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1.
Endocytosis is an essential process by which cells internalize a piece of plasma membrane and material from the outside. In cells with turgor, pressure opposes membrane deformations, and increases the amount of force that has to be generated by the endocytic machinery. To determine this force, and calculate the shape of the membrane, we used physical theory to model an elastic surface under pressure. Accurate fits of experimental profiles are obtained assuming that the coated membrane is highly rigid and preferentially curved at the endocytic site. The forces required from the actin machinery peaks at the onset of deformation, indicating that once invagination has been initiated, endocytosis is unlikely to stall before completion. Coat proteins do not lower the initiation force but may affect the process by the curvature they induce. In the presence of isotropic curvature inducers, pulling the tip of the invagination can trigger the formation of a neck at the base of the invagination. Hence direct neck constriction by actin may not be required, while its pulling role is essential. Finally, the theory shows that anisotropic curvature effectors stabilize membrane invaginations, and the loss of crescent-shaped BAR domain proteins such as Rvs167 could therefore trigger membrane scission. 相似文献
2.
Actin in membrane trafficking 总被引:6,自引:0,他引:6
Lanzetti L 《Current opinion in cell biology》2007,19(4):453-458
Actin cytoskeleton remodeling provides the forces required for a variety of cellular processes based on membrane dynamics, such as endocytosis, exocytosis, and vesicular trafficking at the Golgi. All these events are coordinated by networks of associated proteins, and some of them are functionally connected with cell migration. The site and the duration of actin polymerization, in connection with vesicle budding and fusion, are tightly controlled by both small GTPases and the large GTPase dynamin. Recent advances in the understanding of the mechanisms coupling actin dynamics with membrane trafficking at the cell surface have been brought by the combined studies of actin polymerizing factors and of the endocytic/exocytic machinery. 相似文献
3.
Helen E. Burston Lymarie Maldonado-Báez Michael Davey Benjamen Montpetit Cayetana Schluter Beverly Wendland Elizabeth Conibear 《The Journal of cell biology》2009,185(6):1097-1110
Endocytosis of receptors at the plasma membrane is controlled by a complex mechanism that includes clathrin, adaptors, and actin regulators. Many of these proteins are conserved in yeast yet lack observable mutant phenotypes, which suggests that yeast endocytosis may be subject to different regulatory mechanisms. Here, we have systematically defined genes required for internalization using a quantitative genome-wide screen that monitors localization of the yeast vesicle-associated membrane protein (VAMP)/synaptobrevin homologue Snc1. Genetic interaction mapping was used to place these genes into functional modules containing known and novel endocytic regulators, and cargo selectivity was evaluated by an array-based comparative analysis. We demonstrate that clathrin and the yeast AP180 clathrin adaptor proteins have a cargo-specific role in Snc1 internalization. We additionally identify low dye binding 17 (LDB17) as a novel conserved component of the endocytic machinery. Ldb17 is recruited to cortical actin patches before actin polymerization and regulates normal coat dynamics and actin assembly. Our findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic internalization. 相似文献
4.
The cell membrane deforms during endocytosis to surround extracellular material and draw it into the cell. Results of experiments on endocytosis in yeast show general agreement that 1) actin polymerizes into a network of filaments exerting active forces on the membrane to deform it, and 2) the large-scale membrane deformation is tubular in shape. In contrast, there are three competing proposals for precisely how the actin filament network organizes itself to drive the deformation. We use variational approaches and numerical simulations to address this competition by analyzing a meso-scale model of actin-mediated endocytosis in yeast. The meso-scale model breaks up the invagination process into three stages: 1) initiation, where clathrin interacts with the membrane via adaptor proteins; 2) elongation, where the membrane is then further deformed by polymerizing actin filaments; and 3) pinch-off. Our results suggest that the pinch-off mechanism may be assisted by a pearling-like instability. We rule out two of the three competing proposals for the organization of the actin filament network during the elongation stage. These two proposals could be important in the pinch-off stage, however, where additional actin polymerization helps break off the vesicle. Implications and comparisons with earlier modeling of endocytosis in yeast are discussed. 相似文献
5.
External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane 总被引:1,自引:0,他引:1
Galic M Jeong S Tsai FC Joubert LM Wu YI Hahn KM Cui Y Meyer T 《Nature cell biology》2012,14(8):874-881
Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endocytosis by associating with curved sections of the plasma membrane. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the plasma membrane or indirectly by plasma-membrane-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally induced inward deformation of the plasma membrane by cone-shaped nanostructures (nanocones) and internally induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved plasma membrane. Markedly, live-cell imaging in adherent cells showed selective recruitment of full-length N-BAR proteins and isolated N-BAR domains to plasma membrane sub-regions above nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by nanocones. We further showed that N-BAR domains are periodically recruited to curved plasma membrane sites during local lamellipodia retraction in the front of migrating cells. Recruitment required myosin-II-generated force applied to plasma-membrane-connected actin cables. Together, our results show that N-BAR domains can be directly recruited to the plasma membrane by external push or internal pull forces that locally curve the plasma membrane. 相似文献
6.
Charlotte Kaplan Sam J. Kenny Xuyan Chen Johannes Schneberg Ewa Sitarska Alba Diz-Muoz Matthew Akamatsu Ke Xu David G. Drubin 《Molecular biology of the cell》2022,33(6)
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. 相似文献
7.
Dynamic plasma membrane rearrangements occur during many cellular processes including endocytosis, morphogenesis, and migration. Actin polymerization together with proteins that directly deform membranes, such as the BAR superfamily proteins, is essential for generation of membrane invaginations during endocytosis. Importantly, recent studies revealed that direct membrane deformation contributes also to the formation of plasma membrane protrusions such as filopodia and lamellipodia. Inverse BAR (I-BAR) domain proteins bind phosphoinositide-rich membrane with high affinity and generate negative membrane curvature to induce plasma membrane protrusions. I-BAR domain proteins, such as IRSp53, MIM, ABBA, and IRTKS also harbor many protein-protein interaction modules that link them to actin dynamics. Thus, I-BAR domain proteins may connect direct membrane deformation to actin polymerization in cell morphogenesis and migration. 相似文献
8.
During clathrin-mediated endocytosis, a patch of flat plasma membrane is deformed into a vesicle. In walled cells, such as plants and fungi, the turgor pressure is high and pushes the membrane against the cell wall, thus hindering membrane internalization. In this work, we study how a patch of membrane is deformed against turgor pressure by force and by curvature-generating proteins. We show that a large amount of force is needed to merely start deforming the membrane and an even larger force is needed to pull a membrane tube. The magnitude of these forces strongly depends on how the base of the membrane is constrained and how the membrane is coated with curvature-generating proteins. In particular, these forces can be reduced by partially, but not fully, coating the membrane patch with curvature-generating proteins. Our theoretical results show excellent agreement with experimental data. 相似文献
9.
Gur Fabrikant Soumya Gupta G.V. Shivashankar Michael?M. Kozlov 《Biophysical journal》2013,105(6):1316-1323
Deformations of cell nuclei accompany a number of essential intracellular processes. Although evidence is being accumulated on the primary role actin structures play in controlling the shape of the nucleus, the mechanisms behind this phenomenon remain unknown. Here, we consider theoretically a specific paradigm of nuclear deformation, and a related actin rearrangement, in T cells stimulated by contact with a bead covered by surrogate antigens. We suggest that the nucleus is deformed by the elastic forces developed within a cylindrical layer of polymerized actin, which is generated as a result of the receptor-mediated T-cell activation. We substantiate this proposal with a theoretical analysis of mutual deformations in the actin layer and the nucleus, which recovers the experimentally observed nuclear shapes. Furthermore, we evaluate the forces developed by the actin polymerization that drives the nuclear deformation. The model predicts the mode of actin polymerization generated by the surrogate antigens covering a bead and the values of the elastic moduli of the nuclear shell. We provide a qualitative experimental support for the model assumptions by visualizing the stages of nuclear shape change and the corresponding evolution of the cortical actin. 相似文献
10.
Deformations of cell nuclei accompany a number of essential intracellular processes. Although evidence is being accumulated on the primary role actin structures play in controlling the shape of the nucleus, the mechanisms behind this phenomenon remain unknown. Here, we consider theoretically a specific paradigm of nuclear deformation, and a related actin rearrangement, in T cells stimulated by contact with a bead covered by surrogate antigens. We suggest that the nucleus is deformed by the elastic forces developed within a cylindrical layer of polymerized actin, which is generated as a result of the receptor-mediated T-cell activation. We substantiate this proposal with a theoretical analysis of mutual deformations in the actin layer and the nucleus, which recovers the experimentally observed nuclear shapes. Furthermore, we evaluate the forces developed by the actin polymerization that drives the nuclear deformation. The model predicts the mode of actin polymerization generated by the surrogate antigens covering a bead and the values of the elastic moduli of the nuclear shell. We provide a qualitative experimental support for the model assumptions by visualizing the stages of nuclear shape change and the corresponding evolution of the cortical actin. 相似文献
11.
The Sla1 adaptor‐clathrin interaction regulates coat formation and progression of endocytosis
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Thomas O. Tolsma Lena M. Cuevas Santiago M. Di Pietro 《Traffic (Copenhagen, Denmark)》2018,19(6):446-462
Clathrin‐mediated endocytosis is a fundamental transport pathway that depends on numerous protein‐protein interactions. Testing the importance of the adaptor protein‐clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin‐binding motif (sla1AAA) that disrupt clathrin binding. Live‐cell imaging showed an impaired Sla1‐clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3‐dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1‐clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending. 相似文献
12.
Cellular movements are produced by forces. Typically, cytoskeletal proteins such as microtubules and actin filaments generate forces via polymerization or in conjunction with molecular motors. However, the fertilization of a Limulus polyphemus egg involves a third type of actin-based cellular engine--a biological spring. During the acrosome reaction, a 60-microm long coiled and twisted bundle of actin filaments straightens and extends from a sperm cell, penetrating the vitelline layer surrounding the egg. A subtle overtwist of 0.2 degrees /subunit underlies the mechanochemical basis for the extension of this actin spring. Upon calcium activation, this conformational strain energy is converted to mechanical work, generating the force required to extend the bundle through the vitelline layer. In this article, we stall the extension of the acrosome bundle in agarose gels of different concentrations. From the stall forces, we estimate a maximum force of 2 nN and a puncturing pressure of 1.6 MPa. We show the maximum force of extension is three times larger than the force required to puncture the vitelline layer. Thus, the elastic strain energy stored in the acrosome bundle is more than sufficient to power the acrosome reaction through the egg envelope. 相似文献
13.
Stamnes M 《Current opinion in cell biology》2002,14(4):428-433
Although the actin cytoskeleton is widely believed to play an important role in intracellular protein transport, this role is poorly understood. Recently, progress has been made toward identifying specific actin-binding proteins and signaling molecules involved in regulating actin structures that function in the secretory pathway. Studies on coat protomer I (COPI)-mediated transport at the Golgi apparatus and on clathrin-mediated endocytosis have been particularly informative in identifying such mechanisms. Important similarities between actin regulation at the Golgi and at the plasma membrane have been uncovered. The studies reveal that ADP-ribosylation factor and vesicle coat proteins are able to act through the Rho-family GTP-binding proteins, Cdc42 and Rac, and several specific actin-binding proteins to direct actin assembly through the Arp2/3 complex. Efficient function of the secretory pathway is likely to require precise temporal regulation among transport-vesicle assembly, vesicle scission, and the targeting machinery. It is proposed that numerous actin regulatory mechanisms and the connections between actin signaling and vesicle-coat formation are employed to provide such temporal regulation. 相似文献
14.
《European journal of cell biology》2022,101(4):151267
In clathrin-mediated endocytosis, a principal membrane trafficking route of all eukaryotic cells, forces are applied to invaginate the plasma membrane and form endocytic vesicles. These forces are provided by specific endocytic proteins and the polymerizing actin cytoskeleton. One of the best-studied endocytic systems is endocytosis in yeast, known for its simplicity, experimental amenability, and overall similarity to human endocytosis. Importantly, the yeast endocytic protein machinery generates and transmits tremendous force to bend the plasma membrane, making this system beneficial for mechanistic studies of cellular force-driven membrane reshaping. This review summarizes important protein players, molecular functions, applied forces, and open questions and perspectives of this robust, actin-powered membrane-remodeling protein machine. 相似文献
15.
Actin polymerization is the driving force for a large number of cellular processes. Formation of lamellipodia and filopodia at the leading edge of motile cells requires actin polymerization induced mechanical deformation of the plasma membrane. To generate different types of membrane protrusions, the mechanical properties of actin filaments can be constrained by interacting proteins. A striking example of such constraint is the buckling of actin filaments generated in vitro by the cooperative effect of a processive actin nucleating factor (formin) and a molecular motor (myosin II). We developed a physical model based on equations for an elastic rod that accounts for actin filament buckling. Both ends of the rod were maintained in a fixed position in space and we considered three sets of boundary conditions. The model qualitatively and quantitatively reproduces the shape distribution of actin filaments. We found that actin polymerization counterpoises a force in the range 0.4-1.6 pN for moderate end-to-end distance (approximately 1 microm) and could be as large as 10 pN for shorter distances. If the actin rod attachment includes a spring, we discovered that the stiffness must be in the range 0.1-1.2 pN/nm to account for the observed buckling. 相似文献
16.
Clathrin is important for normal actin dynamics and progression of Sla2p-containing patches during endocytosis in yeast 总被引:1,自引:0,他引:1
Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to the cortex prior to Sla2p. In the absence of clathrin, normal numbers of Sla2p patches form, but many do not internalize or are dramatically delayed in completion of endocytosis. Patches that do internalize receive Sla1p late, which is followed by Abp1, which appears near the end of Sla2p lifetime. In addition, clathrin mutants develop actin comet tails, suggesting an important function in actin patch organization/dynamics. Similar to its mammalian counterparts, the light chain (LC) subunit of yeast clathrin interacts directly with the coiled-coil domain of Sla2p. A mutant of Sla2p that no longer interacts with LC (sla2Delta376-573) results in delayed progression of endocytic patches and aberrant actin dynamics. These data demonstrate an important role for clathrin in organization and progression of early endocytic patches to the late stages of endocytosis. 相似文献
17.
Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins 总被引:11,自引:0,他引:11
Cell membranes undergo continuous curvature changes as a result of membrane trafficking and cell motility. Deformations are achieved both by forces extrinsic to the membrane as well as by structural modifications in the bilayer or at the bilayer surface that favor the acquisition of curvature. We report here that a family of proteins previously implicated in the regulation of the actin cytoskeleton also have powerful lipid bilayer-deforming properties via an N-terminal module (F-BAR) similar to the BAR domain. Several such proteins, like a subset of BAR domain proteins, bind to dynamin, a GTPase implicated in endocytosis and actin dynamics, via SH3 domains. The ability of BAR and F-BAR domain proteins to induce tubular invaginations of the plasma membrane is enhanced by disruption of the actin cytoskeleton and is antagonized by dynamin. These results suggest a close interplay between the mechanisms that control actin dynamics and those that mediate plasma membrane invagination and fission. 相似文献
18.
Merrifield CJ 《Trends in cell biology》2004,14(7):352-358
Endocytosis is characterized by movement and precisely controlled changes in membrane geometry during vesicle formation. Recent developments in live-cell imaging have enabled such movements to be monitored in vivo and correlated with the recruitment and dismissal of fluorescently labeled proteins. This experimental strategy has revealed the sequential recruitment of proteins that are involved in actin polymerization, and actin to single sites of endocytosis in both yeast and mammalian cells. Actin polymerization is correlated with the inward movements of endocytic organelles, which suggests that actin polymerization has a conserved role in this process. In this article, I will discuss three models for the role of actin polymerization in endocytosis. 相似文献
19.
A pathway for association of receptors, adaptors, and actin during endocytic internalization 总被引:42,自引:0,他引:42
In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition. Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility. Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions. Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly. In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes. Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization. Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved. 相似文献
20.
Several findings suggest that actin-mediated motility can play a role in clathrin-mediated endocytosis but it remains unclear whether and when key proteins required for this process are recruited to endocytic sites. Here we investigate this question in live Swiss 3T3 cells using two-colour evanescent field (EF) microscopy. We find that Arp3, a component of the Arp2/3 complex, appears transiently while single clathrin-coated pits internalize. There is also additional recruitment of Neural-Wiskott Aldrich Syndrome Protein (N-WASP), a known activator of the Arp2/3 complex. Both proteins appear at about the same time as actin. We suggest that N-WASP and the Arp2/3 complex trigger actin polymerization during a late step in clathrin-mediated endocytosis, and propel clathrin-coated pits or vesicles from the plasma membrane into the cytoplasm. 相似文献