Volume microscopic analysis of membrane contact sites in mouse kidney renal proximal tubule epithelial cells

Abstract

Subcellular organelles communicate with one another and with the plasma membrane (PM). A wide variety of membrane contact sites (MCS) tether subcellular structures to one another and mediate inter-organelle communication and exchange. The nature, distribution and function of membrane contact sites within the segment-specific architecture of renal epithelial cells has only just begun to be elucidated. We have used advanced imaging techniques, including Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and machine learning-based segmentation, to create a comprehensive, high resolution, 3D and quantifiable volume microscopic atlas of the ultrastructure of proximal tubule epithelial cells of the mouse kidney. We applied this data set to identify and quantify MCS between the endoplasmic reticulum and mitochondria and the PM in these cells. FIB-SEM was performed on a 50 μm by 50 μm by 44 μm block of tissue prepared from the renal cortex of a perfusion fixed 12-week-old male mouse. OsO4-stained tissue was embedded in Durcupan and a 3D X-ray tomogram of the block was used to select a volume of interest that included extended lengths of multiple proximal tubule (PT). The volume was imaged by a customized enhanced FIB-SEM system. The X-Y scanning resolution was set to 4-nm, and 4-nm FIB milling was used to expose surfaces for successive imaging. Generation of the complete image stack required ~11,000 cycles of scanning/FIB ablation over ~4 weeks of uninterrupted imaging. Segmentation of the resultant image set was performed with Zeiss Arivis Pro technology. The boundary box for reconstruction had the dimensions of 29.2 μm x 24.8 μm x 8 μm with a volume of 5793.280 μm3 comprising of 2000 consecutive images at an isotropic voxel size of 4 nm. Deep learning segmentation teased apart 10 cells with distinct boundaries in the PT structure. The volume of the cells in our clipped volume ranged from 8.79 μm3 to 1438.53 μm3 with a median of 45.20 μm3. In one representative proximal tubule cell the volume of mitochondria makes up 84.91 μm3 (31.62%) of the cell volume and the volume of Endoplasmic Reticulum (ER) makes up 23.92 μm3 (8.91%) of the cell volume. The 3D reconstruction and quantitative analysis revealed that there is one dominant ER object accounting for most of the ER volume in the whole cell: i.e., the largest ER object takes up 22.37 μm3 or 93.5% of the ER volume in the cell. A post deep-learning processing algorithm used 3D object modifications to identify 11,109 3D MCS between mitochondria and ER in the 10 cells. The Mito-ER MCS ranged from 512 nm3 to 354,304 nm3 with a median volume of 1792 nm3. Visually we can see Mito-ER and Mito-PM MCS extending across a region of up to ~45 nm between the two organelles. Taken together, these data show that the basal lateral surfaces of PT cell PMs are generously invested with fenestrated smooth ER, as are mitochondria and peroxisomes. Extensive regions of direct contact between mitochondria and PM are also observed. The biochemical identities of these contacts and their physiological functions are being actively pursued. NIDDK DK072612 NIDDK DK120534. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.