Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feed back on assembly. By manipulating membrane tension and Arp2/3 complex activity, we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions.
Abstract Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.
Abstract Cell migration is a hallmark out-of-equilibrium process in biology. In addition to persistent self-propelled motion, many cells display remarkable adaptive behaviors when they navigate complex environments within the body. Combining theory and experiments, we identify a curvature-sensing mechanism underlying obstacle avoidance in immune-like cells. The genetic perturbation of this machinery leads to a reduced capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. We propose that the active polymerization of the actin cytoskeleton at the advancing edge of migrating cells is locally inhibited by the curvature-sensitive BAR protein Snx33 in regions with inward plasma membrane curvature. This coupling between actin and membrane dynamics leads to a mechanochemical instability that generates complex protrusive patterns at the cellular front. Adaptive motility thus arises from two simultaneous curvature-dependent effects, i) the specific reduction of propulsion in regions where external objects deform the plasma membrane and ii) the intrinsic patterning capacity due to the membrane-actin coupling that promotes spontaneous changes in the cell’s protrusions. Our results show how cells utilize actin- and plasma membrane biophysics to sense their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment.
Cell shape and motility are primarily controlled by cellular mechanics. The attachment of the plasma membrane to the underlying actomyosin cortex has been proposed to be important for cellular processes involving membrane deformation. However, little is known about the actual function of membrane-to-cortex attachment (MCA) in cell protrusion formation and migration, in particular in the context of the developing embryo. Here, we use a multidisciplinary approach to study MCA in zebrafish mesoderm and endoderm (mesendoderm) germ layer progenitor cells, which migrate using a combination of different protrusion types, namely, lamellipodia, filopodia, and blebs, during zebrafish gastrulation. By interfering with the activity of molecules linking the cortex to the membrane and measuring resulting changes in MCA by atomic force microscopy, we show that reducing MCA in mesendoderm progenitors increases the proportion of cellular blebs and reduces the directionality of cell migration. We propose that MCA is a key parameter controlling the relative proportions of different cell protrusion types in mesendoderm progenitors, and thus is key in controlling directed migration during gastrulation.
SUMMARY To migrate, divide, and change shape, cells must regulate the mechanics of their periphery. The cell surface is a complex structure that consists of a thin, contractile cortical actin network tethered to the plasma membrane by specialized membrane-to-cortex attachment (MCA) proteins. This active and constantly fluctuating system maintains a delicate mechanochemical state which permits spontaneous polarization and shape change when needed. Combining in silico , in vitro , and in vivo experiments we show how membrane viscosity and MCA protein length regulate cortical dynamics. We reveal a novel mechanism whereby caging of linker proteins in the actin cortex allows for the amplification of small changes in these key parameters, leading to major alterations of cortical contractility. In cells, this mechanism alone gives rise to symmetry breaking phenomena, suggesting that local changes in lipid composition, in combination with the choice of MCA proteins, contribute to the regulation of cellular morphogenesis and function.
In this work, we quantify the mechanical properties of the extra-cellular matrix (ECM) in live zebrafish using Brillouin microscopy. Optimization of the imaging conditions and parameters, combined with careful spectral analysis, allows us to resolve the thin ECM and distinguish its Brillouin frequency shift, a proxy for mechanical properties, from the surrounding tissue. High-resolution mechanical mapping further enables the direct measurement of the thickness of the ECM label-free and in-vivo. We find the ECM to be ~500 nm thick, and in very good agreement with electron microscopy quantification. Our results open the door for future studies that aim to investigate the role of ECM mechanics for zebrafish morphogenesis and axis elongation.
During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MS ALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell–derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell–derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.
