@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 {69812, title = {Vimentin filament transport and organization revealed by single-particle tracking and 3D FIB-SEM}, journal = {Journal of Cell Biology}, volume = {224}, year = {2025}, month = {2025 Apr 03}, pages = {e202406054}, abstract = {

Vimentin intermediate filaments (VIFs) form complex, tightly packed networks; due to this density, traditional imaging approaches cannot discern single-filament behavior. To address this, we developed and validated a sparse vimentin-SunTag labeling strategy, enabling single-particle tracking of individual VIFs and providing a sensitive, unbiased, and quantitative method for measuring global VIF motility. Using this approach, we define the steady-state VIF motility rate, showing a constant \~{}8\% of VIFs undergo directed microtubule-based motion irrespective of subcellular location or local filament density. Significantly, our single-particle tracking approach revealed uncorrelated motion of individual VIFs within bundles, an observation seemingly at odds with conventional models of tightly cross-linked bundles. To address this, we acquired high-resolution focused ion beam scanning electron microscopy volumes of vitreously frozen cells and reconstructed three-dimensional VIF bundles, finding that they form only loosely organized, semi-coherent structures from which single VIFs frequently emerge to locally engage neighboring microtubules. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

bioRxiv Preprint:\ https://doi.org/10.1101/2024.06.10.598346

}, issn = {0021-9525}, doi = {10.1083/jcb.202406054}, url = {https://doi.org/10.1083/jcb.202406054}, author = {Renganathan, Bhuvanasundar and Moore, Andrew S. and Yeo, Wei-Hong and Petruncio, Alyson and Ackerman, David and Weigel, Aubrey V. and Team, The CellMap and Pasolli, H. Amalia and Xu, C. Shan and Shtengel, Gleb and Hess, Harald F. and Serpinskaya, Anna S. and Zhang, Hao F. and Lippincott-Schwartz, Jennifer and Gelfand, Vladimir I.} } @article {69491, title = {Transport and Organization of Individual Vimentin Filaments Within Dense Networks Revealed by Single Particle Tracking and 3D FIB-SEM}, journal = {bioRxiv}, year = {2024}, month = {2024 Jun 10}, abstract = {

Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a 50 um3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

}, doi = {10.1101/2024.06.10.598346}, url = {https://www.biorxiv.org/content/early/2024/06/10/2024.06.10.598346}, author = {Renganathan, Bhuvanasundar and Moore, Andrew S. and Yeo, Wei-Hong and Petruncio, Alyson and Ackerman, David and Wiegel, Aubrey and CellMap Team and Pasolli, H. Amalia and Xu, C. Shan and Hess, Harald F and Serpinskaya, Anna S. and Zhang, Hao F. and Lippincott-Schwartz, Jennifer and Gelfand, Vladimir I.} } @article {64823, title = {GCIB-SEM: A path to 10 nm isotropic imaging of cubic millimeter volumes.}, journal = {bioRxiv}, year = {2019}, month = {2019 Feb 28}, abstract = {Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) generates 3D datasets optimally suited for segmentation of cell ultrastructure and automated connectome tracing but is limited to small fields of view and is therefore incompatible with the new generation of ultrafast multibeam SEMs. In contrast, section-based techniques are multibeam-compatible but are limited in z-resolution making automatic segmentation of cellular ultrastructure difficult. Here we demonstrate a novel 3D electron microscopy technique, Gas Cluster Ion Beam SEM (GCIB-SEM), in which top-down, wide-area ion milling is performed on a series of thick sections, acquiring < 10 nm isotropic datasets of each which are then stitched together to span the full sectioned volume. Based on our results, incorporating GCIB-SEM into existing single beam and multibeam SEM workflows should be straightforward and should dramatically increase reliability while simultaneously improving z-resolution by a factor of 3 or more.}, doi = {10.1101/563239}, author = {Hayworth, Kenneth J and Peale, David R and Januszewski, Michal and Knott, Graham and Lu, Zhiyuan and Xu, C. Shan and Hess, Harald F} }