@article {69169, title = {Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.}, journal = {Nature}, volume = {626}, year = {2024}, month = {2024 Jan 24}, pages = {169-176}, abstract = {

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

}, keywords = {Amyotrophic Lateral Sclerosis, Endoplasmic Reticulum, Humans, Mitochondria, Mitochondrial Membranes, Signal Transduction, Vesicular Transport Proteins}, issn = {1476-4687}, doi = {10.1038/s41586-023-06956-y}, author = {Obara, Christopher J and Nixon-Abell, Jonathon and Moore, Andrew S and Riccio, Federica and Hoffman, David P and Shtengel, Gleb and Xu, C Shan and Schaefer, Kathy and Pasolli, H Amalia and Masson, Jean-Baptiste and Hess, Harald F and Calderon, Christopher P and Blackstone, Craig and Lippincott-Schwartz, Jennifer} } @article {67844, title = {ER proteins decipher the tubulin code to regulate organelle distribution.}, journal = {Nature}, volume = {601}, year = {2022}, month = {2022 Jan 01}, pages = {132-138}, abstract = {

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions. 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 cytoplasm, forming abundant contacts with other organelles. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer. 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.

}, issn = {1476-4687}, doi = {10.1038/s41586-021-04204-9}, author = {Zheng, Pengli and Obara, Christopher J and Szczesna, Ewa and Nixon-Abell, Jonathon and Mahalingan, Kishore K and Roll-Mecak, Antonina and Lippincott-Schwartz, Jennifer and Blackstone, Craig} } @article {67988, title = {Transverse endoplasmic reticulum expansion in hereditary spastic paraplegia corticospinal axons.}, journal = {Human Molecular Genetics}, year = {2022}, month = {2022 Mar 26}, abstract = {

Hereditary spastic paraplegias (HSPs) comprise a large group of inherited neurologic disorders affecting the longest corticospinal axons (SPG1-86 plus others), with shared manifestations of lower extremity spasticity and gait impairment. Common autosomal dominant HSPs are caused by mutations in genes encoding the microtubule-severing ATPase spastin (SPAST; SPG4), the membrane-bound GTPase atlastin-1 (ATL1; SPG3A), and the reticulon-like, microtubule-binding protein REEP1 (REEP1; SPG31). These proteins bind one another and function in shaping the tubular endoplasmic reticulum (ER) network. Typically, mouse models of HSPs have mild, later-onset phenotypes, possibly reflecting far shorter lengths of their corticospinal axons relative to humans. Here, we have generated a robust, double mutant mouse model of HSP in which atlastin-1 is genetically modified with a K80A knock-in (KI) missense change that abolishes its GTPase activity, while its binding partner Reep1 is knocked out. Atl1KI/KI/Reep1-/- mice exhibit early-onset and rapidly progressive declines in several motor function tests. Also, ER in mutant corticospinal axons dramatically expands transversely and periodically in a mutation dosage-dependent manner to create a ladder-like appearance, based on reconstructions of focused ion beam-scanning electron microscopy datasets using machine learning-based auto-segmentation. In lockstep with changes in ER morphology, axonal mitochondria are fragmented and proportions of hypophosphorylated neurofilament H and M subunits are dramatically increased in Atl1KI/KI/Reep1-/- spinal cord. Co-occurrence of these findings links ER morphology changes to alterations in mitochondrial morphology and cytoskeletal organization. Atl1KI/KI/Reep1-/- mice represent an early-onset rodent HSP model with robust behavioral and cellular readouts for testing novel therapies.

}, issn = {1460-2083}, doi = {10.1093/hmg/ddac072}, author = {Zhu, Peng-Peng and Hung, Hui-Fang and Batchenkova, Natalia and Nixon-Abell, Jonathon and Henderson, James and Zheng, Pengli and Renvois{\'e}, Benoit and Pang, Song and Xu, C Shan and Saalfeld, Stephan and Funke, Jan and Xie, Yuxiang and Svara, Fabian and Hess, Harald F and Blackstone, Craig} } @article {68298, title = {Transverse endoplasmic reticulum expansion in hereditary spastic paraplegia corticospinal axons.}, journal = {Human Molecular Genetics}, volume = {31}, year = {2022}, month = {2022 Aug 23}, pages = {2779-2795}, abstract = {

Hereditary spastic paraplegias (HSPs) comprise a large group of inherited neurologic disorders affecting the longest corticospinal axons (SPG1-86 plus others), with shared manifestations of lower extremity spasticity and gait impairment. Common autosomal dominant HSPs are caused by mutations in genes encoding the microtubule-severing ATPase spastin (SPAST; SPG4), the membrane-bound GTPase atlastin-1 (ATL1; SPG3A) and the reticulon-like, microtubule-binding protein REEP1 (REEP1; SPG31). These proteins bind one another and function in shaping the tubular endoplasmic reticulum (ER) network. Typically, mouse models of HSPs have mild, later onset phenotypes, possibly reflecting far shorter lengths of their corticospinal axons relative to humans. Here, we have generated a robust, double mutant mouse model of HSP in which atlastin-1 is genetically modified with a K80A knock-in (KI) missense change that abolishes its GTPase activity, whereas its binding partner Reep1 is knocked out. Atl1KI/KI/Reep1-/- mice exhibit early onset and rapidly progressive declines in several motor function tests. Also, ER in mutant corticospinal axons dramatically expands transversely and periodically in a mutation dosage-dependent manner to create a ladder-like appearance, on the basis of reconstructions of focused ion beam-scanning electron microscopy datasets using machine learning-based auto-segmentation. In lockstep with changes in ER morphology, axonal mitochondria are fragmented and proportions of hypophosphorylated neurofilament H and M subunits are dramatically increased in Atl1KI/KI/Reep1-/- spinal cord. Co-occurrence of these findings links ER morphology changes to alterations in mitochondrial morphology and cytoskeletal organization. Atl1KI/KI/Reep1-/- mice represent an early onset rodent HSP model with robust behavioral and cellular readouts for testing novel therapies.

}, keywords = {Animals, Axons, Disease Models, Animal, Endoplasmic Reticulum, GTP Phosphohydrolases, Humans, Membrane Proteins, Membrane Transport Proteins, Mice, Mice, Knockout, Mutation, Spastic Paraplegia, Hereditary, Spastin}, issn = {1460-2083}, doi = {10.1093/hmg/ddac072}, author = {Zhu, Peng-Peng and Hung, Hui-Fang and Batchenkova, Natalia and Nixon-Abell, Jonathon and Henderson, James and Zheng, Pengli and Renvois{\'e}, Benoit and Pang, Song and Xu, C Shan and Saalfeld, Stephan and Funke, Jan and Xie, Yuxiang and Svara, Fabian and Hess, Harald F and Blackstone, Craig} } @article {64795, title = {Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III.}, journal = {Journal of Cell Biology}, volume = {218}, year = {2019}, month = {2019 Jun 21}, pages = {2583-99}, abstract = {

Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two inter-related mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1 and CHMP1B modifying LD membrane morphology. Furthermore, M1 Spastin, IST1 and CHMP1B are all required to relieve LDs of lipid peroxidation. The roles of M1 Spastin in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may help explain the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.

}, doi = {10.1101/544023}, author = {Chang, Chi-Lun and Weigel, Aubrey V and Ioannou, Maria S and Pasolli, H Amalia and Xu, C Shan and Peale, David R and Shtengel, Gleb and Freeman, Melanie and Hess, Harald F and Blackstone, Craig and Lippincott-Schwartz, Jennifer} } @article {47969, title = {Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER.}, journal = {Science (New York, N.Y.)}, volume = {354}, year = {2016}, month = {2016 Oct 28}, pages = {433-46}, abstract = {

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that plays crucial roles in numerous cellular functions. We used emerging superresolution imaging technologies to clarify the morphology and dynamics of the peripheral ER, which contacts and modulates most other intracellular organelles. Peripheral components of the ER have classically been described as comprising both tubules and flat sheets. We show that this system consists almost exclusively of tubules at varying densities, including structures that we term ER matrices. Conventional optical imaging technologies had led to misidentification of these structures as sheets because of the dense clustering of tubular junctions and a previously uncharacterized rapid form of ER motion. The existence of ER matrices explains previous confounding evidence that had indicated the occurrence of ER {\textquotedblleft}sheet{\textquotedblright} proliferation after overexpression of tubular junction{\textendash}forming proteins.

}, doi = {10.1126/science.aaf3928}, author = {Nixon-Abell, Jonathon and Obara, Christopher J and Weigel, Aubrey V and Li, Dong and Legant, Wesley R and Xu, CShan and Pasolli, H Amalia and Harvey, Kirsten and Hess, Harald F and Betzig, Eric and Blackstone, Craig and Lippincott-Schwartz, Jennifer} }