Explore the vast range of titles available.

Mechanics has been a central focus of physical biology in the past decade. In comparison, how cells manage their size is less understood. Here, we show that a parameter central to both the physics and the physiology of the cell, its volume, depends on a mechano-osmotic coupling. We found that cells change their volume depending on the rate at which they change shape, when they spontaneously spread or when they are externally deformed. Cells undergo slow deformation at constant volume, while fast deformation leads to volume loss. We propose a mechanosensitive pump and leak model to explain this phenomenon. Our model and experiments suggest that volume modulation depends on the state of the actin cortex and the coupling of ion fluxes to membrane tension. This mechano-osmotic coupling defines a membrane tension homeostasis module constantly at work in cells, causing volume fluctuations associated with fast cell shape changes, with potential consequences on cellular physiology.

Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, Bin1 and caveolae composed of caveolin-3 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Bin1 expression lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations results in defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1 dysfunction.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

Recent super-resolution microscopy studies have unveiled a periodic scaffold of actin rings regularly spaced by spectrins under the plasma membrane of axons. However, ultrastructural details are unknown, limiting a molecular and mechanistic understanding of these enigmatic structures. Here, we combine platinum-replica electron and optical super-resolution microscopy to investigate the cortical cytoskeleton of axons at the ultrastructural level. Immunogold labeling and correlative super-resolution/electron microscopy allow us to unambiguously resolve actin rings as braids made of two long, intertwined actin filaments connected by a dense mesh of aligned spectrins. This molecular arrangement contrasts with the currently assumed model of actin rings made of short, capped actin filaments. Along the proximal axon, we resolved the presence of phospho-myosin light chain and the scaffold connection with microtubules via ankyrin G. We propose that braided rings explain the observed stability of the actin-spectrin scaffold and ultimately participate in preserving the axon integrity. The ultrastructural details of the periodic scaffold of actin rings under the plasma membrane of axons remain unknown. Here, the authors combine platinum-replica electron and optical super-resolution microscopy and resolve actin rings as braids made of two long, intertwined actin filaments connected by a dense mesh of aligned spectrins.

Mechanotransduction is a process by which cells sense the mechanical properties of their surrounding environment and adapt accordingly to perform cellular functions such as adhesion, migration and differentiation. Integrin-mediated focal adhesions are major sites of mechanotransduction and their connection with the actomyosin network is crucial for mechanosensing as well as for the generation and transmission of forces onto the substrate. Despite having emerged as major regulators of cell adhesion and migration, the contribution of microtubules to mechanotransduction still remains elusive. Here, we show that talin- and actomyosin-dependent mechanosensing of substrate rigidity controls microtubule acetylation (a tubulin post-translational modification) by promoting the recruitment of α-tubulin acetyltransferase 1 (αTAT1) to focal adhesions. Microtubule acetylation tunes the mechanosensitivity of focal adhesions and Yes-associated protein (YAP) translocation. Microtubule acetylation, in turn, promotes the release of the guanine nucleotide exchange factor GEF-H1 from microtubules to activate RhoA, actomyosin contractility and traction forces. Our results reveal a fundamental crosstalk between microtubules and actin in mechanotransduction that contributes to mechanosensitive cell adhesion and migration. Substrate-rigidity-dependent microtubule acetylation is now shown to be triggered by mechanosensing at focal adhesions, and in turn controls the mechanosensitivity of Yes-associated protein (YAP) translocation, focal adhesion distribution, actomyosin contractility and cell migration.

Unraveling the mechanisms that govern the formation and function of invadopodia is essential towards the prevention of cancer spread. Here, we characterize the ultrastructural organization, dynamics and mechanical properties of collagenotytic invadopodia forming at the interface between breast cancer cells and a physiologic fibrillary type I collagen matrix. Our study highlights an uncovered role for MT1-MMP in directing invadopodia assembly independent of its proteolytic activity. Electron microscopy analysis reveals a polymerized Arp2/3 actin network at the concave side of the curved invadopodia in association with the collagen fibers. Actin polymerization is shown to produce pushing forces that repel the confining matrix fibers, and requires MT1-MMP matrix-degradative activity to widen the matrix pores and generate the invasive pathway. A theoretical model is proposed whereby pushing forces result from actin assembly and frictional forces in the actin meshwork due to the curved geometry of the matrix fibers that counterbalance resisting forces by the collagen fibers. The mechanism of force production by invadopodia is unclear. Here, the authors show that cell surface MT1-MMP when in contact with collagen, induces Arp2/3 branched actin polymerisation on the concave side of invadopodia, which generates a pushing force along with collagen cleavage by MT1-MMP to invade.

The hallmark of non-selective autophagy is the formation of cup-shaped phagophores that capture bulk cytoplasm. The process is accompanied by the conjugation of LC3B to phagophores by an E3 ligase complex comprising ATG12–ATG5 and ATG16L1. Here we combined two complementary reconstitution approaches to reveal the function of LC3B and its ligase complex during phagophore expansion. We found that LC3B forms together with ATG12–ATG5–ATG16L1 a membrane coat that remodels flat membranes into cups that closely resemble phagophores. Mechanistically, we revealed that cup formation strictly depends on a close collaboration between LC3B and ATG16L1. Moreover, only LC3B, but no other member of the ATG8 protein family, promotes cup formation. ATG16L1 truncates that lacked the C-terminal membrane binding domain catalyzed LC3B lipidation but failed to assemble coats, did not promote cup formation and inhibited the biogenesis of non-selective autophagosomes. Our results thus demonstrate that ATG16L1 and LC3B induce and stabilize the characteristic cup-like shape of phagophores. Autophagy degrades cellular waste by engulfing it in phagophore membranes and delivering it to lysosomes for degradation. Here Mohan and colleagues identified a type of membrane coat that assembles on phagophores to guide their expansion.

“Frustrated” coated pits associate with collagen fibers and appear to promote cell adhesion in three-dimensional collagen networks. Clathrin-coated pits are well known to be involved in receptor-mediated endocytosis. Independent of their role in endocytosis, Elkhatib et al. observed that clathrin-coated structures strongly accumulated along collagen fibers in migrating cells. Clathrin-coated structures assembled on and then partially wrapped around and pinched the fibers. In a three-dimensional (3D) network, this mechanism provided multiple anchoring points along cellular protrusions. In the absence of clathrin-coated structures, protrusions were shorter and migration was impaired. This mode of adhesion may cooperate with classical focal adhesions to help cancer cells move in a 3D environment. Science , this issue p. eaal4713 Migrating cells often use focal adhesions in order to move. Focal adhesions are less prominent in cells migrating in three-dimensional (3D) as compared with 2D environments. We looked for alternative adhesion structures supporting cell migration. We analyzed the dynamics of clathrin-coated pits in cells migrating in a 3D environment of collagen fibers. Both topological cues and local engagement of integrins triggered the accumulation of clathrin-coated structures on fibers. Clathrin/adaptor protein 2 (AP-2) lattices pinched collagen fibers by adopting a tube-like morphology and regulated adhesion to fibers in an endocytosis-independent manner. During migration, tubular clathrin/AP-2 lattices stabilized cellular protrusions by providing anchoring points to collagen fibers. Thus, tubular clathrin/AP-2 lattices promote cell adhesion that, in coordination with focal adhesions, supports cell migration in 3D.

Extracellular Vesicles (EVs) are natural communication vectors involved in many physiological processes. Significant efforts have aimed to harness EVs for therapeutic delivery, with key challenges being control and enhancement of EV-mediated delivery steps. We and others have developed strategies to improve cargo loading and enhance EV content delivery using viral or non-viral fusogens. However, few targeting solutions have been proposed. Here, we present a versatile system for precise EV targeting to specific cell types, enabling quantitative assessment of targeting efficiency via luminescence and fluorescence. EVs are genetically engineered to express a chimeric adapter protein anchored by a glycosylphosphatidylinositol (GPI) anchor. This protein includes a fluorescent/luminescent domain for detection and a streptavidin domain recruit biotinylated antibodies or ligands specific to cell-surface antigens or receptors. We validated this platform with three different combinations of ligand/target cells, demonstrating up to 40-fold increase in EV uptake. This adaptable system promises to provide a comprehensive solution for targeted therapeutic delivery using EV-based vectors.

Sarcomeres are the basic contractile units within cardiac myocytes, and the collective shortening of sarcomeres aligned along myofibrils generates the force driving the heartbeat. The alignment of the individual sarcomeres is important for proper force generation, and misaligned sarcomeres are associated with diseases, including cardiomyopathies and COVID-19. The actin bundling protein, α-actinin-2, localizes to the ‘Z-Bodies” of sarcomere precursors and the ‘Z-Lines’ of sarcomeres, and has been used previously to assess sarcomere assembly and maintenance. Previous measurements of α-actinin-2 organization have been largely accomplished manually, which is time-consuming and has hampered research progress. Here, we introduce sarcApp, an image analysis tool that quantifies several components of the cardiac sarcomere and their alignment in muscle cells and tissue. We first developed sarcApp to utilize deep learning-based segmentation and real space quantification to measure α-actinin-2 structures and determine the organization of both precursors and sarcomeres/myofibrils. We then expanded sarcApp to analyze ‘M-Lines’ using the localization of myomesin and a protein that connects the Z-Lines to the M-Line (titin). sarcApp produces 33 distinct measurements per cell and 24 per myofibril that allow for precise quantification of changes in sarcomeres, myofibrils, and their precursors. We validated this system with perturbations to sarcomere assembly. We found perturbations that affected Z-Lines and M-Lines differently, suggesting that they may be regulated independently during sarcomere assembly.