Generation Of Pulling Forces Research Articles

Mechanistic insights into actin force generation during vesicle formation from cryo-electron tomography.

Actin assembly provides force for a multitude of cellular processes. Compared to actin-assembly-based force production during cell migration, relatively little is understood about how actin assembly generates pulling forces for vesicle formation. Here, cryo-electron tomography identified actin filament number, organization, and orientation during clathrin-mediated endocytosis in human SK-MEL-2 cells, showing that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating ameasured branch angle indicate that sufficient force to drive membrane internalization is generated throughpolymerization and that assembly is triggered from ∼4 founding "mother" filaments, consistent with tomography data. Hip1R actin filament anchoring points are present along the entire endocytic invagination, where simulations show that it is key to pulling force generation, and along the neck, where it targets filament growth and makes internalization more robust. Actin organization described here allowed direct translation of structure to mechanism with broad implications for other actin-driven processes.

Modeling Pulling Force Generation by Ensembles of Polymerizing Actin Filaments

Actin polymerization in yeast is the primary mechanism for overcoming the large turgor pressure that opposes clathrin-mediated endocytosis. While generation of pushing forces by actin polymerization is fairly well understood, it is not clear how actin polymerization produces pulling forces. Previous work using a finite element approach has suggested that continuous spatial variation of the polymerization rate can generate pulling forces1. We extend this work by performing calculations on a discrete system of actin filaments with all subunits explicitly treated. We stochastically simulate growing arrays of up to 200 semiflexible actin filaments in a square network, where each filament interacts with the membrane via an interaction potential that has both attractive and repulsive components. The crucial protein Sla2, which binds actin filaments to the membrane, is assumed to contribute a strongly attractive component to the potential. The polymerization and depolymerization rates depend on the filament-membrane gap. We model the elasticity of the actin network by linear springs connecting adjacent filaments to each other. The inner filaments of the array are taken to correspond to a Sla2 patch. They are thus bound more strongly to the membrane, and grow more slowly. The simulation results show that the outer filaments push on the membrane, while the inner filaments pull on it. We calculate the total pulling force as a function of several model parameters, including the potential depths, the free filament on- and off-rates, the numbers of fast- and slow-growing filaments, the filament tip stiffness, and the network rigidity. [1] A. E. Carlsson and P. V. Bayly, Biophys. J. 106:1596-1606(2014).

Two populations of cytoplasmic dynein contribute to spindle positioning in C. elegans embryos.

The position of the mitotic spindle is tightly controlled in animal cells as it determines the plane and orientation of cell division. Contacts between cytoplasmic dynein and astral microtubules (MTs) at the cell cortex generate pulling forces that position the spindle. An evolutionarily conserved Gα-GPR-1/2Pins/LGN-LIN-5Mud/NuMA cortical complex interacts with dynein and is required for pulling force generation, but the dynamics of this process remain unclear. In this study, by fluorescently labeling endogenous proteins in Caenorhabditis elegans embryos, we show that dynein exists in two distinct cortical populations. One population directly depends on LIN-5, whereas the other is concentrated at MT plus ends and depends on end-binding (EB) proteins. Knockout mutants lacking all EBs are viable and fertile and display normal pulling forces and spindle positioning. However, EB protein-dependent dynein plus end tracking was found to contribute to force generation in embryos with a partially perturbed dynein function, indicating the existence of two mechanisms that together create a highly robust force-generating system.

Multisite Phosphorylation of NuMA-Related LIN-5 Controls Mitotic Spindle Positioning in C. elegans.

During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα–LGN–NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5–GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation.

往復式及び回転式シェーバーによる人工毛切断挙動観察

The shaver is a popular electronic utility for male to cut their beard, hair and so on. It is roughly divided two shaving system as oscillating or rotating motion of an inner blade. The cutting hairs mechanism is also assumed that inner blade edge just cut hairs at the moment of contact between inner blade and hairs. However, the hairs were likely to pull into the shaver and a pulling force generated which we felt in use. The detail cutting motion and pulling force was observed and measured to clarify the differences between oscillation and rotation type by a high speed camera. Each shaving system showed same tendency that the inner blade penetrated to the hair at the first, then both the inner blade and hair moved together to the outer blade. Finally, the inner and outer blade cut the hair by scissor action with pulling force generation simultaneously. As regards oscillation shaver, it was indicated that the inner blade hit the hair several times before the hair was completely cut. The maximum pulling force was approximately 0.15 N and the average was 0.04 which was higher than rotation type of 0.13 N as maximum and 0.03 N as average. Finally, the relation between the inner blade speed and pulling force of rotating type shaver was investigated, and the rotating type showed lower pulling force generation nevertheless of its lower inner blade speed (1.18 m/s) than oscillation type (2.3 m/s).

F-actin asymmetry and the endoplasmic reticulum–associated TCC-1 protein contribute to stereotypic spindle movements in theCaenorhabditis elegansembryo

The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα-GPR-LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.

Cortical localization of the Gα protein GPA-16 requires RIC-8 function duringC. elegansasymmetric cell division

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the Galpha proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using co-immunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the Gbeta protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two Galpha proteins can activate the same pathway and that their dual presence is normally needed to counter Gbetagamma. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA-16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between Galpha proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.