, we employed coarse grained molecular simulations of different MT lattices and at varying concentration of severing enzymes. Comparison of results from our simulations with data from in-vitro severing assays shows that the cooperative removal of protofilament fragments, at increased concentration of severing enzymes, is a likely MT destabilization scenario. Moreover, we found that optimization of energetics is not a strong requirement if severing proceeds entirely by an unfoldase mechanism. |
Wednesday, March 4, 2020 9:12AM - 9:24AM |
L20.00005: Dynamic stability of actin cytoskeletal networks with mixed geometries Pasha Tabatabai, Laura Lanier, Michael Murrell Cells dynamically remodel their cytoskeleton while simultaneously utilizing it to carry out vital tasks such as migration and division. In cells, multiple distinct actin structures coexist, and it is unclear how this coexistence of multiple dynamic networks affects the stability of each network. To this end, we re-engineer a dynamic cytoskeletal system using a minimal set of purified proteins to simultaneously nucleate filamentous actin networks containing mixtures of both branched Arp2/3 and linear formin networks. With this system, we investigate the effect of coexistence of networks with different geometries and growth rates on steady-state structure. Additionally, we explore the influence of both filament severing activity and molecular motor activity on network stability. |
Wednesday, March 4, 2020 9:24AM - 9:36AM |
L20.00006: Actin crosslinking controls mobility, microtubule crosslinkers control co-localization in a composite cytoskeletal network Leila Farhadi, Michael Rust, Moumita Das, Rae M Robertson-Anderson, Jennifer L Ross Actin and microtubule filaments are cytoskeletal biopolymers with various vital roles in the cell. Despite decades of studying them separately, they have recently been shown to interact in networks mechanically and chemically. Here, we are interested in the composite network mechanics and mobility as the actin and microtubules are increasingly crosslinked. We use biotin-NeutrAvidin crosslinkers to irreversibly crosslink actin and MAP65, an antiparallel microtubule crosslinker to bundle microtubules. Cytoskeleton networks are imaged over time using fluorescent microscopy and the mobility characteristics are measured. We find that actin crosslinkers tune the mobility of this composite network, while microtubule crosslinkers can control the co-localization of actin and microtubules. |
Wednesday, March 4, 2020 9:36AM - 9:48AM |
L20.00007: Avalanches in simulations of branched actomyosin networks with the Arp2/3 complex James Liman, Carlos Bueno, Yossi Eliaz, Nicholas Schafer, Neal Waxham, Peter G Wolynes, Herbert Levine, Margaret Cheung Actomyosin networks are ubiquitous in biology. They provide structure to cells and are involved in cell movement, growth, and division. The dynamics of actomyosin networks are active processes and are greatly influenced by actin binding-proteins (ABPs). These ABPs include both motor proteins (non-muscle myosin IIA heavy chain (NMIIA)) and cross-linker proteins (α-actinin). Another important ABP, the Arp2/3 complex, nucleates branched filaments thereby influencing the topology of the network. In this work, we simulate the spatiotemporal configurations of actomyosin networks with and without the Arp2/3 complex. The simulations show that the branched actomyosin networks that include the Arp2/3 complex exhibit sporadic convulsive movements, which we call avalanches, that release built-up stress in the network. We then identify and characterize these avalanches. The characteristics of these avalanches observed in the simulations are consistent with the recent experimental observation of “cytoquakes”. |
Wednesday, March 4, 2020 9:48AM - 10:00AM |
L20.00008: A dynamic contractile F-actin network reconstituted in Xenopus egg extract Jianguo Zhao, Christoph F. Schmidt The actin cytoskeleton in most animal cells is a highly dynamic multi-component network that rapidly turns over, adapts, generates forces and moves. The continuous remodelling of the cytoskeleton and spontaneous flows have been extensively visualized in a variety of cellular processes, including intracellular transport, cell migration and division. It remains unclear how the spatiotemporal interactions of the various components self-organize to give rise to global F-actin flows. We reconstituted a fully active model cytoskeleton in water-in-oil emulsion droplets containing xenopus egg extract. We observed steady state directed flows and non-equilibrium phase separation. We analysed the flow and examined the contractile stress generated by actin-myosin interactions. |
Wednesday, March 4, 2020 10:00AM - 10:36AM |
L20.00009: Controlling ‘cell’ size and shape to elucidate the mechanics of microtubule aster positioning Invited Speaker: Jay Gatlin The microtubule (MT) cytoskeleton plays critically important roles in numerous eukaryotic cellular functions, and it does so across a functionally diverse and morphologically disparate range of cell types. In these roles, MT assemblies must adopt distinct cell cycle-dependent morphologies and physical dimensions to perform specific functions. During interphase, the MT network takes the form of a radial astral array (aster) that functions to center the nucleus, a by proxy the mitotic spindle, which ultimately dictates the position of the cell division plane. The mechanical underpinnings of this positioning phenomenon remain elusive despite its fundamental importance to both symmetric and asymmetric vision and intensive study. To address this gap in our collective understanding, we have combined photo-labile hydrogels with cell-free extracts in a new experimental platform that affords exquisite control of “cell” shape and volume. By observing the behavior/dynamics of MT asters confined in hydrogel micro-containers of different geometries, we have elucidated the relative contribution of MT-based pushing forces to aster positioning and have begun to characterize the length scales over which they operate. |
Wednesday, March 4, 2020 10:36AM - 10:48AM |
L20.00010: Active Composites of Actin and Kinesin-driven Microtubules John Berezney, Seth Fraden, Zvonimir Dogic Two major structural proteins, actin and microtubules, form multiple co-existing and interpenetrating filamentous protein networks within the cell cytoplasm. The out-of-equilibrium active reorganization of these structures by molecular motors is necessary for basic physiological processes such as cell division, cell motility, and environmental sensing. While the passive structure and mechanics of such materials have been well documented, the effects of their steady-state out-of-equilibrium reorganization is a site of current research. To demonstrate some of the mechanics governing the active reorganization of these materials, we have built a polymer blend of kinesin-driven microtubule networks which reorganize a passive entangled actin network. We find both the mechanics of the actin network as well as its initial structure can have dramatic effects on the steady-state behavior of the system. To capture the range of behaviors, we build a state diagram which captures the non-equilibrium phenomena we observe. |
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