Session B07: Physics of Cytoskeleton I

How immune cells respond to physical cues – the role of cytoskeletal dynamics

The activation of lymphocytes, an essential step in the adaptive immune response, involves the binding of specialized receptors with antigens. This results in large-scale dynamics and reorganization of the cytoskeleton, and assembly of receptors into signaling microclusters, which are critical for immune cell activation and formation of the immune synapse. The cytoskeleton plays distinct roles in the exertion of mechanical stresses that drive receptor activation, spatial reorganization and assembly of signaling proteins in lymphocytes. Forces exerted by T cells on the antigen presenting surface arise due to the dynamics of actin polymerization, myosin motor activity and microtubule dynamics. Force fluctuation analysis reveals distinct contributions of these cytoskeletal elements to force generation in T cells. Our results also indicate a mechanical coupling between the actomyosin and microtubule systems whereby different actin structures influence microtubule dynamics in distinct ways. Microtubule growth dynamics at the IS are differentially modulated by distinct actin nucleators. Furthermore, microtubule filament dynamics and shape deformations are actively modulated by actin polymerization and myosin activity in the immune synapse. While antigens binding to T cell receptors are the primary drivers of lymphocyte signaling, additional factors serve to modulate activation. I will summarize our studies that reveal how physical cues such as stiffness and topography modulate T cell activation by regulating the coordinated dynamics of the acto-myosin and microtubule cytoskeleton. Finally, I will discuss our recent work that reveals how cell-secreted chemicals or cytokines control actin and microtubule dynamics and cellular force generation in killer T lymphocytes. Together, these studies reveal how different regulators of signaling work in concert with the cytoskeleton to control the immune response.   

Identifying mechanisms of non-muscle myosin II filament assembly and amplification

The majority of contractile forces in non-muscle cells are generated by non-muscle myosin 2-driven contraction of actin networks. High-resolution imaging in the lamella of migrating fibroblasts revealed myosin filaments appear and amplify by an actin-dependent partitioning process, feeding into larger actomyosin networks.Our goals were to delineate mechanisms of new filament establishment, and define how filament partitioning enables the formation of contractile networks. We fail to observe calcium- or Rho-mediated activation in the immediate vicinity of a new myosin filament, suggesting monomer activation occurs with low spatial precision. However, consistent with published work, we observe filament appearance following leading edge retraction. Appearance stalls when actin dynamics are pharmacologically halted, but rescued by myosin monomer release from posterior stress fibers via ROCK inhibition. This suggests that filament formation is a low-precision, stochastic event dependent on regional increases in available monomer. We then used a molecular counting method to determine the mechanism of partitioning after filaments are established. Our in vitro, fixed-cell, and live-cell imaging suggests that 1) in vitro single filaments are ~30 monomers, 2) live-cell bipolar structures can be upward of a single filament (>>30 monomers), and 3) multiple filaments are present during partitioning. Taken together, we propose a model whereby increased actin network density during retraction events acts as a kinetic trap for myosin monomers, resulting in filament nucleation. Then, established myosin filaments act as a diffusion trap for assembly-competent monomers and possibly other cytoplasmic filaments, resulting in sub-resolution stacks and clusters. Established filaments and filament clusters then mature into a contractile network to enable cell migration. We anticipate similar mechanisms exist in more complex contractile processes throughout cell and tissue biology.   

In vitro light-controlled patterns and force generation for synthetic cytoskeletal networks

The cytoskeleton of a cell enables its shape and motility. To understand how patterns and associated forces may emerge in a simple cytoskeletal protein system, we synthesize, express, and purify a cortical protein (Tcb2) from the ciliated protozoan Tetrahymena. Tcb2 is a 25 kDa calcium-binding protein containing four EF-hand loops. By using caged calcium, we use light as a trigger to control the spatio-temporal assembly and dis-assembly of these in-vitro Tcb2 networks, both in confined microscopic geometries as well as in liquid droplets. We discuss the emergent patterns (reversible and irreversible) and the resultant complex fluid flow fields generated by the contractile network in response to different optical field structures. Through simple reaction-diffusion models, we discuss the associated physical mechanisms controlling the assembly and dis-assembly of these networks. By understanding simple contractile force-generating protein networks, our work offers insight into design and control of artificial cytoskeletons in synthetic and living cells.   

The Effect of Ionic Strength in Microtubule Tactoid Formation

Cell division via mitosis is one of the most important biological processes to sustain life. Microtubules, along with their associated proteins, crosslinkers, and enzymes, play a crucial role in the formation of the mitotic spindle. Previous studies on self-organization of microtubules showed that, in presence of polymer crowding agents, microtubules self-organize into bundles. Introducing certain concentrations of an antiparallel crosslinker, MAP65, in the system resulted in the bundles coalescing into homogenous, birefringent tactoids. Unlike the meiotic spindle and tactoids observed with actin filaments, which have been shown to have properties of liquid crystals, microtubule tactoids are solids. In an effort to investigate the mechanism of the organization and possibly alter the interactions to reduce the solidity of the tactoids, we altered the ionic strength of the buffer when forming tactoids in the presence of MAP65. We varied the concentrations of NaCl, KCl, and MgCl₂. The salt concentration affected the MT-MAP65 and MAP65-MAP65 bonds at different rates. Both of these interactions altered the formation of tactoids.   

The Effects of Ionic Strength in MAP65 Binding to Microtubules

Previously we have shown that, microtubules can form tactoids in the presence of antiparallel crosslinker, MAP65. In order to determine the relative binding strength of MAP65 to microtubules compared to MAP65 dimerization, we have performed single-molecule experiments on MAP65 binding to microtubules in the presence of altered ionic strength. Specifically we have used NaCl in buffer to determine the binding time and diffusion of single MAP65 on microtubules using total internal reflection (TIRF) microscopy.   

Motor-free Contractility in Active Gels

Force generation in animal cells is usually due to molecular motors. Recent experimental evidences, however, have implied that contractility can be generated in the absence of motors. In this talk, we propose a motor-free mechanism that generates contraction in biopolymer networks. Unlike motors which rely on polar substrate for their functions, our mechanism is based on the asymmetric force-extension relation of semiflexible biopolymers. Together with active binding and unbinding of crosslinkers, this asymmetry leads to a nonthermal, ratchet-like process that generate steady-state contraction. Our mechanism may provide an explanation for the motor-independent contractility observed in living cells.   

Tunable Non-Equilibrium Dynamics of Motor-Driven Cytoskeleton Composites

Motor-driven scaffolds of actin filaments and microtubules display rich non-equilibrium dynamics and structure that are critical to mesoscale cellular behavior. Numerous studies have investigated the dynamics of actomyosin networks, active kinesin-microtubule systems, and steady-state actin-microtubule composites. Yet, in cells all of these components interact to enable the diverse processes and properties that cells exhibit. Here, we create in vitro actin-microtubule networks driven by myosin-II minifilaments and quad-headed kinesin clusters, and use multi-color confocal microscopy to image the active composites. To characterize the non-equilibrium dynamics of actin and microtubules in the composites, we use differential dynamic microscopy (DDM) to quantify filament contraction and flow as the composites dynamically restructure. We find that myosin and kinesin activity lead to dramatically different dynamics of both filaments, and that static crosslinking of either filament changes the patterns and rates of motor-driven restructuring.    

Actin-microtubule networks as time-varying rigidly percolating double networks

The cytoskeleton is a dynamic composite scaffold made of networks of different types of biopolymers, primarily actin filaments and microtubules, and motor proteins. Here we present results from Langevin Dynamics simulations of a model that combines two structure-function frameworks: a double network (DN) made of a network of stiff filaments (microtubules) interacting with a network of flexible filaments (actin), and rigidity percolation theory. We present results for the collective time-evolving mechanical response of these composites with and without active elements (myosin and kinesin motors), and obtain a phase diagram in the space of concentrations of actin, microtubules, and motors. Our results can provide mechanistic insights into the contractility and restructuring of active cytoskeletal composites and the rational design of materials inspired by them.   

Competition between severing and tubulin induced repair of microtubules

Cytoskeletal structures undergo dynamic changes in size to aid in cell polarization, motility, and intracellular transport. Such changes require a rapid turnover of cytoskeleton proteins but how various cytoskeleton-associated proteins coordinate to achieve that is not well understood. Recent experiments have addressed this question by adding key cytoskeleton-associated proteins to cytoskeletal filaments in-vitro and have reported novel synergistic effects. Free pool of tubulin had previously been thought to primarily influence assembly of microtubules, however new experiments indicate a novel effect on their disassembly – free tubulin pool can repair nanoscale damages created by severing proteins. Based on this observation, we propose a model for microtubule severing as a competition between the processes of damage spreading and repair. Using theory and simulations, we demonstrate that this model is in quantitative agreement with recent in vitro experiments, and predict the existence of a critical tubulin concentration above which severing becomes rare but fast. We find that length - control via this new model of severing is more sensitive to changes in the concentration of tubulin and severing protein, and leads to a dramatically increased dynamic range. Our work describes how the concerted action of multiple microtubule associated proteins produces novel dynamical properties of microtubules, which are not seen when studying the action of proteins individually.   

Laser ablation reveals how the mitotic spindle reshapes the fission yeast nucleus

When eukaryotic cells divide, the mitotic spindle, a self-assembling microtubule-based machine, segregates chromosomes to each daughter cell. In organisms with closed cell division, such as the fission yeast S. pombe, the nuclear envelope remains intact. Thus, the spindle must accomplish its feat from under an additional mechanical constraint. In the case of S. pombe, spindle elongation is accompanied by a series of nuclear shape changes. The nucleus begins as a single spheroid, passes through peanut- and then barbell-shaped forms, and pinches off into two new daughter nuclei. While spindle elongation clearly helps drive these shape changes, how the nuclear envelope and spindle coordinate is not clear. To test the mechanical interactions between the envelope and spindle, we use targeted laser ablation to sever the spindle and to rupture the envelope, and examine the ensuing responses by live cell confocal fluorescence microscopy. We combine the targeted damage of laser ablation with genetic and biochemical perturbations that further affect mechanics, such as by increasing or decreasing envelope membrane tension or by altering spindle elongation dynamics. These readouts allow us to begin mapping how the spindle and nuclear envelope shape each other as the cell divides.   

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The cytoskeleton is a dynamic network of proteins, including semiflexible actin filaments, rigid microtubules, crosslinking proteins, and myosin and kinesin motors, that enable key processes in the cell such as growth, movement, and cell division. While active actomyosin and kinesin-microtubule systems have been extensively studied in recent years, how interactions between actin and microtubules impact cytoskeletal restructuring by myosin and kinesin remains poorly understood. Here, we investigate the active dynamics and restructuring of actin-microtubule composites driven by kinesin motors and myosin II minifilaments. We combine spatial image autocorrelation and particle image velocimetry to characterize time-varying network structure and velocity flow fields during motor activity. We find that myosin activity causes network contraction into microscale foci whereas kinesin motors induce large-scale flow and bundling. Crosslinking of actin or microtubules tunes the flow rate and the resulting network structure. These findings provide insight into how the different components of the cytoskeleton work in concert to enable diverse cellular processes and structures, and advance their use as a tunable active matter system.