Bulletin of the American Physical Society
APS March Meeting 2021
Volume 66, Number 1
Monday–Friday, March 15–19, 2021; Virtual; Time Zone: Central Daylight Time, USA
Session C11: Physics of Cytoskeleton Across Scales IFocus Live
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Sponsoring Units: DBIO DSOFT Chair: Carlos Bueno, Rice Univ; Dimitrios Vavylonis, Lehigh University |
Monday, March 15, 2021 3:00PM - 3:12PM Live |
C11.00001: Tension-dependent Myosin Dynamics on Contractile Actomyosin Structures Wen-hung Chou, David R Kovar, Margaret Gardel The actomyosin cytoskeleton powers dynamic cellular processes by generating mechanical tension. Myosin dynamics must be regulated to maintain proper mechanical tension. Tension itself has been proposed to regulate myosin dynamics. Although in vitro studies have shown that myosin detaches slower from F-actin under resisting load, it is unclear whether mechanical tension can regulate myosin dynamics in cells. In this work, we assess myosin turnover dynamics on stress fibers with fluorescence recovery after photobleaching (FRAP) experiments. We reduce global cellular tension with pharmacological inhibitors at non-saturating doses, which decreases the total traction force while preserving the overall actomyosin architecture. Myosin recovers much faster after photobleaching under reduced tension, suggesting that myosin dissociates faster. The smaller immobile fraction suggests that non-exchanging myosin molecules become dynamic when tension is reduced. Indeed, quasi-sarcomeric myosin bands along individual stress fibers are more disordered and dynamic under reduced tension. Furthermore, we directly modulate tension on stress fibers by inducing strain sites. Myosin FRAP recovery adjacent to the strain site would shed light on the role of mechanical tension regulating myosin dynamics. |
Monday, March 15, 2021 3:12PM - 3:24PM Live |
C11.00002: Investigating AGT-DNA covalent and non-covalent interactions in methyl-induced DNA damage repair Rajendra Koirala, Rudramani Pokhrel, Prabin Baral, Purushottam Babu Tiwari, Prem Prasad Chapagain, Narayan Adhikari Methylation induced DNA base-pairing damage, one of the major causes of cancer, can be repaired by O6-alkylguanine-DNA alkyltransferase (AGT). During this process, AGT forms a transient covalent bonding with DNA and then transfers the methyl group from the methylated GUA7 to CYS145. Herein, we modeled the covalent complex of the AGT-DNA system and then investigated structural features of the complex using molecular dynamics (MD) simulations. Utilizing the umbrella sampling method, we also investigated the free energy change between the non-covalent complexes before (pre-repair complex) and after (post-repair complex) the methyl transfer. Our analysis shows that residues THR95, TYR114, and SER151 in AGT consistently form hydrogen bonding with THY9, THY23, and methylated GUA7 (or repaired GUA7) in DNA suggesting the importance of these residues/nucleotides in the AGT-DNA complex formation. In addition, calculation of change in free energies also shows that the pre-transfer complex with methylated GUA7 is more favorable in the catalytic cavity compared to the post-transfer complex with repaired Guanine (GUA7). Our process will not only helpful in computational studies of DNA repair mechanism but also in exploration of cancer therapeutics targeting the AGT-DNA complexes. |
Monday, March 15, 2021 3:24PM - 3:36PM Live |
C11.00003: Force and Power Generation by Contractile Actomyosin Gels Johannes Flommersfeld, David Brückner, Haiyang Jia, Petra Schwille, Chase Broedersz Contractile actomyosin gels are a central source of active cellular force generation. However, the principles underlying the active generation of forces and mechanical power in such gels remain elusive. Specifically, it is unclear how the short time-scale motor dynamics controls the large-scale dynamics and thermodynamics of active stress generation. Here, we introduce a novel experimental system which couples reconstituted actomyosin networks to 3D-printed elastic scaffolds, enabling a dynamic quantification of the generated forces. We observe that the contraction is characterized by a dramatic acceleration, which we show using a model to be due to the load-dependent kinetics of the myosin motors. Thus, we find how the microscopic properties of molecular motors control the macroscopic contraction dynamics. Our model provides insights on how interplay between network mechanics and motor dynamics impacts the large-scale dynamics and thermodynamics of contractile actomyosin gels. |
Monday, March 15, 2021 3:36PM - 3:48PM Live |
C11.00004: Dissecting cytoquakes in the actomyosin cortex using super-resolved micropost arrays Shankar Sivarajan, Yu Shi, Katherine M Xiang, John Crocker, Daniel H Reich The actomyosin network in living cells is an active matter system whose dynamics are responsible for a wide range of cellular behavior. Recent experiments have shown that the cellular cortex exhibits active fluctuations punctuated by rearrangements with large step-like displacements whose size distribution and spatial and temporal correlations resemble those seen in earthquakes and avalanches [1]. We present detailed analysis of the dynamics and size distributions of these cortical "cytoquake" phenomena, via subnanometer particle tracking with poly(dimethylsiloxane) micropost array detectors. We find that, for multiple cell types and substrate stiffnesses, the cortical fluctuations show heavy-tailed distributions whose apparent scaling behavior suggests that a single physical process may account for the dynamics over a wide range of size and time scales. |
Monday, March 15, 2021 3:48PM - 4:00PM Live |
C11.00005: Elasticity from entanglements in branched actin Martin Lenz, Mehdi Bouzid, Cesar Valencia Gallardo, Giuseppe Foffi, Julien Heuvingh, Olivia du Roure Branched actin networks exert pushing forces in eukaryotic cells, and adapt their stiffness to their environment. To understand the microscopic underpinnings of their response, we show using high-sensitivity micromanipulation experiments, numerical simulations and theory that unlike usual crosslinked networks, branched actin is dominated by the proliferation of interfilament contacts under compression. The tree-like topology of the networks make them particularly prone to developing growth-induced entanglements, and is thus key to their active adaptive mechanics. |
Monday, March 15, 2021 4:00PM - 4:12PM Live |
C11.00006: Discrete mechanical model of lamellipodial actin networks David M Rutkowski, Dimitrios Vavylonis Determining how mechanical forces and actin filament turnover coordinate within the lamellipodium is important for understanding cell migration. Continuum models have investigated the stress profile of lamellipodial actin networks including around focal adhesions. However, the forces and deformations of individual actin filaments important in lamellipodial mechanics have largely not been considered. We developed a filament-level computational model of an actin network undergoing retrograde flow simulated via 3d Brownian dynamics. Retrograde flow is maintained by both pushing forces at the leading edge (due to actin polymerization) and pulling forces at the back (due to molecular motors). Connectivity between actin filaments is maintained by bonds representing the Arp 2/3 complex and actin filament crosslinkers. Remodeling of the network occurs via the addition of actin filaments near the leading edge and via filament and severing. We investigate how several parameters affect the stress distribution, network deformation and retrograde flow speed of the actin network, including focal adhesion strength and crosslinking. The model reproduces actin arcs and filopodial bundle as well reduction in retrograde flow speed under cytochalasin D. |
Monday, March 15, 2021 4:12PM - 4:24PM Live |
C11.00007: Exploring the impact of Arp2/3 concentration on actomyosin dynamics Chengxuan Li, James Liman, Yossi Eliaz, Margaret Cheung Actin binding proteins facilitate the structural re-organization of actomyosin networks which underpins the shape changes in living cells. We explore the effect of actin-related proteins 2/3 (Arp2/3) complex, an actin nucleator and brancher, on actomyosin network structures and dynamics. Using a coarse-grained active network model, we show that such actomyosin networks with high concentrations of Arp2/3 complexes show inhibited dynamics because of the saturation of nucleation sites on actin filaments by the Arp2/3 complexes, while low Arp2/3 concentrations aggravate contractility of the networks with hallmarks of short contraction time and small actin clusters. At intermediate Arp2/3 concentrations, sudden collapses of actin clusters in the networks, or “avalanches”, occur. We have implemented graph theory to quantify the higher-order organization inside actomyosin networks, powerful to visualize the hierarchy of the complex networks as well as to extract unprecedented insights on the dynamics of actomyosin networks that can be validated experimentally. |
Monday, March 15, 2021 4:24PM - 4:36PM Live |
C11.00008: Regulation of actin turnover by myosin activity and network architecture Danielle Scheff, Margaret Gardel Actin remodeling through turnover, in which filaments continuously depolymerize from one end while repolymerizing on the other, is essential for the survival of most cells. However, the regulation of this process through actin architecture and myosin activity is poorly studied. Here, we investigate the ability of myosin activity and actin architecture to control turnover. Using a minimal system to recreate turnover in vitro, we observe that severing by the protein cofilin increases the turnover rate in our networks. We additionally find that myosin mediated severing is sufficient to increase the rate of actin turnover, even in systems without cofilin. Preventing filament depolymerization or reducing myosin-mediated buckling filaments reduces this effect, suggesting that the increased rate relies on actin severing and disassembly. This increase in turnover, however, is dependent on actin structure. When actin is bundled, cofilin mediated turnover vanishes. Remarkably, bundling does not impact myosin mediated severing, and myosin is able to increase turnover even in these networks. These results suggest that not only can myosin regulate turnover of actin filaments, but also that different methods of disassembly might be needed to remodel actin depending on its local structure. |
Monday, March 15, 2021 4:36PM - 4:48PM Live |
C11.00009: Dynamic actin waves in primary rat cortical astrocytes sense environmental cues Kate O'Neill, Emanuela Saracino, Barbara Barile, Nicholas Mennona, Spandan Pathak, Maria Grazia Mola, Grazia Paola Nicchia, Valentina Benfenati, Wolfgang Losert Recent work has highlighted the importance of the brain's non-neuronal cells in cognitive function. Astrocytes, one type of these non-neuronal glial cells, have physiological roles ranging from maintaining brain homeostasis to modulating neuronal communication. Astrocytes perform these functions by regulating the flux of ions and water molecules across their membranes and by communicating with other astrocytes within the brain’s networks. These functions require dynamic reorganization of the cytoskeleton in response to changes in the brain’s microenvironments, but these dynamics have not been well studied in astrocytes, especially on shorter time scales of seconds to minutes. To this end, we use live confocal microscopy to capture actin dynamics in live astrocytes under control and “triggering” conditions after astrocytes have been transduced with actin-GFP. We then employ optical flow to analyze these actin dynamics by tracking changes in fluorescence. We also study the persistence of these dynamics and identify small regions of especially high activity, which we call functional microdomains. We propose that actin dynamics are a local manifestation of astrocytes’ global homeostatic response to changes in the extracellular microenvironment. |
Monday, March 15, 2021 4:48PM - 5:00PM Live |
C11.00010: Model of dendritic actin network with distributed turnover and structural remodeling Danielle Holz, Aaron R Hall, Dimitrios Vavylonis The dendritic network of actin filaments provides the force for lamellipodial protrusions, driven by polymerization and branch generation by the Arp2/3 complex. Electron microscopy has revealed a network structure that varies with distance to the leading edge: a dense brushwork composed of short filaments near the leading edge is followed by longer and more linear filaments near the center and rear. Single molecule imaging experiments have shown that actin assembles throughout the lamellipodium and frequent disassembly within a few seconds after incorporation into the filament network. To investigate the mechanisms behind network remodeling, we created a three-dimensional stochastic model at the filament level that includes mechanisms for polymerization, depolymerization, branching, capping, uncapping, severing, oligomer diffusion, annealing, and debranching. We find that incorporating filament severing, enhanced near barbed ends, can explain the single molecule actin lifetime distribution and provide stable lamellipodia, as long as the oligomer fragments reanneal to free barbed or pointed ends with rate constants comparable to in vitro measurements. We thus propose a unified mechanism that fits a diverse set of basic lamellipodia phenomenology. |
Monday, March 15, 2021 5:00PM - 5:36PM Live |
C11.00011: The dual role of calcium/calmodulin-dependent kinase II in transducing calcium signals and reorganizing actomyosin networks Invited Speaker: Margaret Cheung Calcium/calmodulin-dependent kinase II (CaMKII) plays a key role in the plasticity of dendritic spines. Calcium signals (Ca2+) cause calcium-calmodulin (CaM) to active CaMKII, which leads to remodeling of the actin filament network in the spine. In this presentation, I will first focus on the binding mechanism of CaM and its binding target, which requires mutually- and conformationally-induced changes in both participants Then, I will reveal how a target mechanistically tunes CaM’s affinity for Ca2+ by examining interactions with neurogranin (Ng) and CaM-dependent kinase II (CaMKII). These two targets are biochemically known to tune CaM’s affinity for Ca2+ in opposite directions in postsynaptic neuronal cells. I will further discuss the active role of holoenzyme CaM/CaMKII in transmitting chemical reactions at a molecular scale to specific mechanical responses at a micron scale by alternating the topology of actomyosin networks. The observation made by a team of collaborators using an integrated approach of experiments, theory, and computation provides a simple mechanism by which the structural dynamics of CaMKII establishes a link between calcium signaling and the morphological plasticity of dendritic spines. |
Monday, March 15, 2021 5:36PM - 5:48PM Live |
C11.00012: Quantifying mechano-chemical coupling between RhoA and the actomyosin cortex in vivo Melis Tekant, Gary Choi, Alasdair Hastewell, Alexandru Bacanu, Jorn Dunkel, Nikta Fakhri Spatiotemporal symmetry-breaking transitions in biochemical patterns are essential in triggering morphological changes during the developmental processes. Cell and tissue-scale deformations is achieved through intra-cellular force networks that translate localized biochemical signals into effective mechanical stresses that determine the global shape dynamics. However, the mechanochemical coupling between the biochemical patterns and the resulting stress patterns is not well understood. Here, we quantify the local coupling between membrane-bound Rho-GTP concentration fields and the mechanical deformations of the actomyosin cortex in the starfish oocytes during meiosis. We generate various Rho-GTP dynamic patterns by overexpressing the Rho activator and map the resulting stress patterns via tracking endogenous probe particles embedded in the cell cortex. This method provides a novel approach to probe the local coupling between biochemistry and mechanics. |
Monday, March 15, 2021 5:48PM - 6:00PM Live |
C11.00013: Reconstituting an active cytoskeleton in a droplet Jianguo Zhao, Charlie Duclut, Rahil Golipour, An Pham, Behzad Golshaei, Chonglin Guan, James L Harden, Frank Jülicher, Christoph F. Schmidt The actin cytoskeleton is a viscoelastic network of semiflexible actin polymers that dominates many biological processes such as cell locomotion, division, spreading, organelle transport and positioning. It is a dissipative structure that is maintained in and can switch between dynamic steady states through regulated balanced “fluxes”, i.e. spatial movements, polymerization, depolymerization, forming and breaking crosslinks. While equilibrium actin networks have been studied extensively, a dynamic cytoskeleton resembling that of living cells has been impossible to reconstruct from isolated components. We here use Xenopus laevis egg extract to reconstitute dynamic actin networks in water-in-oil emulsion droplets with sizes comparable to cells. We observe convergent steady-state 3D flow of the actin network towards the droplet center, driven by myosin and maintained by constant actin polymerization and depolymerization. Lipid vesicles and other aggregates get concentrated to form an inclusion in the center of the droplets. We constructed a hydrodynamic model of a contracting network with turn-over that can explain the measured actin velocity and density profiles and the resulting stress field, that we probe by local UV laser cutting and magnetic beads. |
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