Possible origins of macroscopic left-right asymmetry in organisms

I consider the microscopic mechanisms by which a particular left-right (L/R) asymmetry is generated at the organism level from the microscopic handedness of cytoskeletal molecules. In light of a fundamental symmetry principle, the typical pattern-formation mechanisms of diffusion plus regulation cannot implement the "right-hand rule"; at the microscopic level, the cell's cytoskeleton of chiral filaments seems always to be involved, usually in collective states driven by polymerization forces or molecular motors. It seems particularly easy for handedness to emerge in a shear or rotation in the background of an effectively two-dimensional system, such as the cell membrane or a layer of cells, as this requires no pre-existing axis apart from the layer normal. I detail a scenario involving actin/myosin layers in snails and in C. elegans, and also one about the microtubule layer in plant cells. I also survey the other examples that I am aware of, such as the emergence of handedness such as the emergence of handedness in neurons, in eukaryote cell motility, and in non-flagellated bacteria.Comment: 42 pages, 6 figures, resubmitted to J. Stat. Phys. special issue. Major rewrite, rearranged sections/subsections, new Fig 3 + 6, new physics in Sec 2.4 and 3.4.1, added Sec 5 and subsections of Sec

Possible mechanisms for initiating macroscopic left-right asymmetry in developing organisms

How might systematic left-right (L/R) asymmetry of the body plan originate in multicellular animals (and plants)? Somehow, the microscopic handedness of biological molecules must be brought up to macroscopic scales. Basic symmetry principles suggest that the usual "biological" mechanisms -- diffusion and gene regulation -- are insufficient to implement the "right-hand rule" defining a third body axis from the other two. Instead, on the cellular level, "physical" mechanisms (forces and collective dynamic states) are needed involving the long stiff fibers of the cytoskeleton. I discuss some possible scenarios; only in the case of vertebrate internal organs is the answer currently known (and even that is in dispute).Comment: 9 pp latex, 6 figures. Proc. Landau 100 Memorial Conf. (Chernogolovka, June 2008); to appear AIP Conf. series. (v2: added 4 ref's + revised Sec 2.2.

Stochastic Ratcheting on a Funneled Energy Landscape is Necessary for Highly Efficient Contractility of Actomyosin Force Dipoles

Current understanding of how contractility emerges in disordered actomyosin networks of non-muscle cells is still largely based on the intuition derived from earlier works on muscle contractility. This view, however, largely overlooks the free energy gain following passive cross-linker binding, which, even in the absence of active fluctuations, provides a thermodynamic drive towards highly overlapping filamentous states. In this work, we shed light on this phenomenon, showing that passive cross-linkers, when considered in the context of two anti-parallel filaments, generate noticeable contractile forces. However, as binding free energy of cross-linkers is increased, a sharp onset of kinetic arrest follows, greatly diminishing effectiveness of this contractility mechanism, allowing the network to contract only with weakly resisting tensions at its boundary. We have carried out stochastic simulations elucidating this mechanism, followed by a mean-field treatment that predicts how contractile forces asymptotically scale at small and large binding energies, respectively. Furthermore, when considering an active contractile filament pair, based on non-muscle myosin II, we found that the non-processive nature of these motors leads to highly inefficient force generation, due to recoil slippage of the overlap during periods when the motor is dissociated. However, we discovered that passive cross-linkers can serve as a structural ratchet during these unbound motor time spans, resulting in vast force amplification. Our results shed light on the non-equilibrium effects of transiently binding proteins in biological active matter, as observed in the non-muscle actin cytoskeleton, showing that highly efficient contractile force dipoles result from synergy of passive cross-linker and active motor dynamics, via a ratcheting mechanism on a funneled energy landscape.Comment: 13 pages, 6 figure

Doctor of Philosophy

dissertationCells encounter mechanical cues from the environment to which they sense and respond. The actin cytoskeleton is the main network that can not only sense mechanical changes, but can also reorganize in response. Actin stress fibers are predominant in cultured fibroblast cells and are load-bearing structures of the cell. Here, in collaboration with others, I have investigated the mechanisms of stress fiber strain response and remodeling using fluorescently-labeled cytoskeletal proteins and live cell microscopy, traction force microscopy, and genetic manipulation to assess these mechanisms. High resolution image acquisition and analysis have provided novel insight into the mechanosensitivity of actin stress fibers. Specifically, the actin-associated protein zyxin has been implicated in an actin repair mechanism with mechanical consequences. We discovered a novel zyxin-mediated actin repair mechanism that restored structural and mechanical integrity to stress fibers following a hyperleongation event in a single stress fiber sarcomere. We also discovered that while these spontaneously occurring hyperelongation events impact single sarcomeres along a stress fiber, they coincide with compensatory shortening in the near-by regions of stress fiber sarcomeres, suggesting there is active remodeling that occurs in actin stress fibers in order to maintain the structure and mechanical homeostasis in live cells. Lastly, we designed a computational model to test whether actin and myosin-based mechanical changes drive some of these dynamic changes in stress fibers. We discovered that variable differences in actin stiffness and myosin contractility may be the main factors in spontaneous changes in iv stress fiber sarcomere length. The findings presented in this dissertation have made exciting contributions to the field of actin cytoskeletal dynamics, and will provide groundwork to future studies dissecting the role of actin-associated proteins in structural and mechanical homeostasis in stress fibers

On the microstructure of active cellular processes

Eukaryotic cells use a multitude of protein machines to regulate their own structure. In this thesis, we study how the geometrical arrangement of these interacting microscopic active elements sculpt the cell's own internal microstructure and its membrane enclosure.We first focus on the mechanisms generating actomyosin contractility, a crucial driver of cell motion and organization. We question the current position of highly organized, sarcomeric contractility as the only possible mechanism to drive contractility. We propose an alternative mechanism, and show that only it can account for the observed contractility of disordered actomyosin assemblies. It moreover yields qualitatively new effects in intracellular force transmission, including stress reversal and amplification, consistent with experimentally observations in fiber networks.We next elucidate some of the mechanisms through which the cell deforms and cuts its own membrane, thus enabling exchanges with the extracellular medium as well as between its internal compartments. We find that the function of the proteins responsible for this remodeling is strongly influenced by the mechanics of the membrane, and use these effects to elucidate the modes of operation of proteins clathrin and dynamin, as well as of protein complex ESCRT-III

Cytoskeleton and Cell Motility

The present article is an invited contribution to the Encyclopedia of Complexity and System Science, Robert A. Meyers Ed., Springer New York (2009). It is a review of the biophysical mechanisms that underly cell motility. It mainly focuses on the eukaryotic cytoskeleton and cell-motility mechanisms. Bacterial motility as well as the composition of the prokaryotic cytoskeleton is only briefly mentioned. The article is organized as follows. In Section III, I first present an overview of the diversity of cellular motility mechanisms, which might at first glance be categorized into two different types of behaviors, namely "swimming" and "crawling". Intracellular transport, mitosis - or cell division - as well as other extensions of cell motility that rely on the same essential machinery are briefly sketched. In Section IV, I introduce the molecular machinery that underlies cell motility - the cytoskeleton - as well as its interactions with the external environment of the cell and its main regulatory pathways. Sections IV D to IV F are more detailed in their biochemical presentations; readers primarily interested in the theoretical modeling of cell motility might want to skip these sections in a first reading. I then describe the motility mechanisms that rely essentially on polymerization-depolymerization dynamics of cytoskeleton filaments in Section V, and the ones that rely essentially on the activity of motor proteins in Section VI. Finally, Section VII is devoted to the description of the integrated approaches that have been developed recently to try to understand the cooperative phenomena that underly self-organization of the cell cytoskeleton as a whole.Comment: 31 pages, 16 figures, 295 reference

From Molecular Signal Activation to Locomotion: An Integrated, Multiscale Analysis of Cell Motility on Defined Matrices

The adhesion, mechanics, and motility of eukaryotic cells are highly sensitive to the ligand density and stiffness of the extracellular matrix (ECM). This relationship bears profound implications for stem cell engineering, tumor invasion and metastasis. Yet, our quantitative understanding of how ECM biophysical properties, mechanotransductive signals, and assembly of contractile and adhesive structures collude to control these cell behaviors remains extremely limited. Here we present a novel multiscale model of cell migration on ECMs of defined biophysical properties that integrates local activation of biochemical signals with adhesion and force generation at the cell-ECM interface. We capture the mechanosensitivity of individual cellular components by dynamically coupling ECM properties to the activation of Rho and Rac GTPases in specific portions of the cell with actomyosin contractility, cell-ECM adhesion bond formation and rupture, and process extension and retraction. We show that our framework is capable of recreating key experimentally-observed features of the relationship between cell migration and ECM biophysical properties. In particular, our model predicts for the first time recently reported transitions from filopodial to “stick-slip” to gliding motility on ECMs of increasing stiffness, previously observed dependences of migration speed on ECM stiffness and ligand density, and high-resolution measurements of mechanosensitive protrusion dynamics during cell motility we newly obtained for this study. It also relates the biphasic dependence of cell migration speed on ECM stiffness to the tendency of the cell to polarize. By enabling the investigation of experimentally-inaccessible microscale relationships between mechanotransductive signaling, adhesion, and motility, our model offers new insight into how these factors interact with one another to produce complex migration patterns across a variety of ECM conditions

Energetics of Biological Mechanics and Dynamics

Living matter is a class of soft matter systems that maintains itself away from thermodynamic equilibrium by the continual consumption of chemical energy. Indi- vidual proteins consume energy and break detailed balance to drive active force generation by molecular motors, force-dependent binding kinetics, and chemically driven (dis)assembly. These non-equilibrium dynamics propagate across heterogeneous structures to drive essential life processes such as replication, migration, and shape change at the scale of both single cells and multicellular tissues. While much work has been done to understand the molecular processes underlying each individual non-equilibrium behaviors, we lack a general understanding of how the microscopic breaking of detailed balance translates to large-scale cellular behaviors and materials properties.Using the tools of non-equilibrium thermodynamics, this thesis examines this question by measuring energy dissipation during dynamical and mechanical phase transitions seen in experiments, simulations, and theoretical models of biological materials. We choose the actomyosin cytoskeleton, a network composed of polymeric proteins (actin) that are driven away from thermodynamic equilibrium by the activity of molecular motors (myosin), as our model system. Actomyosin contains the three types of non-equilibrium driving we will focus on: force generation, non-equilibrium binding kinetics, and active (dis)assembly. At the subcellular level, analysis of actin filament motions in experiments shows that energy dissipated through bending controls the transition between stable and contractile steady states. Using simulations, we show that non-equilibrium binding kinetics of molecular motors controls a fluid-solid phase transition characterized by thermodynamic quantities with opposite symmetries under time-reversal. At the cellular level, we develop new tools for measuring irreversibility in spatiotemporal dynamics to analyze the energetic costs of oscillations and synchronization of a model biochemical oscillator inspired by (dis)assembly driven actomyosin dynamics. Throughout this thesis, we show that a cell’s distance from equilibrium, quantified by energy dissipation, tunes its mechanical properties and dynamics. This provides a framework to unify disparate biological function through the lens of non-equilibrium thermodynamics