@article {69351, title = {COPII with ALG2 and ESCRTs control lysosome-dependent microautophagy of ER exit sites.}, journal = {Dev Cell}, year = {2024}, month = {2024 Apr 06}, abstract = {

Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca\ is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed\ by\ super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism\ was ESCRT dependent and required\ ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In\ vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress.

}, issn = {1878-1551}, doi = {10.1016/j.devcel.2024.03.027}, author = {Liao, Ya-Cheng and Pang, Song and Li, Wei-Ping and Shtengel, Gleb and Choi, Heejun and Schaefer, Kathy and Xu, C Shan and Lippincott-Schwartz, Jennifer} } @article {69602, title = {Data Release: High-Resolution Imaging and Segmentation of P7 Mouse Tissue Microarchitecture Using FIB-SEM and Machine Learning}, journal = {bioRxiv}, year = {2024}, month = {2024 Sep 07}, abstract = {

This report presents a comprehensive data release exploring the tissue microarchitecture of P7 aged mice using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) combined with machine learning-based segmentations of nuclei. The study includes high-resolution 3D volumes and nucleus segmentations for seven vital tissues{\textemdash}pancreas, liver, kidney, heart, thymus, hippocampus, and skin{\textemdash}from a single mouse. The detailed datasets are openly accessible on OpenOrganelle.org, providing a valuable resource for the scientific community to support further research and collaboration.

}, doi = {10.1101/2024.09.05.611438}, url = {https://www.doi.org/10.1101/2024.09.05.611438}, author = {Ackerman, David and Avetissian, Emma and Bleck, Christopher K. E. and Bogovic, John A. and Innerberger, Michael and Korff, Wyatt and Li, Wei-Ping and Lu, Zhiyuan and Petruncio, Alyson and Preibisch, Stephan and Qiu, Wei and Rhoades, Jeff and Saalfeld, Stephan and Silva, Malan and Trautman, Eric T. and Vorimo, Rebecca and Weigel, Aubrey and Yu, Zhiheng and Zubov, Yurii and ,} } @article {69402, title = {NMDAR-mediated activation of pannexin1 channels contributes to the detonator properties of hippocampal mossy fiber synapses.}, journal = {iScience}, volume = {27}, year = {2024}, month = {2024 Apr 06}, pages = {109681}, abstract = {

Pannexins are large-pore ion channels expressed throughout the mammalian brain that participate in various neuropathologies; however, their physiological roles remain obscure. Here, we report that pannexin1 channels (Panx1) can be synaptically activated under physiological recording conditions in rodent acute hippocampal slices. Specifically, NMDA receptor (NMDAR)-mediated responses at the mossy fiber to CA3 pyramidal cell synapse were followed by a slow postsynaptic inward current that could activate CA3 pyramidal cells but was absent in Panx1 knockout mice. Immunoelectron microscopy revealed that Panx1 was localized near the postsynaptic density. Further, Panx1-mediated currents were potentiated by metabotropic receptors and bidirectionally modulated by burst-timing-dependent plasticity of NMDAR-mediated transmission. Lastly, Panx1 channels were preferentially recruited when NMDAR activation enters a supralinear regime, resulting in temporally delayed burst-firing. Thus, Panx1 can contribute to synaptic amplification and broadening the temporal associativity window for co-activated pyramidal cells, thereby supporting the auto-associative functions of the CA3 region.

}, issn = {2589-0042}, doi = {10.1016/j.isci.2024.109681}, author = {Rangel-Sandoval, Cinthia and Soula, Marisol and Li, Wei-Ping and Castillo, Pablo E and Hunt, David L} } @article {67668, title = {Optimization of Fluorescent Proteins and Techniques for In-resin Correlative Light and Electron Microscopy}, journal = {Microscopy and Microanalysis}, volume = {26}, year = {2020}, month = {07/2020}, pages = {1036{\textendash}1039}, abstract = {

Correlative light and electron microscopy (CLEM) combines the power of electron microscopy, with its excellent resolution and contrast, with that of fluorescence imaging, which allows the staining of specific molecules, organelles, and cell populations. Fluorescence imaging is also readily compatible with live cells and behaving animals, facilitating real-time visualization of cellular processes, potentially followed by electron microscopic reconstruction. Super-resolution single-molecule localization microscopy is a relatively new modality that harnesses the ability of some fluorophores to photoconvert, through which localization precision better than Abbe{\textquoteright}s diffraction limit is achieved through iterative high-resolution localization of single-molecule emitters. Here we describe our lab{\textquoteright}s recent progress in the development of reagents and techniques for super-resolution single-molecule localization CLEM and their applications to biological problems.

}, doi = {10.1017/S143192762001675X}, author = {Paez-Segala, Maria and Wang, Yalin and Iyer, Nirmala and Li, Wei-Ping and Rivlin, Patricia and Looger, Loren} } @article {67669, title = {Development and Applications of Fluorescent Proteins for Correlative Light and Electron Microscopy}, journal = {Microscopy and Microanalysis}, volume = {24}, year = {2018}, month = {01/2018}, pages = {2318 - 2319}, abstract = {

Recent advances in super-resolution microscopy have pushed the resolution limit of light microscopy closer to that of electron microscopy. However, as they invariably rely on fluorescence, light microscopy techniques only visualize whatever gets labeled. On the other hand, while electron microscopy reveals cellular structures at the highest resolution, it offers no specificity. The information gap between the two imaging modalities can only be bridged by correlative light and electron microscopy (CLEM). Previously we have developed a probe (mEos4) whose fluorescence and photoconversion survive 0.5-1\% OsO4 fixation, allowing super-resolution visualization of organelles and fused proteins in the context of resinembedded ultrastructure in both transmission EM (TEM) and scanning EM (SEM) [1,2].

}, issn = {1431-9276}, doi = {10.1017/S1431927618012072}, url = {https://www.cambridge.org/core/product/identifier/S1431927618012072/type/journal_article}, author = {Paez-Segala, Maria G. and Wang, Yalin and Iyer, Nirmala and Li, Wei-Ping and Rivlin, Patricia K. and Looger, Loren L.} } @article {46576, title = {High-performance probes for light and electron microscopy.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Apr 27}, pages = {568-76}, abstract = {

We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These {\textquoteright}spaghetti monster{\textquoteright} fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3365}, author = {Viswanathan, Sarada and Williams, Megan E. and Bloss, Erik B. and Stasevich, Timothy J. and Speer, Colenso M. and Nern, Aljoscha and Pfeiffer, Barret D. and Hooks, Bryan M. and Li, Wei-Ping and English, Brian P. and Tian, Teresa and Henry, Gilbert L. and Macklin, John J. and Patel, Ronak and Gerfen, Charles R and Zhuang, Xiaowei and Wang, Yalin and Rubin, Gerald M and Looger, Loren L} } @article {45696, title = {A genetically specified connectomics approach applied to long-range feeding regulatory circuits.}, journal = {Nature Neuroscience}, volume = {17}, year = {2014}, month = {2014 Dec}, pages = {1830-9}, abstract = {

Synaptic connectivity and molecular composition provide a blueprint for information processing in neural circuits. Detailed structural analysis of neural circuits requires nanometer resolution, which can be obtained with serial-section electron microscopy. However, this technique remains challenging for reconstructing molecularly defined synapses. We used a genetically encoded synaptic marker for electron microscopy (GESEM) based on intra-vesicular generation of electron-dense labeling in axonal boutons. This approach allowed the identification of synapses from Cre recombinase-expressing or GAL4-expressing neurons in the mouse and fly with excellent preservation of ultrastructure. We applied this tool to visualize long-range connectivity of AGRP and POMC neurons in the mouse, two molecularly defined hypothalamic populations that are important for feeding behavior. Combining selective ultrastructural reconstruction of neuropil with functional and viral circuit mapping, we characterized some basic features of circuit organization for axon projections of these cell types. Our findings demonstrate that GESEM labeling enables long-range connectomics with molecularly defined cell types.

}, issn = {1546-1726}, doi = {10.1038/nn.3854}, author = {Atasoy, Deniz and Betley, J Nicholas and Li, Wei-Ping and Su, Helen H and Sertel, Sinem M and Scheffer, Louis K and Simpson, Julie H and Fetter, Richard D and Sternson, Scott M} }