Sorting of lipids and proteins is a key process allowing eukaryotic cells to execute efficient and accurate intracellular transport and to maintain membrane homeostasis. It occurs during the formation of highly curved transport intermediates that shuttle between cell compartments. Protein sorting is reasonably well described, but lipid sorting is much less understood. Lipid sorting has been proposed to be mediated by a physical mechanism based on the coupling between membrane composition and high curvature of the transport intermediates. To test this hypothesis, we have performed a combination of fluorescence and force measurements on membrane tubes of controlled diameters pulled from giant unilamellar vesicles. A model based on membrane elasticity and nonideal solution theory has also been developed to explain our results. We quantitatively show, using 2 independent approaches, that a difference in lipid composition can build up between a curved and a noncurved membrane. Importantly, and consistent with our theory, lipid sorting occurs only if the system is close to a demixing point. Remarkably, this process is amplified when even a low fraction of lipids is clustered upon cholera toxin binding. This can be explained by the reduction of the entropic penalty of lipid sorting when some lipids are bound together by the toxin. Our results show that curvature-induced lipid sorting results from the collective behavior of lipids and is even amplified in the presence of lipid-clustering proteins. In addition, they suggest a generic mechanism by which proteins can facilitate lipid segregation in vivo. cholera toxin ͉ giant unilamellar vesicle ͉ membrane curvature ͉ membrane nanotube ͉ optical tweezers L ipids and proteins are not homogeneously distributed among cell membranes (1). How intracellular trafficking maintains the composition differences between the various membrane compartments of the cell is still poorly understood. In many cases, trafficking intermediates are tubular structures with radii typically in the range of 50 nm (2). It has been suggested that lipid sorting could be mediated by a physical mechanism based on the coupling between membrane composition and the high curvature of these intermediates (refs. 3-6; and see refs. 7-12 for theoretical papers) and on solid substrate topography (13). Added lipid or protein dyes have been shown to be sorted into or out of highly curved regions but without a systematic study of the influence of the underlying membrane composition on the sorting process (3,14). It is crucial from a biological point of view to know whether the native membrane lipids themselves can be sorted by curvature and if so, whether or not the mechanism is robust for variable membrane compositions. In fact, a recent theoretical paper predicts a weak effect of curvature on membrane composition because of the overwhelming cost of mixing entropy, suggesting that curvature does not significantly contribute to sorting (15). In contrast, we demonstrate in the present work that cooperative behavior be...
We present a detailed analysis of the micropipet experiments recently reported by J-B. Manneville et al., [Phys. Rev. Lett. 82, 4356 (1999)], including a derivation of the expected behavior of the membrane tension as a function of the areal strain in the case of an active membrane, i.e., containing a nonequilibrium noise source. We give a general expression, which takes into account the effect of active centers both directly on the membrane and on the embedding fluid dynamics, keeping track of the coupling between the density of active centers and the membrane curvature. The data of the micropipet experiments are well reproduced by our expressions. In particular, we show that a natural choice of the parameters quantifying the strength of the active noise explains both the large amplitude of the observed effects and its remarkable insensitivity to the active-center density in the investigated range.
Cell polarization is essential in a wide range of biological processes such as morphogenesis, asymmetric division, and directed migration. In this study, we show that two tumor suppressor proteins, adenomatous polyposis coli (APC) and Dlg1-SAP97, are required for the polarization of migrating astrocytes. Activation of the Par6–PKCζ complex by Cdc42 at the leading edge of migrating cells promotes both the localized association of APC with microtubule plus ends and the assembly of Dlg-containing puncta in the plasma membrane. Biochemical analysis and total internal reflection fluorescence microscopy reveal that the subsequent physical interaction between APC and Dlg1 is required for polarization of the microtubule cytoskeleton.
The generation of membrane curvature in intracellular traffic involves many proteins that can curve lipid bilayers. Among these, dynamin-like proteins were shown to deform membranes into tubules, and thus far are the only proteins known to mechanically drive membrane fission. Because dynamin forms a helical coat circling a membrane tubule, its polymerization is thought to be responsible for this membrane deformation. Here we show that the force generated by dynamin polymerization, 18 pN, is sufficient to deform membranes yet can still be counteracted by high membrane tension. Importantly, we observe that at low dynamin concentration, polymer nucleation strongly depends on membrane curvature. This suggests that dynamin may be precisely recruited to membrane buds' necks because of their high curvature. To understand this curvature dependence, we developed a theory based on the competition between dynamin polymerization and membrane mechanical deformation. This curvature control of dynamin polymerization is predicted for a specific range of concentrations (∼0.1-10 μM), which corresponds to our measurements. More generally, we expect that any protein that binds or self-assembles onto membranes in a curvature-coupled way should behave in a qualitatively similar manner, but with its own specific range of concentration.force | nucleation | fission | endocytosis | dynamin-like proteins M embrane remodeling is an essential task of proteins involved in membrane traffic (1, 2). Dynamin is a large GTPase that has been shown to polymerize into a helical collar at the neck of endocytic buds (3), where it subsequently plays a key role in the formation of endocytic vesicles through fission (4-8). This function is fundamental, as the knockout of the dynamin neuronal isoform leads to striking defects in synapse organization and results in a strong dysfunction of neuronal activity (9). The recruitment of dynamin to endocytic buds is thought to depend on the local synthesis of phosphatidylinositol(4,5)bisphosphate (PIP 2 ), as dynamin has a PIP 2 binding pleckstrin homology (PH) domain (10). Dynamin is recruited late in clathrin-coated vesicle formation, as seen by total internal reflection fluorescence (TIRF) microscopy (11-13). Because PIP 2 is also responsible for the binding of clathrin coats, it is expected to be present at the clathrin bud from the beginning of its formation. Thus, another explanation was suggested: Proteins that interact with dynamin and possess a curvature-sensing Bin-Amphiphysin-Rvs (BAR) domain (such as endophilin and amphiphysin) were proposed to sense the high curvature of the neck and recruit dynamin (14). Because the curvature of the neck is increasing during clathrin-coated vesicle formation to finally reach the range needed for BAR recruitment, this could explain the arrival of dynamin at the very late stage of clathrin-coated vesicle formation. However, in solution, dynamin spontaneously associates into helices and rings (15) with an internal radius of ≈10 nm. Dynamin can also polymerize around pre...
Solid tumours are often first diagnosed by palpation, suggesting that the tumour is more rigid than its surrounding environment. Paradoxically, individual cancer cells appear to be softer than their healthy counterparts. In this review, we first list the physiological reasons indicating that cancer cells may be more deformable than normal cells. Next, we describe the biophysical tools that have been developed in recent years to characterise and model cancer cell mechanics. By reviewing the experimental studies that compared the mechanics of individual normal and cancer cells, we argue that cancer cells can indeed be considered as softer than normal cells. We then focus on the intracellular elements that could be responsible for the softening of cancer cells. Finally, we ask whether the mechanical differences between normal and cancer cells can be used as diagnostic or prognostic markers of cancer progression.
Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)γ1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC’s activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier.
The last step of cell division, cytokinesis, produces two daughter cells that remain connected by an intercellular bridge. This state often represents the longest stage of the division process. Severing the bridge (abscission) requires a well-described series of molecular events, but the trigger for abscission remains unknown. We found that pulling forces exerted by daughter cells on the intercellular bridge appear to regulate abscission. Counterintuitively, these forces prolonged connection, whereas a release of tension induced abscission. Tension release triggered the assembly of ESCRT-III (endosomal sorting complex required for transport-III), which was followed by membrane fission. This mechanism may allow daughter cells to remain connected until they have settled in their final locations, a process potentially important for tissue organization and morphogenesis.
SummaryMembrane 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.
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