Matrix rigidity sensing regulates a large variety of cellular processes and has important implications for tissue development and disease. However, how cells probe matrix rigidity, and hence respond to it, remains unclear. Here, we show that rigidity sensing and adaptation emerge naturally from actin cytoskeleton remodelling. Our in vitro experiments and theoretical modelling demonstrate a biphasic rheology of the actin cytoskeleton, which transitions from fluid on soft substrates to solid on stiffer ones. Furthermore, we find that increasing substrate stiffness correlates with the emergence of an orientational order in actin stress fibres, which exhibit an isotropic to nematic transition that we characterize quantitatively in the framework of active matter theory. These findings imply mechanisms mediated by a large-scale reinforcement of actin structures under stress, which could be the mechanical drivers of substrate stiffness-dependent cell shape changes and cell polarity.

Membrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly.

Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1(mut/mut) patient with healthy MALT1(+/mut) family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway-first promoting activation via the CBM--then triggering HOIL1-dependent negative-feedback termination, preventing reactivation.

When B cells encounter membrane-bound antigens, the formation and coalescence of B cell antigen receptor (BCR) microclusters amplifies BCR signaling. The ability of B cells to probe the surface of antigen-presenting cells (APCs) and respond to APC-bound antigens requires remodeling of the actin cytoskeleton. Initial BCR signaling stimulates actin-related protein (Arp) 2/3 complex-dependent actin polymerization, which drives B cell spreading as well as the centripetal movement and coalescence of BCR microclusters at the B cell-APC synapse. Sustained actin polymerization depends on concomitant actin filament depolymerization, which enables the recycling of actin monomers and Arp2/3 complexes. Cofilin-mediated severing of actin filaments is a rate-limiting step in the morphological changes that occur during immune synapse formation. Hence, regulators of cofilin activity such as WD repeat-containing protein 1 (Wdr1), LIM domain kinase (LIMK), and coactosin-like 1 (Cotl1) may also be essential for actin-dependent processes in B cells. Wdr1 enhances cofilin-mediated actin disassembly. Conversely, Cotl1 competes with cofilin for binding to actin and LIMK phosphorylates cofilin and prevents it from binding to actin filaments. We now show that Wdr1 and LIMK have distinct roles in BCR-induced assembly of the peripheral actin structures that drive B cell spreading, and that cofilin, Wdr1, and LIMK all contribute to the actin-dependent amplification of BCR signaling at the immune synapse. Depleting Cotl1 had no effect on these processes. Thus, the Wdr1-LIMK-cofilin axis is critical for BCR-induced actin remodeling and for B cell responses to APC-bound antigens.

Signaling by the B cell antigen receptor (BCR) initiates actin remodeling. The assembly of branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, allowing B cells to form membrane protrusions that can scan the surface of antigen-presenting cells (APCs). The resulting Arp2/3 complex-dependent actin retrograde flow promotes the centripetal movement and progressive coalescence of BCR microclusters, which amplifies BCR signaling. Glia maturation factor γ (GMFγ) is an actin disassembly-protein that releases Arp2/3 complex-nucleated actin filaments from actin networks. By doing so, GMFγ could either oppose the actions of the Arp2/3 complex or support Arp2/3 complex-nucleated actin polymerization by contributing to the recycling of actin monomers and Arp2/3 complexes. We now show that reducing the levels of GMFγ in human B cell lines via transfection with a specific siRNA impairs the ability of B cells to spread on antigen-coated surfaces, decreases the velocity of actin retrograde flow, diminishes the coalescence of BCR microclusters into a central cluster at the B cell-APC contact site, and decreases APC-induced BCR signaling. These effects of depleting GMFγ are similar to what occurs when the Arp2/3 complex is inhibited. This suggests that GMFγ cooperates with the Arp2/3 complex to support BCR-induced actin remodeling and amplify BCR signaling at the immune synapse.

BAR domain proteins contribute to membrane deformation in diverse cellular processes. The inverted-BAR (I-BAR) protein IRSp53, for instance, is found on the inner leaflet of the tubular membrane of filopodia; however its role in the formation of these structures is incompletely understood. Here we develop an original assay in which proteins are encapsulated in giant unilamellar vesicles connected to membrane nanotubes. Our results demonstrate that I-BAR dimers sense negative membrane curvature. Experiment and theory reveal that the I-BAR displays a non-monotonic sorting with curvature, and expands the tube at high imposed tension while constricting it at low tension. Strikingly, at low protein density and tension, protein-rich domains appear along the tube. This peculiar behaviour is due to the shallow intrinsic curvature of I-BAR dimers. It allows constriction of weakly curved membranes coupled to local protein enrichment at biologically relevant conditions. This might explain how IRSp53 contributes in vivo to the initiation of filopodia.

Axons span extreme distances and are subject to significant stretch deformations during limb movements or sudden head movements, especially during impacts. Yet, axon biomechanics, and its relation to the ultrastructure that allows axons to withstand mechanical stress, is poorly understood. Using a custom developed force apparatus, we demonstrate that chick dorsal root ganglion axons exhibit a tension buffering or strain-softening response, where its steady state elastic modulus decreases with increasing strain. We then explore the contributions from the various cytoskeletal components of the axon to show that the recently discovered membrane-associated actin-spectrin scaffold plays a prominent mechanical role. Finally, using a theoretical model, we argue that the actin-spectrin skeleton acts as an axonal tension buffer by reversibly unfolding repeat domains of the spectrin tetramers to release excess mechanical stress. Our results revise the current viewpoint that microtubules and their associated proteins are the only significant load-bearing elements in axons.

Cell migration over heterogeneous substrates during wound healing or morphogenetic processes leads to shape changes driven by different organizations of the actin cytoskeleton and by functional changes including lamellipodial protrusions and contractile actin cables. Cells distinguish between cell-sized positive and negative curvatures in their physical environment by forming protrusions at positive ones and actin cables at negative ones; however, the cellular mechanisms remain unclear. Here, we report that concave edges promote polarized actin structures with actin flow directed towards the cell edge, in contrast to well-documented retrograde flow at convex edges. Anterograde flow and contractility induce a tension anisotropy gradient. A polarized actin network is formed, accompanied by a local polymerization-depolymerization gradient, together with leading-edge contractile actin cables in the front. These cables extend onto non-adherent regions while still maintaining contact with the substrate through focal adhesions. The contraction and dynamic reorganization of this actin structure allows forward movements enabling cell migration over non-adherent regions on the substrate. These versatile functional structures may help cells sense and navigate their environment by adapting to external geometric and mechanical cues.

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.

Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole-a protuberance of the zygote's vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces.