@article {70013, title = {Compartmentalization of the Endoplasmic Reticulum in Mouse Kidney Proximal Tubule Epithelial Cells}, journal = {Physiology}, volume = {40}, year = {2025}, month = {2025 May 12}, pages = {1280}, abstract = {

The renal proximal tubule plays a critical role in water and solute reabsorption. Recently we generated a high resolution 3D, quantifiable volume microscopic identification of the ultrastructure of kidney Proximal Tubule (PT) cells using enhanced Focused Ion Beam Scanning Electron Microscopy (eFIB-SEM) and machine learning-based segmentation approaches. This analysis revealed that, in a volume of 70x60x177 {\textmu}m3 of mouse kidney tissue, the mean volume of PT cells is 1980.25 {\textmu}m3 {\textpm} 491.28 μm3. In an analysis of 25 PT cells, mitochondria (MITO) and endoplasmic reticulum (ER) accounted for an average of 26.4\% and 6.3\% of cell volume, respectively. Importantly, 91\% of the total ER volume appeared to be comprised of a single contiguous ER structure as determined by tracing the ER surface. Using semi-thin sections (0.5 {\textmu}m) of mouse kidney and antibodies directed against ER proteins we assessed the functional compartmentalization of the ER in PT cells by immunofluorescence microscopy. We find that ER proteins that participate in maintaining ER structure and lipid exchange, such as CLIMP-63 and VAP-A, localize to regions of the ER that are in close apposition to the basolateral plasma membrane (BL PM) of the PT cell. This distribution is confirmed by co-staining with an antibody directed against the Na, K-ATPase, a marker of the BL PM. In contrast, regions of the ER that are involved in calcium ion storage, as detected by staining for the SERCA calcium ATPase, are distributed broadly through the cytoplasm in the area of the cell that is rich in MITO. Staining for mitofilin, a MITO outer membrane protein, confirmed the abundance and distribution of the MITO in all of the PT cells. PDI, a protein that regulates proper folding and maturation of newly synthesized proteins in the lumen of the ER, resides primarily in portions of the ER that surround the nucleus and extend into the apical regions of the cell. PDI is mostly absent from the BL portions of the PT cells. Interestingly, calreticulin, which participates both in ER calcium storage and newly synthesized protein folding and quality control processes, is heavily concentrated in the subapical region of the cell. Using the machine learning algorithm to segment the lumen of the seemingly continuous ER structure demonstrates that, within the limit of resolution of this technique, continuity of ER lumens is limited to discrete patches. The defined distributions of these ER markers demonstrates that the extensive ER network in proximal tubule cells is divided into subdomains with distinct functional capacities and properties. NIH-RC2 DK120534. RDP and EML conributed equally. OAW and MJC contributed equally. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

}, doi = {10.1152/physiol.2025.40.S1.1280}, url = {https://doi.org/10.1152/physiol.2025.40.S1.1280}, author = {Reyna-Neyra, Andrea and Pandya, Raj D. and Lackner, Emily M. and Pang, Song and Li, Wei-Ping and Xu, C. Shan and Zugates, Christopher and Burdyniuk, Mariia and Pandya, Vraj D. and Weisz, Ora A. and Caplan, Michael J.} } @article {70147, title = {Illuminating Renal Proximal Tubule Architecture through High-Resolution Volume EM and Machine Learning Analysis.}, journal = {J Am Soc Nephrol}, year = {2025}, month = {2025 Oct 06}, abstract = {

BACKGROUND: Kidney epithelial cells perform complex vectorial fluid and solute transport at high volumes and rapid rates. Their structural organization both reflects and enables these sophisticated physiological functions. However, our understanding of the nanoscale spatial organization and intracellular ultrastructure that underlies these crucial cellular functions remains limited.

METHODS: To address this knowledge gap, we generated and reconstructed an extensive electron microscopic dataset of renal proximal tubule (PT) epithelial cells at isotropic resolutions down to 4nm. We employed artificial intelligence-based segmentation tools to identify, trace, and measure all major subcellular components. We complemented this analysis with immunofluorescence microscopy to connect subcellular architecture to biochemical function.

RESULTS: Our ultrastructural analysis revealed complex organization of membrane-bound compartments in proximal tubule cells. The apical endocytic system featured deep invaginations connected to an anastomosing meshwork of dense apical tubules, rather than discrete structures. The endoplasmic reticulum displayed distinct structural domains: fenestrated sheets in the basolateral region and smaller, disconnected clusters in the subapical region. We identified, quantified, and visualized membrane contact sites between endoplasmic reticulum, plasma membrane, mitochondria, and apical endocytic compartments. Immunofluorescence microscopy demonstrated distinct localization patterns for endoplasmic reticulum resident proteins at mitochondrial and plasma membrane interfaces.

CONCLUSIONS: This study provides novel insights into proximal tubule cell organization, revealing specialized compartmentalization and unexpected connections between membrane-bound organelles. We identified previously uncharacterized structures, including mitochondria-plasma membrane bridges and an interconnected endocytic meshwork, suggesting mechanisms for efficient energy distribution, cargo processing and structural support. Morphological differences between 4nm and 8nm datasets indicate subsegment-specific specializations within the proximal tubule. This comprehensive open-source dataset provides a foundation for understanding how subcellular architecture supports specialized epithelial function in health and disease.

}, issn = {1533-3450}, doi = {10.1681/ASN.0000000884}, url = {https://doi.org/10.1681/asn.0000000884}, author = {Pandya, Raj D and Lackner, Emily M and Xu, C Shan and Zugates, Christopher and Burdyniuk, Mariia and Reyna-Neyra, Andrea and Pandya, Vraj D and Li, Wei-Ping and Pang, Song and Weisz, Ora A and Caplan, Michael J} } @article {69882, title = {Molecular organization of central cholinergic synapses.}, journal = {Proc Natl Acad Sci U S A}, volume = {122}, year = {2025}, month = {2025 Apr 29}, pages = {e2422173122}, abstract = {

Synapses have undergone significant diversification and adaptation, contributing to the complexity of the central nervous system. Understanding their molecular architecture is essential for deciphering the brain{\textquoteright}s functional evolution. While nicotinic acetylcholine receptors (nAchRs) are widely distributed across metazoan brains, their associated protein networks remain poorly characterized. Using in vivo proximity labeling, we generated proteomic maps of subunit-specific nAchR interactomes in developing and mature brains. Our findings reveal a developmental expansion and reconfiguration of the nAchR interactome. Proteome profiling with genetic perturbations showed that removing individual nAchR subunits consistently triggers compensatory shifts in receptor subtypes, highlighting mechanisms of synaptic plasticity. We also identified the Rho-GTPase regulator Still life (Sif) as a key organizer of cholinergic synapses, with loss of Sif disrupting their molecular composition and structural integrity. These results provide molecular insights into the development and plasticity of central cholinergic synapses, advancing our understanding of synaptic identity conservation and divergence.

}, keywords = {Animals, Brain, Cholinergic Neurons, Drosophila melanogaster, Drosophila Proteins, Neuronal Plasticity, Proteome, Proteomics, Receptors, Nicotinic, Synapses}, issn = {1091-6490}, doi = {10.1073/pnas.2422173122}, url = {https://doi.org/10.1073/pnas.2422173122}, author = {Rosenthal, Justin S and Zhang, Dean and Yin, Jun and Long, Caixia and Yang, George and Li, Yan and Lu, Zhiyuan and Li, Wei-Ping and Yu, Zhiheng and Li, Jiefu and Yuan, Quan} } @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 Jeff L Rhoades 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} }