Mitochondria are highly dynamic organelles that mediate essential cell functions such as apoptosis and cell-cycle control in addition to their role as efficient ATP generators. Mitochondrial morphology changes are tightly regulated, and their shape can shift between small, fragmented units and larger networks of elongated mitochondria. We demonstrate that mitochondrial elements become significantly elongated and interconnected shortly after nutrient depletion. This mitochondrial morphological shift depends on the type of starvation, with an additive effect observed when multiple nutrients are depleted simultaneously. We further show that starvation-induced mitochondrial elongation is mediated by downregulation of dynamin-related protein 1 (Drp1) through modulation of two Drp1 phosphorylation sites, leading to unopposed mitochondrial fusion. Finally, we establish that mitochondrial tubulation upon nutrient deprivation protects mitochondria from autophagosomal degradation, which could permit mitochondria to maximize energy production and supply autophagosomal membranes during starvation.autophagy | mitofusin M itochondria are dynamic organelles that mediate many essential cell functions. Depending on the cellular context, mitochondria shift between fragmented and tubular network-like morphologies by means of coordinated fission and fusion (1, 2). Proteins responsible for mitochondrial fusion include the outer mitochondrial membrane proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) (3-5) and the inner membrane protein optic atrophy 1 (Opa1) (6). Fission is mediated by dynamin-related protein 1 (Drp1) (7,8) and its interaction with binding partners Fis1 and/or mitochondrial fission factor (Mff) (9, 10). Multiple mechanisms, including phosphorylation, sumoylation, and ubiquitination, coordinate Drp1 fission capacity (11)(12)(13)(14). Phosphorylation of Drp1 at S616 by Cdk1/cyclin B results in increased Drp1 fission activity (13). Conversely, phosphorylation of Drp1 at S637 by PKA decreases fission by causing Drp1 retention in the cytosol, whereas dephosphorylation of S637 by calcineurin causes Drp1 translocation to the mitochondria and increased mitochondrial fission (11, 15).Mitochondrial morphological dynamics are linked to regulation of many specific cell functions. Changes in mitochondrial cristae and mitochondrial fragmentation, for example, play a vital role in apoptosis (16,17). Ca 2+ transfer (18), cell-cycle regulation (13,19), and mitochondrial quality control (20, 21) are all closely tied to changes in mitochondrial morphology. Furthermore, stress conditions and changes in energy source can induce significant mitochondrial morphological changes (22, 23). Very recently, nutrient starvation was shown to induce mitochondrial elongation and to protect mitochondria from autophagic degradation (24). In addition to the above functions, mitochondria have recently been linked to autophagosome biogenesis during starvation conditions (25), and it is possible that mitochondrial morphological changes play a role in th...

The final stage of cytokinesis is abscission, the cutting of the narrow membrane bridge connecting two daughter cells. The endosomal sorting complex required for transport (ESCRT) machinery is required for cytokinesis, and ESCRT-III has membrane scission activity in vitro, but the role of ESCRTs in abscission has been undefined. Here, we use structured illumination microscopy and timelapse imaging to dissect the behavior of ESCRTs during abscission. Our data reveal that the ESCRT-I subunit tumor-susceptibility gene 101 (TSG101) and the ESCRT-III subunit charged multivesicular body protein 4b (CHMP4B) are sequentially recruited to the center of the intercellular bridge, forming a series of cortical rings. Late in cytokinesis, however, CHMP4B is acutely recruited to the narrow constriction site where abscission occurs. The ESCRT disassembly factor vacuolar protein sorting 4 (VPS4) follows CHMP4B to this site, and cell separation occurs immediately. That arrival of ESCRT-III and VPS4 correlates both spatially and temporally with the abscission event suggests a direct role for these proteins in cytokinetic membrane abscission.

The ESCRT (Endosomal Sorting Complex Required for Transport) machinery is required for the scission of membrane necks in processes including the budding of HIV-1, and cytokinesis. An essential step in cytokinesis is recruitment of the ESCRT-I complex and the ESCRT associated protein ALIX to the midbody (the structure that tethers two daughter cells) by the protein CEP55. Biochemical experiments show that peptides from ALIX and the ESCRT-I subunit TSG101 compete for binding to the ESCRT and ALIX binding region (EABR) of CEP55. A 2.0 Å crystal structure of EABR bound to an ALIX peptide shows that EABR forms an aberrant dimeric parallel coiled-coil. Bulky and charged residues at the interface of the two central heptad repeats create asymmetry and a single binding site for an ALIX or TSG101 peptide. Both ALIX and ESCRT-I are required for cytokinesis, suggesting that multiple CEP55 dimers are required for function, Cytokinesis, the division of the cytoplasm, is the final step of the M phase of the cell cycle. Cytokinesis begins with the formation of the contractile ring, which drives the growth of the cleavage furrow. Vesicle trafficking components, including the exocyst complex and SNAREs, deliver the additional membrane needed for the cleavage furrow to grow (1-3). When the extension of the furrow ends, the contractile ring disassembles, and a structure known as the midbody remains as the final tether between the two daughter cells. The last step in cytokinesis, the cleavage of the plasma membrane at the midbody, is referred to as abscission. The mechanism of abscission became clearer with the discovery that the midbody protein CEP55 (4-6) recruits two key components of the ESCRT machinery (7-11), the ESCRT-I complex and ALIX (12,13). The role of ALIX and ESCRT-I in abscission appears to be to recruit ESCRT-III subunits, which are required for normal midbody morphology (14) and are widely believed to have a membrane scission activity (15).Deletion analysis of ALIX mapped the interaction with CEP55 to a putative unstructured Prorich sequence near its C-terminus (12,13). Similarly, the TSG101 subunit of ESCRT-I interacts via an unstructured linker between its ubiquitin-binding UEV domain and the region that forms the core complex with other ESCRT-I subunits (12,13). CEP55 is a predominantly coiled-coil protein that otherwise lacks familiar protein:protein interaction domains. The predicted coiledcoil of CEP55 is interrupted near the middle by a ∼60 residue region that has been suggested to serve as a hinger between the N-and C-terminal coiled coil regions (13). Remarkably, thisThe ESCRT machinery, which is required for membrane abscission in cytokinesis, is targeted to the midbody by a coiled-coil in CEP55 that is built around an unusual charged core.

The endosomal sorting complex required for transport (ESCRT)-III complex, capable of polymerization and remodeling, participates in abscission of the intercellular membrane bridge connecting two daughter cells at the end of cytokinesis. Here, we integrate quantitative imaging of ESCRT-III during cytokinetic abscission with biophysical properties of ESCRT-III complexes to formulate and test a computational model for ESCRT-mediated cytokinetic abscission. We propose that cytokinetic abscission is driven by an ESCRT-III fission complex, which arises from ESCRT-III polymerization at the edge of the cytokinetic midbody structure, located at the center of the intercellular bridge. Formation of the fission complex is completed by remodeling and breakage of the ESCRT-III polymer assisted by VPS4. Subsequent spontaneous constriction of the fission complex generates bending deformation of the intercellular bridge membrane. The related membrane elastic force propels the fission complex along the intercellular bridge away from the midbody until it reaches an equilibrium position, determining the scission site. Membrane attachment to the dome-like end-cap of the fission complex drives membrane fission, completing the abscission process. We substantiate the model by theoretical analysis of the membrane elastic energy and by experimental verification of the major model assumptions.

The ESCRT machinery mediates membrane fission in a variety of processes in cells. According to current models, ESCRT-III proteins drive membrane fission by assembling into helical filaments on membranes. Here, we used 3D STORM imaging of endogenous ESCRT-III component IST1 to reveal the evolution of the structural organization of ESCRT-III in mammalian cytokinetic abscission. Using this approach, ESCRT-III ring and spiral assemblies were resolved and characterized at different stages of abscission. Visualization of IST1 structures in cells lacking the microtubule-severing enzyme spastin and in cells depleted of specific ESCRT-III components or the ATPase VPS4 demonstrated the contribution of these components to the organization and function of ESCRTs in cells. This work provides direct evidence that ESCRT-III proteins form helical filaments to mediate their function in cells and raises new mechanistic scenarios for ESCRT-driven cytokinetic abscission.

BackgroundGlycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential.ObjectivesThis study sought to compare compounds interfering with platelet GPVI–atherosclerotic plaque interaction to improve current antiatherothrombotic therapy.MethodsHuman atherosclerotic plaque–induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers.ResultsGPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc–free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release.ConclusionsAnti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences have relevance for clinical trials targeting GPVI-collagen interaction combined with established antiplatelet therapies in patients with spontaneous plaque rupture or intervention-associated plaque injury.

Epithelial cells grown in three dimensional (3D) cultures of extracellular matrix differentiate into a multicellular structure of polarized cells. This process shares many characteristics with the physiological development of an epithelial tissue and the formation of polarity in epithelial cells. Imaging 3D cultures of polarized epithelial cells is therefore a powerful tool to study epithelial architecture and morphogenesis under close to physiological conditions. The new generation of confocal microscopes allows live cell imaging of fluorescently tagged molecules in these cultures. This opens up new opportunities for studying how molecules behave and are distinguished asymmetrically within a 3D setting. This unit discusses technical aspects for culturing and imaging MDCK 3D culture for both fixed 3D cultures and live cell imaging.

Mammalian cell abscission was recently shown to be driven by the ESCRT machinery, but the mechanism has not been fully resolved. This work identifies the ESCRT components ESCRT-II and CHMP6 as essential components of ESCRT-mediated abscission and introduces a new approach for inhibition of abscission using the first 52 amino acids of CHMP6.