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.

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feed back on assembly. By manipulating membrane tension and Arp2/3 complex activity we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

12Autophagy is an essential physiological process by which eukaryotic cells degrade and recycle 13 cellular materials. Although the biochemical hierarchies of the mammalian autophagy pathway 14 have been identified, questions remain regarding the sequence, subcellular location, and 15 structural requirements of autophagosome formation. Here, we characterize the structural 16 organization of key components of the mammalian autophagic initiation machinery at ~20 nm 17 spatial resolution via three-color, three-dimensional super-resolution fluorescence microscopy. 18 We thus show that upon cell starvation, FIP200, a large structural protein of the ULK1 complex 19 with no direct yeast homolog, scaffolds the formation of cup-like structures located at SEC12-20 enriched remodeled ER-exit sites prior to LC3 lipidation. This cup scaffold, then, provides a 21 structural asymmetry to enforce the directional recruitment of downstream components, 22 including the Atg12-Atg5-Atg16 complex, WIPI2, and LC3, to the cup inside. Moreover, we 23 provide evidence that the early autophagic machinery is recruited in its entirety to these cup 24 structures prior to LC3 lipidation, and gradually disperses and dissociates on the outer face of the 25 phagophore membrane during elongation. We thus shed new light on the physical process of 26 mammalian autophagic initiation and development at the nanometer-scale. 28Macroautophagy, hereafter autophagy, is an essential catabolic process by which eukaryotic cells 29 degrade and recycle organelles and proteins 1-8 . The general mechanism of autophagy begins with 30 the activation of the ULK1 complex by mTORC1 in response to stresses. Subsequent recruitment 31 and activation of the PI3K complex, PtdIns3P effectors (WIPI proteins), and the Atg12-Atg5- 32Atg16 complex enable the lipidation of LC3, which drives the formation of a cup-shaped 33 membrane structure known as the isolation membrane, or phagophore. The phagophore engulfs 34 cargo and closes before fusing with lysosome for cargo degradation. Together, the phagophore 35 membrane with its associated protein machinery constitutes the autophagosome. 36Although the biochemical hierarchy of autophagic machinery has been studied 37 extensively 1,4 , open questions remain, especially regarding the spatiotemporal dynamics of 38 autophagosome formation 6-8 . In particular, although the overall mechanism of autophagy is 39 conserved across eukaryotes, autophagy initiation in yeast occurs at a single location (the pre-40 autophagosomal structure) whereas mammalian autophagosomes form throughout the cytosol, 41 likely scaffolded by the ER or related tubulovesicular networks 4,[9][10][11] . Meanwhile, many proteins 42 essential for autophagy in mammals have no direct homologs in yeasts 1,3,7 . Understanding the 43 functions of these proteins, particularly regarding their role in the structural organization of early 44 autophagosomes, is of critical importance. 45FIP200, also known as RB1CC1, is a component of the mammalian ULK1 complex, and 46 ...

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