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The endosomal sorting complex required for transport (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 human immunodeficiency virus 1, and for membrane abscission in cytokinesis. In Saccharomyces cerevisiae, ESCRT-III consists of Vps20, Snf7, Vps24 and Vps2 (also known as Did4), which assemble in that order and require the ATPase Vps4 for their disassembly. In this study, the ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Here we show that three subunits of ESCRT-III, Vps20, Snf7 and Vps24, are 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 ESCRT machinery is essential for several fundamental cellular pathways (1-7). The biogenesis of lysosomes involves the maturation of early endosomes into multivesicular bodies (MVBs) (1,2,5). In this pathway, portions [...]

Author(s): Alex M. Valm [1]; Sarah Cohen [1]; Wesley R. Legant [2]; Justin Melunis [3]; Uri Hershberg [3, 4]; Eric Wait [5]; Andrew R. Cohen [5]; Michael W. Davidson [6]; [...]

Author(s): John F. Presley [1, 2]; Theresa H. Ward [1]; Andrea C. Pfeifer [1]; Eric D. Siggia [3]; Robert D. Phair [4]; Jennifer Lippincott-Schwartz (corresponding author) [1] Cytosolic coat proteins [...]

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180–microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.The endoplasmic reticulum proteins CLIMP63, kinectin and p180 bind preferentially to subsets of microtubules with different post-translational modifications, thereby linking the ‘tubulin code’ to the intracellular distribution of membrane organelles. [ABSTRACT FROM AUTHOR]

Cells contain hundreds of organelles and macromolecular assemblies. Obtaining a complete understanding of their intricate organization requires the nanometre-level, three-dimensional reconstruction of whole cells, which is only feasible with robust and scalable automatic methods. Here, to support the development of such methods, we annotated up to 35 different cellular organelle classes—ranging from endoplasmic reticulum to microtubules to ribosomes—in diverse sample volumes from multiple cell types imaged at a near-isotropic resolution of 4 nm per voxel with focused ion beam scanning electron microscopy (FIB-SEM)1. We trained deep learning architectures to segment these structures in 4 nm and 8 nm per voxel FIB-SEM volumes, validated their performance and showed that automatic reconstructions can be used to directly quantify previously inaccessible metrics including spatial interactions between cellular components. We also show that such reconstructions can be used to automatically register light and electron microscopy images for correlative studies. We have created an open data and open-source web repository, ‘OpenOrganelle’, to share the data, computer code and trained models, which will enable scientists everywhere to query and further improve automatic reconstruction of these datasets.Focused ion beam scanning electron microscopy (FIB-SEM) combined with deep-learning-based segmentation is used to produce three-dimensional reconstructions of complete cells and tissues, in which up to 35 different organelle classes are annotated. [ABSTRACT FROM AUTHOR]

Proteins from the endoplasmic reticulum (ER) are channelled through the Golgi complex before being transported to their final destinations. It is now possible to follow the entire life history of a single transport intermediates in living cells, through new florescent tagging techniques. ER-to-Golgi transport has been visualized through the use of tso45 VSVG, a viral glycoprotein, tagged with green fluorescent protein.