COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.DOI: http://dx.doi.org/10.7554/eLife.26691.001

Phagosome maturation is defined as the process by which phagosomes fuse sequentially with endosomes and lysosomes to acquire an acidic pH and hydrolases that degrade ingested particles. While the essential role of actin cytoskeleton remodeling during particle internalization is well established, its role during the later stages of phagosome maturation remains largely unknown. We have previously shown that purified mature phagosomes assemble F-actin at their membrane, and that the ezrin-radixin-moesin (ERM) proteins ezrin and moesin participate in this process. Moreover, we provided evidence that actin assembly on purified phagosomes stimulates their fusion with late endocytic compartments in vitro. In this study, we further investigated the role of ezrin in phagosome maturation. We engineered a structurally open form of ezrin and demonstrated that ezrin binds directly to the actin assembly promoting factor N-WASP (Neural WiskottAldrich Syndrome Protein) by its FERM domain. Using a cell-free system, we found that ezrin stimulates F-actin assembly on purified phagosomes by recruiting the N-WASP-Arp2/3 machinery. Accordingly, we showed that the down-regulation of ezrin activity in macrophages by a dominant-negative approach caused reduced F-actin accumulation on maturing phagosomes. Furthermore, using fluorescence and electron microscopy, we found that ezrin is required for the efficient fusion between phagosomes and lysosomes. Live-cell imaging analysis supported the notion that ezrin is necessary for the fusogenic process itself, promoting the transfer of the lysosome content into the phagosomal lumen.

During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

14During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of 15 pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial 16 membrane and thereby permeabilizing it. However, it remains unclear how outer 17 membrane openings form. Here, we combined different correlative microscopy and 18 electron cryo-tomography approaches to visualize the effects of Bax activity on 19 mitochondria in human cells. Our data show that Bax clusters localize near outer 20 membrane ruptures of highly variable size. Bax clusters contain structural elements 21 suggesting a higher-order organization of their components. Furthermore, unfolding of 22 inner membrane cristae is coupled to changes in the supramolecular assembly of ATP 23 synthases, particularly pronounced at membrane segments exposed to the cytosol by 24 ruptures. Based on our results, we propose a comprehensive model in which molecular 25 reorganizations of the inner membrane and sequestration of outer membrane 26

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments.

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During brain development, human axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly inaccessible to imaging at molecular resolution in a near-native context. Here we apply cryo-correlative light microscopy and electron tomography to growing axonal tracts from human cerebral organoids. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These data provide an unprecedented resource and reveal a molecular architecture that helps explain the unique biology of growing human axons.