Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. Around 200 activated Arp2/3 complexes are required for robust internalization. A newly developed molecule-counting method determined that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Simulations predict that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed by cryo-electron tomography, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints.
C. elegans transition zone structures are dispensable for axoneme assembly but are required for cell–matrix interactions during neurite extension, revealing an unexpected role for the transition zone in cell adhesion.
Centrioles are known to be essential for cilia assembly. However, their contribution has not been clearly defined. Serwas et al. show that centrioles degenerate early in C. elegans ciliogenesis. Ciliary structures are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles.
Carboxysomes are capsid-like, CO 2-fixing organelles that are present in all cyanobacteria and some chemoautotrophs and that substantially contribute to global primary production. They are composed of a selectively permeable protein shell that encapsulates Rubisco, the principal CO 2-fixing enzyme, and carbonic anhydrase. As the centerpiece of the carbonconcentrating mechanism, by packaging enzymes that collectively enhance catalysis, the carboxysome shell enables the generation of a locally elevated concentration of substrate CO 2 and the prevention of CO 2 escape. A functional carboxysome consisting of an intact shell and cargo is essential for cyanobacterial growth under ambient CO 2 concentrations. Using cryoelectron microscopy, we have determined the structure of a recombinantly produced simplified b-carboxysome shell. The structure reveals the sidedness and the specific interactions between the carboxysome shell proteins. The model provides insight into the structural basis of selective permeability of the carboxysome shell and can be used to design modifications to investigate the mechanisms of cargo encapsulation and other physiochemical properties such as permeability. Notably, the permeability properties are of great interest for modeling and evaluating this carbon-concentrating mechanism in metabolic engineering. Moreover, we find striking similarity between the carboxysome shell and the structurally characterized, evolutionarily distant metabolosome shell, implying universal architectural principles for bacterial microcompartment shells. Carboxysomes are bacterial microcompartments (BMCs) that encapsulate Rubisco and carbonic anhydrase (CA) in a selectively permeable protein shell.
words)Force generation due to actin assembly is a fundamental aspect of membrane sculpting for many essential processes. In this work, we use a multiscale computational model constrained by experimental measurements to show that a minimal branched actin network is sufficient to internalize endocytic pits against physiological membrane tension. A parameter sweep identified the number of Arp2/3 complexes as particularly important for robust internalization, which prompted the development of a molecule-counting method in live mammalian cells. Using this method, we found that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Our simulations also revealed that actin networks self-organize in a radial branched array with barbed filament ends oriented to grow toward the base of the pit, and that the distribution of linker proteins around the endocytic pit is critical for this organization. Surprisingly, our model predicted that long actin filaments bend from their attachment sites in the coat to the base of the pit and store elastic energy that can be harnessed to drive endocytosis. This prediction was validated using cryo-electron tomography on cells, which revealed the presence of bent actin filaments along the endocytic site. Furthermore, we predict that under elevated membrane tension, the self-organized actin network directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, our study reveals that spatially constrained actin filament assembly utilizes an adaptive mechanism that enables endocytosis under varying physical constraints.
Actin filament assembly provides force during clathrin-mediated endocytosis. Here, cryo-electron tomography analysis of actin filament number, organization, and orientation during clathrin-mediated endocytosis in intact human cells revealed that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating a measured branch angle of 68 +/- 9° indicate that sufficient force to drive endocytosis can be generated through polymerization, and that assembly is triggered from 4 +/- 2 founding "mother" filaments, consistent with the tomography data. The actin-binding protein Hip1R decorates the entire endocytic invagination, including the neck region. Simulations showed that the unexpected Hip1R neck localization targets filament growth to this region, improving internalization efficiency and robustness. Actin cytoskeleton organization described here allowed direct translation of structural information to mechanism.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.