Mitochondria undergo fission-fusion events that render these organelles highly dynamic in cells. We report a relationship between mitochondrial form and cell cycle control at the G1-S boundary. Mitochondria convert from isolated, fragmented elements into a hyperfused, giant network at G1-S transition. The network is electrically continuous and has greater ATP output than mitochondria at any other cell cycle stage. Depolarizing mitochondria at early G1 to prevent these changes causes cell cycle progression into S phase to be blocked. Inducing mitochondrial hyperfusion by acute inhibition of dynamin-related protein-1 (DRP1) causes quiescent cells maintained without growth factors to begin replicating their DNA and coincides with buildup of cyclin E, the cyclin responsible for G1-to-S phase progression. Prolonged or untimely formation of hyperfused mitochondria, through chronic inhibition of DRP1, causes defects in mitotic chromosome alignment and S-phase entry characteristic of cyclin E overexpression. These findings suggest a hyperfused mitochondrial system with specialized properties at G1-S is linked to cyclin E buildup for regulation of G1-to-S progression.cell cycle ͉ mitochondrial morphology ͉ dynamin-related protein 1

The ESCRT system is essential for multivesicular body biogenesis, in which cargo sorting is coupled to the invagination and scission of intralumenal vesicles. The ESCRTs are also needed for budding of enveloped viruses including HIV-1, and for membrane abscission in cytokinesis. In yeast, ESCRT-III consists of Vps20, Snf7, Vps24, and Vps2, which assemble in that order, and require the ATPase Vps4 for their disassembly. The ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Three subunits of ESCRT-III, Vps20, Snf7, and Vps24, were sufficient to detach intralumenal vesicles. Vps2, the ESCRT-III subunit responsible for recruiting Vps4, and the ATPase activity of Vps4 were required for ESCRT-III recycling and supported additional rounds of budding. The minimum set of ESCRT-III and Vps4 proteins capable of multiple cycles of vesicle detachment corresponds to the ancient set of ESCRT proteins conserved from archaea to animals.The Endosomal Sorting Complex Required for Transport (ESCRT) machinery is essential for several fundamental cellular pathways [1][2][3][4][5][6][7] . The biogenesis of lysosomes involves the maturation of early endosomes into multivesicular bodies (MVBs) 1,2,5 . In this pathway, portions of the limiting membrane of the endosome invaginate and then detach into the lumen of the endosome, forming intralumenal vesicles (ILVs). The MVBs then fuse with the lysosome and the ILVs and their contents are degraded. Cell surface receptors destined for downregulation and some lysosomal resident enzymes are sorted into this pathway following their covalent ubiquitination. The ESCRTs are involved both in sorting of ubiquitinated cargo into ILVs and in the morphogenesis of the ILVs themselves 1, 2, 5, 7 . The ESCRT complexes are also required for the release of nascent HIV-1 virions from the plasma membrane of human cells [8][9][10] . The ESCRTs are needed for cytokinesis in both animal cells 11,12 and a subset of the archaea 13, 14 .The detachment of ILVs from the endosomal limiting membrane involves the scission of the narrow membrane neck connecting the nascent vesicle with the limiting membrane ( Supplementary Fig. 1). This process is topologically opposite to the mechanism of membrane cleavage by dynamin family proteins 15 , which coat the outer surface of membrane tubules and cleave from the outside. Cleavage of the membrane neck connecting a nascent virion to the plasma membrane or connecting two daughter also occurs from the membrane surface (Supplementary Fig. 1). The conserved and ubiquitous involvement of ESCRTs in the inwardly directed cleavage of membrane necks has led to the view that ESCRTs are likely to have membrane scission activity. Thus far the absence of an ESCRT scission assay reconstituted entirely from purified proteins and lipids has prevented a direct test of this hypothesis, and has more broadly impeded progress in the ESCRT field.The ESCRTs consist of the ESCRT-0, -I, -...

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers1. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect2–4, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin5,6. Clathrin-independent endocytic events are often less reliant on dynamin7, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we found that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which both are clathrin-independent endocytic cargoes8. In controlled in vitro systems, endoA2 reshapes membranes prior to scission. Furthermore, we demonstrate that endoA2, dynamin, and actin contribute in parallel to the scission of Shiga toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings finally highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling force-driven dynamic scission.

Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of β1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.