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The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is controversial, and dedicated factors governing the morphology of the mitotic ER have remained elusive. Here, we describe the ER membrane proteins REEP3 and REEP4 as major determinants of ER morphology in metaphase cells. REEP3/4 are specifically required for generating the high-curvature morphology of mitotic ER and promote ER tubulation through their reticulon homology domains (RHDs). This ER-shaping activity of REEP3/4 is distinct from their previously described function to clear ER from metaphase chromatin. We further show that related REEP proteins do not contribute to mitotic ER shaping and provide evidence that the REEP3/4 carboxyterminus mediates regulation of the proteins. These findings confirm that ER converts to higher curvature during mitosis, identify REEP3/4 as specific and crucial morphogenic factors mediating ER tubulation during mitosis, and define the first cell cycle-specific role for RHD proteins.

International audience; MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles.

Dynamic actin filament arrays localize to polygonal spaces between ER sheets and tubules and regulate lateral movement and transformations of sheets, that is, sheet persistence, and thereby the characteristic ER network architecture in cultured mammalian cells. Myosin 1c has a role in creating and/or maintaining ER-associated actin arrays., The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

During mitosis, ER network reorganization can lead to packing of the ER into tight concentric layers at the cell cortex and occurs in tandem with rounding of the cell. Morphometric and 3D EM analysis shows that in addition to reorganization, ER sheets undergo transformation toward more fenestrated and tubular forms before anaphase in mammalian cells., The endoplasmic reticulum (ER) is both structurally and functionally complex, consisting of a dynamic network of interconnected sheets and tubules. To achieve a more comprehensive view of ER organization in interphase and mitotic cells and to address a discrepancy in the field (i.e., whether ER sheets persist, or are transformed to tubules, during mitosis), we analyzed the ER in four different mammalian cell lines using live-cell imaging, high-resolution electron microscopy, and three dimensional electron microscopy. In interphase cells, we found great variation in network organization and sheet structures among different cell lines. In mitotic cells, we show that the ER undergoes both spatial reorganization and structural transformation of sheets toward more fenestrated and tubular forms. However, the extent of spatial reorganization and sheet-to-tubule transformation varies among cell lines. Fenestration and tubulation of the ER correlates with a reduced number of membrane-bound ribosomes.

The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied inSaccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm. In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids. The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine. Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM. The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network. In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance. Nucleoids did not colocalize with dynamin-related protein 1, Drp1, a protein of the mitochondrial fission machinery.