Physics Ph.D. Candidate, Washington University in St. Louis
Pulling forces in endocytosis are being generated by actin polymerization.
In the process of endocytosis, by which the cell plasma membrane invaginate inward in order to bring the essential extracellular proteins into the cytoplasm, there are many different proteins that play their roles. Among them actin monomers are the main players; As previous super-resolution microscope experiments have shown, the membrane curving does not initiate until the actin monomers arrive at the endocytic patch. Actin monomers form filamentous long structures in an area of about 200 nm squared wide beneath the membrane, where the same filaments exerts pushing and pulling forces on the membrane. The mechanism behind the generation of the pushing force by the growth of an actin filament (actin polymerization) is pretty well-understood and modeled; The famous Brownian Ratchet model is an example of a simple picture on how such polymerization generates force to push on an obstacle ahead. However, we know that in endocytosis, the membrane's curvature is progressing inward to form a drop shape pit that will get pinched off eventually into a vesicle, carrying proteins into the cell cytoplasm. Therefore, at the center of this endocytic patch, pulling forces are being exerted on the membrane to derive the invagination. Studying the mechanism behind the generation of these pulling forces, caused by the growth of the actin filaments, is the main focus of my research. Our hypothesis is that the retrograde movement of the entire actin network in the endocytic patch is the force behind the curvature generation in the form that we see on the cell membrane. Recent experimental findings confirm the existence of a specific protein called Sla2, that acts as a glue connecting protein that holds the slower growing filaments at the center of the endocytic patch and keeps them connected to the membrane (like a hook, that in physical terms is a potential well) Consequently, the retrograde motion of the whole actin network based on the actin polymerization, can naturally lead to the membrane curvature and endocytosis.
Abstract: To elucidate the nature of load sharing in the growth of multiple biopolymers, we perform stochastic simulations of the growth of biopolymer bundles against obstacles under a broad range of conditions and varying assumptions. The obstacle motion due to thermal fluctuations is treated explicitly. We assume the "Perfect Brownian Ratchet" (PBR) model, in which the polymerization rate equals the free-filament rate as soon as the filament-obstacle distance exceeds the monomer size. Accurate closed-form formulas are obtained for the case of a rapidly moving obstacle. We find the following: (1) load sharing is usually sub-perfect in the sense that polymerization is slower than for a single filament carrying the same average force; (2) the sub-perfect behavior becomes significant at a total force proportional to the logarithm or the square root of the number of filaments, depending on the alignment of the filaments; (3) for the special case of slow barrier diffusion and low opposing force, an enhanced obstacle velocity for an increasing number of filaments is possible; (4) the obstacle velocity is very sensitive to the alignment of the filaments in the bundle, with a staggered alignment being an order of magnitude faster than an unstaggered one at forces of only 0.5 pN per filament for 20 filaments; (5) for large numbers of filaments, the power is maximized at a force well below 1 pN per filament; (6) for intermediate values of the obstacle diffusion coefficient, the shape of the force velocity relation is very similar to that for rapid obstacle diffusion.
Pub.: 10 Dec '14, Pinned: 29 Jun '17
Abstract: Membrane deformation during endocytosis in yeast is driven by local, templated assembly of a sequence of proteins including polymerized actin and curvature-generating coat proteins such as clathrin. Actin polymerization is required for successful endocytosis, but it is not known by what mechanisms actin polymerization generates the required pulling forces. To address this issue, we develop a simulation method in which the actin network at the protein patch is modeled as an active gel. The deformation of the gel is treated using a finite-element approach. We explore the effects and interplay of three different types of force driving invagination: 1), forces perpendicular to the membrane, generated by differences between actin polymerization rates at the edge of the patch and those at the center; 2), the inherent curvature of the coat-protein layer; and 3), forces parallel to the membrane that buckle the coat protein layer, generated by an actomyosin contractile ring. We find that with optimistic estimates for the stall stress of actin gel growth and the shear modulus of the actin gel, actin polymerization can generate almost enough force to overcome the turgor pressure. In combination with the other mechanisms, actin polymerization can the force over the critical value.
Pub.: 18 Apr '14, Pinned: 29 Jun '17
Abstract: Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000-10000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.
Pub.: 18 Jun '17, Pinned: 29 Jun '17