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.

We developed a general approach for investigation of how cellular processes become adapted for specific cell types during differentiation. Previous studies reported substantial differences in the morphology and dynamics of clathrin-mediated endocytosis (CME) sites. However, associating specific CME properties with distinct differentiated cell types and determining how these properties are developmentally specified during differentiation have been elusive. Using genome-edited human embryonic stem cells, and isogenic fibroblasts and neuronal progenitor cells derived from them, we established by live-cell imaging and platinum replica transmission electron microscopy that CME site dynamics and ultrastructure on the plasma membrane are precisely reprogrammed during differentiation. Expression levels for the endocytic adaptor protein AP2μ2 were found to underlie dramatic changes in CME dynamics and structure. Additionally, CME dependency on actin assembly and phosphoinositide-3 kinase activity are distinct for each cell type. Collectively, our results demonstrate that key CME properties are reprogrammed during differentiation at least in part through AP2μ2 expression regulation.

Microtubules (MTs) are important for cellular structure, transport of cargoes and segregation of chromosomes and organelles during mitosis. The stochastic growth and shrinkage of MTs, known as dynamic instability, is necessary for these functions. Previous studies to determine how individual MT-associated proteins (MAPs) affect MT dynamics have been performed either through studies, which provide limited opportunity for observation of individual MTs or manipulation of conditions, or studies, which focus either on purified proteins, and therefore lack cellular complexity, or on cell extracts made from genetically intractable organisms. In order to investigate the ensemble activities of all MAPs on MT dynamics using lysates made from a genetically tractable organism, we developed a cell-free assay for budding yeast lysates using total internal reflection fluorescence (TIRF) microscopy. Lysates were prepared from yeast strains expressing GFP-tubulin. MT polymerization from pre-assembled MT seeds adhered to a coverslip was observed in real time. Through use of cell division cycle () and MT depolymerase mutants, we found that MT polymerization and dynamic instability are dependent on the cell cycle state and the activities of specific MAPs.

Conserved proteins drive clathrin-mediated endocytosis (CME), which from yeast to humans involves a burst of actin assembly. To gain mechanistic insights into this process, we performed a side-by-side quantitative comparison of CME in two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae is more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast. In both yeasts, accumulation of ~70 WASp molecules activates the Arp2/3 complex to drive membrane invagination. In contrast to budding yeast, WASp-mediated actin nucleation plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, although the assembly of two zones of actin filaments is specific for fission yeast and not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations with broad implications that are expected to extend from yeast to humans.

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.DOI: http://dx.doi.org/10.7554/eLife.29140.001