SUMMARY Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of “network connectivity” and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions.

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The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model.

Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations.

Cell and tissue morphogenesis depend on the production and spatial organization of tensional forces in the actin cytoskeleton. Actin network architecture is made of distinct modules characterized by specific filament organizations. The assembly of these modules are well described, but their integration in a cellular network is less understood. Here, we investigated the mechanism regulating the interplay between network architecture and the geometry of the extracellular environment of the cell. We found that α-actinin, a filament crosslinker, is essential for network symmetry to be consistent with extracellular microenvironment symmetry. It is required for the interconnection of transverse arcs with radial fibres to ensure an appropriate balance between forces at cell adhesions and across the actin network. Furthermore, this connectivity appeared necessary for the ability of the cell to integrate and to adapt to complex patterns of extracellular cues as they migrate. Our study has unveiled a role of actin filament crosslinking in the spatial integration of mechanical forces that ensures the adaptation of intracellular symmetry axes in accordance with the geometry of extracellular cues. This article has an associated First Person interview with the first author of the paper.

TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. TRIOBP has three isoforms, named TRIOBP-1, TRIOBP-4, and TRIOBP-5. In vitro, TRIOBP isoform 4 (TRI-OBP-4) forms dense F-actin bundles resembling the inner ear hair cell rootlet structure. Deletion of TRIOBP isoforms 4 and 5 leads to hearing loss in mice due to the absence of stereocilia rootlets. The mechanism of actin bundle formation by TRIOBP is not fully understood. The amino acid sequences of TRI-OBP isoforms 4 and 5 contain two repeated motifs, referred to here as R1 and R2. Recent our study demonstrated that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific. Structural analysis of TRIOBP by amino acid sequence showed ID proteins. Thus the second structure of TRIOBP may not have. To investigate the structural property of TRIOBP-4, we analyzed the structure of TRIOBP-4 by using circular dichroism, dynamic light scattering, and fluorescence correlation spectroscopy. Our analysis show that TRIOBP has a beta-sheet, but not alpha-helix. To investigate the structure of tight F-actin bundle structure with TRIOBP, we analyzed 3-D structure of the bundle by using 3-D image analysis from transmitted electron microscope images.

Cell and tissue morphogenesis depend on the production and spatial organization of tensional forces in the actin cytoskeleton. Actin network architecture is complex because it is made of distinct modules in which filaments adopt a variety of organizations. The assembly and dynamics of these modules is well described but the self-organisation rules directing the global network architecture are much less understood. Here we investigated the mechanism regulating the interplay between network architecture and the geometry of cell's extracellular environment. We found that α-actinin, a filament crosslinker, is essential for network symmetry to be consistent with extracellular microenvironment symmetry. It appeared to be required for the interconnection of transverse arcs with radial fibres to ensure an appropriate balance between forces at cell adhesions and across the entire actin network. Furthermore, the connectivity of the actin network appeared necessary for the cell ability to integrate and adapt to complex patterns of extracellular cues as they migrate. Altogether, our study has unveiled a role of actin-filament crosslinking in the physical integration of mechanical forces throughout the entire cell, and the role of this integration in the establishment and adaptation of intracellular symmetry axes in accordance with the geometry of extracellular cues.

Cytoskeletal filaments assemble into dense parallel, antiparallel, or disordered networks, providing a complex environment for active cargo transport and positioning by molecular motors. The interplay between the network architecture and intrinsic motor properties clearly affects transport properties but remains poorly understood. Here, by using surface micropatterns of actin polymerization, we investigate stochastic transport properties of colloidal beads in antiparallel networks of overlapping actin filaments. We found that 200-nm beads coated with myosin Va motors displayed directed movements toward positions where the net polarity of the actin network vanished, accumulating there. The bead distribution was dictated by the spatial profiles of local bead velocity and diffusion coefficient, indicating that a diffusion-drift process was at work. Remarkably, beads coated with heavy–mero-myosin II motors showed a similar behavior. However, although velocity gradients were steeper with myosin II, the much larger bead diffusion observed with this motor resulted in less precise positioning. Our observations are well described by a 3-state model, in which active beads locally sense the net polarity of the network by frequently detaching from and reattaching to the filaments. A stochastic sequence of processive runs and diffusive searches results in a biased random walk. The precision of bead positioning is set by the gradient of net actin polarity in the network and by the run length of the cargo in an attached state. Our results unveiled physical rules for cargo transport and positioning in networks of mixed polarity.