@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 {67753, title = {An open-access volume electron microscopy atlas of whole cells and tissues.}, journal = {Nature}, volume = {599}, year = {2021}, month = {2021 Nov 1}, pages = {147-51}, abstract = {

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels. Owing to advances in the\ precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm\ voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and\ pose questions.

}, issn = {1476-4687}, doi = {10.1038/s41586-021-03992-4}, author = {Xu, C Shan and Pang, Song and Shtengel, Gleb and M{\"u}ller, Andreas and Ritter, Alex T and Hoffman, Huxley K and Takemura, Shin-ya and Lu, Zhiyuan and Pasolli, H Amalia and Iyer, Nirmala and Chung, Jeeyun and Bennett, Davis and Weigel, Aubrey V and Freeman, Melanie and Van Engelenburg, Schuyler B and Walther, Tobias C and Farese, Robert V and Lippincott-Schwartz, Jennifer and Mellman, Ira and Solimena, Michele and Hess, Harald F} } @article {65659, title = {Cell types promoting goosebumps form a niche to regulate hair follicle stem cells.}, journal = {Cell}, volume = {182}, year = {2020}, month = {2020 Aug 6}, pages = {578}, abstract = {

Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2020.06.031}, author = {Shwartz, Yulia and Gonzalez-Celeiro, Meryem and Chen, Chih-Lung and Pasolli, H Amalia and Sheu, Shu-Hsien and Fan, Sabrina Mai-Yi and Shamsi, Farnaz and Assaad, Steven and Lin, Edrick Tai-Yu and Zhang, Bing and Tsai, Pai-Chi and He, Megan and Tseng, Yu-Hua and Lin, Sung-Jan and Hsu, Ya-Chieh} } @article {65281, title = {Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.}, journal = {Science}, volume = {367}, year = {2020}, month = {2020 Jan 17}, abstract = {

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

}, issn = {1095-9203}, doi = {10.1126/science.aaz5357}, author = {Hoffman, David P and Shtengel, Gleb and Xu, C Shan and Campbell, Kirby R and Freeman, Melanie and Wang, Lei and Milkie, Daniel E and Pasolli, H Amalia and Iyer, Nirmala and Bogovic, John A and Stabley, Daniel R and Shirinifard, Abbas and Pang, Song and Peale, David and Schaefer, Kathy and Pomp, Wim and Chang, Chi-Lun and Lippincott-Schwartz, Jennifer and Kirchhausen, Tom and Solecki, David J and Betzig, Eric and Hess, Harald F} } @article {65658, title = {Microdomains form on the luminal face of neuronal extracellular vesicle membranes.}, journal = {Scientific Reports}, volume = {10}, year = {2020}, month = {2020 Jul 20}, pages = {11953}, abstract = {

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology.

}, issn = {2045-2322}, doi = {10.1038/s41598-020-68436-x}, author = {Matthies, Doreen and Lee, Nathanael Y J and Gatera, Ian and Pasolli, H Amalia and Zhao, Xiaowei and Liu, Hui and Walpita, Deepika and Liu, Zhe and Yu, Zhiheng and Ioannou, Maria S} } @article {51795, title = {Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution.}, journal = {Science (New York, N.Y.)}, volume = {363}, year = {2019}, month = {2019 Jan 18}, pages = {eaau8302}, abstract = {

Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire\ Drosophila\ brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

}, doi = {10.1126/science.aau8302}, author = {Gao, Ruixuan and Asano, Shoh M and Upadhyayula, Srigokul and Pisarev, Igor and Milkie, Daniel E and Liu, Tsung-Li and Singh, Ved and Graves, Austin R and Huynh, Grace H and Zhao, Yongxin and Bogovic, John A and Colonell, Jennifer and Ott, Carolyn M and Zugates, Christopher T and Tappan, Susan and Rodriguez, Alfredo and Mosaliganti, Kishore R and Sheu, Shu-Hsien and Pasolli, H Amalia and et al} } @article {64863, title = {A lipid-based partitioning mechanism for selective incorporation of proteins into membranes of HIV particles.}, journal = {Nature Cell Biology}, volume = {21}, year = {2019}, month = {2019 Apr}, pages = {452-461}, abstract = {

Particles that bud off from the cell surface, including viruses and microvesicles, typically have a unique membrane protein composition distinct from that of the originating plasma membrane. This selective protein composition enables viruses to evade the immune response and infect other cells. But how membrane proteins sort into budding viruses such as human immunodeficiency virus (HIV) remains unclear. Proteins could passively distribute into HIV-assembly-site membranes producing compositions resembling pre-existing plasma-membrane domains. Here, we demonstrate that proteins instead sort actively into HIV-assembly-site membranes, generating compositions enriched in cholesterol and sphingolipids that undergo continuous remodeling. Proteins are recruited into and removed from the HIV assembly site through lipid-based partitioning, initiated by oligomerization of the HIV structural protein Gag. Changes in membrane curvature at the assembly site further amplify this sorting process. Thus, a lipid-based sorting mechanism, aided by increasing membrane curvature, generates the unique membrane composition of the HIV surface.

}, issn = {1476-4679}, doi = {10.1038/s41556-019-0300-y}, author = {Sengupta, Prabuddha and Seo, Arnold Y and Pasolli, H Amalia and Song, Yul Eum and Johnson, Marc and Lippincott-Schwartz, Jennifer} } @article {65067, title = {Neuron-astrocyte metabolic coupling protects against activity-induced fatty acid toxicity.}, journal = {Cell}, volume = {177}, year = {2019}, month = {2019 May 30}, pages = {1522-1535.e14}, abstract = {

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial β-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2019.04.001}, author = {Ioannou, Maria S and Jackson, Jesse and Sheu, Shu-Hsien and Chang, Chi-Lun and Weigel, Aubrey V and Liu, Hui and Pasolli, H Amalia and Xu, C Shan and Pang, Song and Matthies, Doreen and Hess, Harald F and Lippincott-Schwartz, Jennifer and Liu, Zhe} } @article {65156, title = {RNA granules hitchhike on lysosomes for long-distance transport, Using annexin A11 as a molecular tether.}, journal = {Cell}, volume = {179}, year = {2019}, month = {2019 Sep 19}, pages = {147-164.e20}, abstract = {

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In\ vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2019.08.050}, author = {Liao, Ya-Cheng and Fernandopulle, Michael S and Wang, Guozhen and Choi, Heejun and Hao, Ling and Drerup, Catherine M and Patel, Rajan and Qamar, Seema and Nixon-Abell, Jonathon and Shen, Yi and Meadows, William and Vendruscolo, Michele and Knowles, Tuomas P J and Nelson, Matthew and Czekalska, Magdalena A and Musteikyte, Greta and Gachechiladze, Mariam A and Stephens, Christina A and Pasolli, H Amalia and Forrest, Lucy R and St George-Hyslop, Peter and Lippincott-Schwartz, Jennifer and Ward, Michael E} } @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 {49584, title = {Stem cells repurpose proliferation to contain a breach in their niche barrier.}, journal = {eLife}, volume = {7}, year = {2018}, month = {2018 Dec 06}, abstract = {

Adult stem cells are responsible for life-long tissue maintenance. They reside in and interact with specialized tissue microenvironments (niches). Using murine hair follicle as a model, we show that when junctional perturbations in the niche disrupt barrier function, adjacent stem cells dramatically change their transcriptome independent of bacterial invasion and become capable of directly signaling to and recruiting immune cells. Additionally, these stem cells elevate cell cycle transcripts which reduce their quiescence threshold, enabling them to selectively proliferate within this microenvironment of immune distress cues. However, rather than mobilizing to fuel new tissue regeneration, these ectopically proliferative stem cells remain within their niche to contain the breach. Together, our findings expose a potential communication relay system that operates from the niche to the stem cells to the immune system and back. The repurposing of proliferation by these stem cells patch the breached barrier, stoke the immune response and restore niche integrity.

}, issn = {2050-084X}, doi = {10.7554/eLife.41661}, author = {Lay, Kenneth and Yuan, Shaopeng and Gur-Cohen, Shiri and Miao, Yuxuan and Han, Tianxiao and Naik, Shruti and Pasolli, H Amalia and Larsen, Samantha B and Fuchs, Elaine} } @article {48346, title = {Stochastic protein labeling enables long-term single molecule observation in vivo.}, journal = {bioRxiv}, year = {2017}, month = {2017 Mar 13}, abstract = {Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control protein copy number in a cell. This system has a dynamic titration range of more than 10,000 fold, enabling sparse labeling of proteins expressed at widely different levels. Combined with fluorescence signal amplification tags, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in live zebrafish. We found that axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor Sox2 samples clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements for a quantitative understanding of complex control of molecular dynamics in vivo.}, doi = {10.1101/116186}, author = {Liu, Hui and Dong, Peng and Ioannou, Maria S and Li, Li and Shea, Jamien and Pasolli, H Amalia and Grimm, Jonathan B and Rivlin, Patricia K and Lavis, Luke D and Koyama, Minoru and Liu, Zhe} } @article {48752, title = {Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {115}, year = {2017}, month = {2017 Jan 09}, pages = {343-8}, abstract = {

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of \>10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact\ zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics\ in vivo.

}, doi = {10.1073/pnas.1713895115}, author = {Liu, Hui and Dong, Peng and Ioannou, Maria S and Li, Li and Shea, Jamien and Pasolli, H Amalia and Grimm, Jonathan B and Rivlin, Patricia K and Lavis, Luke D and Koyama, Minoru and Liu, Zhe} } @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} }