In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition. Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility. Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions. Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly. In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes. Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization. Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved.

Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.

Highlights d Knockdown of Ptbp1 converts Mu ¨ller glia into retinal ganglion cells in mature retinas d Central projections of converted retinal ganglion cells restore visual responses d Induction of neurons with dopaminergic features in PD model mice

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An integrated theoretical model reveals how the chemical and the mechanical aspects of endocytosis are coordinated coherently in yeast cells, driving progression through the endocytic pathway and ensuring efficient vesicle scission in vivo.

The lipid phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) appears to play an important role in endocytosis. However, the timing of its formation and turnover, and its specific functions at different stages during endocytic internalization, have not been established. In this study, Sla2 ANTH-GFP and Sjl2-3GFP were expressed as functional fusion proteins at endogenous levels to quantitatively explore PtdIns(4,5)P2 dynamics during endocytosis in yeast. Our results indicate that PtdIns(4,5)P2 levels increase and decline in conjunction with coat and actin assembly and disassembly, respectively. Live-cell image analysis of endocytic protein dynamics in an sjl1Δ sjl2Δ mutant, which has elevated PtdIns(4,5)P2 levels, revealed that the endocytic machinery is still able to assemble and disassemble dynamically, albeit nonproductively. The defects in the dynamic behavior of the various endocytic proteins in this double mutant suggest that PtdIns(4,5)P2 turnover is required for multiple stages during endocytic vesicle formation. Furthermore, our results indicate that PtdIns(4,5)P2 turnover may act in coordination with the Ark1/Prk1 protein kinases in stimulating disassembly of the endocytic machinery.

ETOC: During yeast endocytic site formation, Ede1p (yeast Eps15), but not clathrin light chain, is important for the recruitment of most other early-arriving proteins to endocytic sites. Cargo and clathrin light chain may play roles in regulating the transition of endocytic sites out of the “intermediate coat” stage of endocytosis.