Type-I myosins promote actin polymerization to drive membrane bending in endocytosis.

Clathrin-mediated endocytosis in budding yeast requires the formation of a dynamic actin network that produces the force to invaginate the plasma membrane against the intracellular turgor pressure. The type-I myosins Myo3 and Myo5 are important for endocytic membrane reshaping, but mechanistic details of their function remain scarce. Here, we studied the function of Myo3 and Myo5 during endocytosis using quantitative live-cell imaging and genetic perturbations. We show that the type-I myosins promote, in a dose-dependent way, the growth and expansion of the actin network, which controls the speed of membrane and coat internalization. We found that this myosin-activity is independent of the actin nucleation promoting activity of myosins, and cannot be compensated for by increasing actin nucleation. Our results suggest a new mechanism for type-I myosins to produce force by promoting actin filament polymerization.

High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding.

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called "MultiCLEM," allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.

The contributions of the actin machinery to endocytic membrane bending and vesicle formation.

Branched and cross-linked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analyzed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thus temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our data show that initiation of membrane bending requires the actin network to be physically linked to the plasma membrane and to be optimally cross-linked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independent of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 s before scission, when the filament nucleation rate drops. From that time point on, invagination growth and vesicle scission are driven by an expansion of the actin network without a proportional increase of net actin amounts. The expansion is sensitive to the amount of filamentous actin and its cross-linking. Our results suggest that the mechanism by which actin reshapes the membrane changes during the progress of endocytosis, possibly adapting to varying force requirements.