Cells change direction of migration by sensing rigidity of environment and traction force, yet its underlying mechanism is unclear. Here, we show that tip actin barbed ends serve as an active "force sensor" at the leading edge. We established a method to visualize intracellular single-molecule fluorescent actin through an elastic culture substrate. We found that immediately after cell edge stretch, actin assembly increased specifically at the lamellipodium tip. The rate of actin assembly increased with increasing stretch speed. Furthermore, tip actin polymerization remained elevated at the subsequent hold step, which was accompanied by a decrease in the load on the tip barbed ends. Stretch-induced tip actin polymerization was still observed without either the WAVE complex or Ena/VASP proteins. The observed relationships between forces and tip actin polymerization are consistent with a force-velocity relationship as predicted by the Brownian ratchet mechanism. Stretch caused extra membrane protrusion with respect to the stretched substrate and increased local tip polymerization by >5% of total cellular actin in 30 s. Our data reveal that augmentation of lamellipodium tip actin assembly is directly coupled to the load decrease, which may serve as a force sensor for directed cell protrusion.

How mechanical stress applied to the actin network modifies actin turnover has attracted considerable attention. Actomyosin exerts the major force on the actin network, which has been implicated in actin stability regulation. However, direct monitoring of immediate changes in F-actin stability on alteration of actomyosin contraction has not been achieved. Here we reexamine myosin regulation of actin stability by using single-molecule speckle analysis of actin. To avoid possible errors attributable to actin-binding probes, we employed DyLight-labeled actin that distributes identical to F-actin in lamellipodia. We performed time-resolved analysis of the effect of blebbistatin on actin turnover. Blebbistatin enhanced actin disassembly in lamellipodia of fish keratocytes and lamellar of Xenopus XTC cells at an early stage of the inhibition, indicating that actomyosin contraction stabilizes cellular F-actin. In addition, our data show a previously unrecognized relationship between the actin network-driving force and the actin turnover rates in lamellipodia. These findings point to the power of direct viewing of molecular behavior in elucidating force regulation of actin filament turnover.

Fluorescent markers that bind endogenous target proteins are frequently employed for quantitative live-cell imaging. To visualize the actin cytoskeleton in live cells, several actin-binding probes have been widely used. Among them, Lifeact is the most popular probe with ideal properties, including fast exchangeable binding kinetics. Because of its fast kinetics, Lifeact is generally believed to distribute evenly throughout cellular actin structures. In this study, however, we demonstrate misdistribution of Lifeact toward the rear of lamellipodia where actin filaments continuously move inward along the retrograde flow. Similarly, phalloidin showed biased misdistribution toward the rear of lamellipodia in live cells. We show evidence of convection-induced misdistribution of actin probes by both experimental data and physical models. Our findings warn about the potential error arising from the use of target-binding probes in quantitative live imaging.

Animal cells that spread onto a surface often rely on actin-rich lamellipodial extensions to execute protrusion. Many cell types recently adhered on a two-dimensional substrate exhibit protrusion and retraction of their lamellipodia, even though the cell is not translating. Travelling waves of protrusion have also been observed, similar to those observed in crawling cells. These regular patterns of protrusion and retraction allow quantitative analysis for comparison to mathematical models. The periodic fluctuations in leading edge position of XTC cells have been linked to excitable actin dynamics using a one-dimensional model of actin dynamics, as a function of arc-length along the cell. In this work we extend this earlier model of actin dynamics into two dimensions (along the arc-length and radial directions of the cell) and include a model membrane that protrudes and retracts in response to the changing number of free barbed ends of actin filaments near the membrane. We show that if the polymerization rate at the barbed ends changes in response to changes in their local concentration at the leading edge and/or the opposing force from the cell membrane, the model can reproduce the patterns of membrane protrusion and retraction seen in experiment. We investigate both Brownian ratchet and switch-like force-velocity relationships between the membrane load forces and actin polymerization rate. The switch-like polymerization dynamics recover the observed patterns of protrusion and retraction as well as the fluctuations in F-actin concentration profiles. The model generates predictions for the behavior of cells after local membrane tension perturbations.

Microtubule (MT) networks play key roles in cell division, intracellular transport, and cell motility. These functions of MT networks occur through interactions between MTs and various associated proteins, notably motor proteins that bundle and slide MTs. Our objective in this study was to address the question of how motors determine the nature of MT networks. We conducted in vitro assays using homotetrameric kinesin Eg5, a motor protein involved in the formation and maintenance of the mitotic spindle. The mixing of Eg5 and MTs produced a range of spatiotemporal dynamics depending on the motor/filament ratio. Low motor/filament ratios produced globally connected static MT networks with sparsely distributed contractile active nodes (motor-accumulating points with radially extending MTs). Increasing the motor/filament ratio facilitated the linking of contractile active nodes and led to a global contraction of the network. When the motor/filament ratio was further increased, densely distributed active nodes formed local clusters and segmented the network into pieces with their strong contractile forces. Altering the properties of the motor through the use of chimeric Eg5, which has kinesin-1 heads, resulted in the generation of many isolated asters. These results suggest that the spatial distribution of contractile active nodes determines the dynamics of MT-motor networks. We then developed a coarse-grained model of MT-motor networks and identified two essential features for reproducing the experimentally observed patterns: an accumulation of motors that form the active nodes necessary to generate contractile forces, and a nonlinear dependency of contractile force on motor densities. Our model also enabled us to characterize the mechanical properties of the contractile network. Our study provides insight into how local motor-MT interactions generate the spatiotemporal dynamics of macroscopic network structures.

Multiciliated cells (MCCs) promote fluid flow through coordinated ciliary beating, which requires properly organized basal bodies (BBs). Airway MCCs have large numbers of BBs, which are uniformly oriented and, as we show here, align linearly. The mechanism for BB alignment is unexplored. To study this mechanism, we developed a long-term and high-resolution live-imaging system and used it to observe green fluorescent protein-centrin2-labeled BBs in cultured mouse tracheal MCCs. During MCC differentiation, the BB array adopted four stereotypical patterns, from a clustering "floret" pattern to the linear "alignment." This alignment process was correlated with BB orientations, revealed by double immunostaining for BBs and their asymmetrically associated basal feet (BF). The BB alignment was disrupted by disturbing apical microtubules with nocodazole and by a BF-depleting Odf2 mutation. We constructed a theoretical model, which indicated that the apical cytoskeleton, acting like a viscoelastic fluid, provides a self-organizing mechanism in tracheal MCCs to align BBs linearly for mucociliary transport.

Microtubules (MTs) in cells form hierarchical network structure and play important roles in various cellular activities. They emerge as the result of the interactions between MTs and various types of MT-binding proteins, and dynamically changes their structures during cell cycles and according to physiological roles of the cell. To obtain an integrative perspective of the MT networks, we performed reconstruction of the MT networks by using MTs and bipolar kinesin, Eg5. Human Eg5 expressed by HEK293 cells moved processively on an MT at ca. 10 nm/s and had ability to bundle MTs in parallel and anti-parallel manners. When MTs and Eg5 were mixed above the critical concentrations of 1 μM and 1 nM, respectively, the mixture generated various types of network architecture depending on the kinesin-MT mixing ratios. At the low ratios (<1:350), the system generates static network staying in the same architecture for >20-hours. Increase to 1:250 made the network contractile with two distinct phases: early slow contraction followed by the abrupt shortening and rupture of the network. Further increase to 1:40, MT networks rapidly contracted into large aggregate immediately after addition of ATP. In contrast, modulation of motor property by using recombinant Eg5 with four heads replaced by faster kinesin (KIF5B, 140 nm/s) heads led to generate a large number of asters. A coarse-grained mathematical model reproduced the network dynamics experimentally observed and delivered a comprehensive picture on the range and diversity of Eg5-MT networks at different spatiotemporal scales. We also calculated the elastic energy stored in the networks, and found that it is accumulated through early contractions in the active network, and can be released into mechanical work during rupturing. This feature might play some roles in the initiation of cell locomotion. (Supported by CREST, JST).

In both randomly moving Dictyostelium and mammalian cells, phosphatidylinositol (3,4,5)-trisphosphate and F-actin are known to propagate as waves at the membrane and act to push out the protruding edge. To date, however, the relationship between the wave geometry and the patterns of amoeboid shape change remains elusive. Here, by using phase map analysis, we show that morphology dynamics of randomly moving Dictyostelium discoideum cells can be characterized by the number, topology, and position of spatial phase singularities, i.e., points that represent organizing centers of rotating waves. A single isolated singularity near the cellular edge induced a rotational protrusion, whereas a pair of singularities supported a symmetric extension. These singularities appeared by strong phase resetting due to de novo nucleation at the back of preexisting waves. Analysis of a theoretical model indicated excitability of the system that is governed by positive feedback from phosphatidylinositol (3,4,5)-trisphosphate to PI3-kinase activation, and we showed experimentally that this requires F-actin. Furthermore, by incorporating membrane deformation into the model, we demonstrated that geometries of competing waves explain most of the observed semiperiodic changes in amoeboid morphology.

Although folding reaetions and fluctuations ofsmall and single domain proteins have been relatively well understood by now, much less is c[ear for folding and functiona] dynamics oflarger and mu]ti-dornain proteins.Here, we present our recent computational work on three case studies of ]arge andlor multi-demain proteins.FiTst, we discuss both folding and conformational dynamies of adenylate kinase, a mode] allosteric protein, which shows rather rich behaviors.Second, we discuss fo]ding-coupled docking in a sp]it GFP.Fina]]y, importance of co-trarislational folding is addressed for a three-domain pretein suf] by computersirnulations. lsF-ol vamopyneefftu :

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