Three-Dimensional Visualization of the Podocyte Actin Network Using Integrated Membrane Extraction, Electron Microscopy, and Machine Learning

Abstract

Background: Actin stress fibers are abundant in cultured cells, but little is known about them in vivo. In podocytes, much evidence suggests that mechanobiologic mechanisms underlie podocyte shape and adhesion in health and in injury, with structural changes to actin stress fibers potentially responsible for pathologic changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. A technology to image the actin cytoskeleton at high resolution is needed to better understand the role of structures such as actin stress fibers in podocytes.

Methods: We developed the first visualization technique capable of resolving the three-dimensional cytoskeletal network in mouse podocytes in detail, while definitively identifying the proteins that comprise this network. This technique integrates membrane extraction, focused ion-beam scanning electron microscopy, and machine learning image segmentation.

Results: Using isolated mouse glomeruli from healthy animals, we observed actin cables and intermediate filaments linking the interdigitated podocyte foot processes to newly described contractile actin structures, located at the periphery of the podocyte cell body. Actin cables within foot processes formed a continuous, mesh-like, electron-dense sheet that incorporated the slit diaphragms.

Conclusions: Our new technique revealed, for the first time, the detailed three-dimensional organization of actin networks in healthy podocytes. In addition to being consistent with the gel compression hypothesis, which posits that foot processes connected by slit diaphragms act together to counterbalance the hydrodynamic forces across the glomerular filtration barrier, our data provide insight into how podocytes respond to mechanical cues from their surrounding environment.

Keywords: actin; cytoskeleton; intermediate filaments; podocyte.

Graphical abstract

Figure 1.

Schematic diagrams show the experimental workflow for the membrane-extraction technique. (1) After anesthesia, mice were perfused through the heart with 4-µm magnetic beads for 3 minutes, after which the kidneys were harvested. (2) (A) Kidneys were minced and (B) passed through a 100-µm cell strainer. (C) Glomeruli were collected by pelleting them in a tube with the assistance of a magnet. (3) Glomeruli were incubated with the membrane-extraction solution for (A) 1.5 minutes before (B) fixing them for 10 minutes in PFA containing membrane-extraction solution. (4) Membrane-extracted glomeruli were used for the appropriate imaging modality.

Figure 2.

SEM of intact and membrane-extracted podocytes show the exposed actin network of the interdigitating foot processes (FPs) after cell membrane removal. (A and B) Low and high magnification SEM images of an intact healthy glomerulus show four podocytes with major processes (MPs) and interdigitating FPs. (C and D) Low and high magnification SEM images of a membrane-extracted healthy glomerulus show a capillary loop with the cytoskeletal filaments of MPs and FPs. Note the podocyte nucleus in the bottom right corner in (C). The zoomed-in image in (D) shows the MPs (arrows), the FPs (arrowheads), and the slit diaphragms in between (asterisks). Note the porous nature of the slit diaphragms. (E and F) Low and high magnification SEM images of a membrane-extracted and fractured healthy capillary show the endothelial cell cytoskeleton (EC-Cytoskeleton, arrows in E), the GBM and FPs (indicated in F) with the slit diaphragms in between (asterisks in F). (G) Low magnification SEM of a membrane-extracted healthy podocyte shows the cell body, the MPs and the FPs. Note the microfilaments surrounding the cell body (arrows). (H and I) QF-DEEM and TEM micrographs of a membrane-extracted healthy capillary wall show the different types of microfilaments in the podocyte FPs and MPs. The microfilaments in the MPs appear as long filaments running above the FPs (arrows in H and I), whereas the FPs contain bundled microfilaments in the QF-DEEM (arrowheads in H) that appear as electron-dense structures on the TEM images (arrowheads in I). (J) Low magnification QF-DEEM image of a membrane-extracted glomerulus shows an en face view of interdigitating FPs (arrows). Note the slit diaphragm areas (asterisks). (K) High magnification image of the boxed area in (J) shows details of the microfilament bundles in the FPs (cyan box, short arrow) and the loose thicker microfilaments above (yellow box, long arrow). Inserts show high magnification views of the microfilaments. (L) Quantification of the thicknesses of the individual filaments in the FPs (represented by the cyan box in [K]) and the long filaments nearby (represented by the yellow box in [K]) are consistent with the first being actin and the latter being intermediate filaments. t test: ****P<0.001.

Figure 3.

Immunolabeling of actin structures in membrane-extracted glomeruli. (A–C and D–F) Z-stack Airyscan super-resolution imaging of a whole membrane-extracted glomerulus using Alexa546-Phallodiin as a stabilizer in the extraction procedure. Superficial (A–C) and deeper (D–F) optical sections of the membrane-extracted glomerulus show phalloidin staining at the glomerular capillary wall areas (A–F, cyan arrows, Cap) and at the periphery of the podocyte cell bodies. Note the phalloidin-positive structures imaged en face in two podocyte cell bodies (green arrows, Pod). (B and E) DIC images show the podocyte cell bodies and the capillary loop areas. (G–H) 3D reconstruction of the Z-stack images in (A–F) shows one podocyte cell body (boxed in G). (H and I) Two 3D views of the reconstructed podocyte cell body (Supplemental Video 1) show the actin cables surrounding the podocyte cell body. (J–M) TEM images of LR-White ultrathin sections of a membrane-extracted glomerulus labeled with an antibody against γ-actin. (J) An en face image of podocyte MP and interdigitating FPs shows actin immunogold labeling only in the FPs (arrows). (K) TEM micrograph shows a capillary wall with the FPs atop the GBM labeled with actin immunogold. Although the microfilaments in the MPs are not labeled, this micrograph demonstrates the electron dense areas in the extracted FPs are actin positive. Note the labeling in the endothelial fenestrae area as well (arrowheads). (L and M) Two high-magnification TEM images show the actin staining in the central actin cables (CA) in the FPs and in the peripheral actin cables (PA) that reach to the slit diaphragm areas. (N–Q) Micrographs showing HMDS-Platinum replica electron microscopy (HMDS-EM) images of membrane-extracted glomeruli labeled with anti–γ-actin immunogold nanoparticles (yellow enhancement). (N) En face HMDS-EM image shows membrane-extracted podocyte FPs labeled with actin immunogold. Note that the slit diaphragms (SDs) bridging adjacent FPs and the GBM areas are both negative for actin immunoGold labeling. (O–Q) Low- and high-magnification HMDS-EM images show membrane-extracted FPs labeled with actin immunogold nanoparticles. (O) Although no staining of GBM, SDs, or intermediate filaments (IFs) was observed, FPs were labeled for actin. (P and Q) High magnification images of the boxed areas in red and yellow in (O) show actin immunogold nanoparticles labeling the actin filaments in the FPs. SDs (asterisks).

Figure 4.

Immunolabeling of IFs in membrane-extracted glomeruli. (A–D) Airyscan images show membrane-extracted glomeruli stained with phalloidin (red), anti-nestin (green), and anti-vimentin (cyan). (A–B) and (C–D) are two different optical sections of the same glomerulus showing partial colocalization of vimentin and nestin in the podocyte cell bodies (Pod) and MPs (arrows) but not in FPs of the capillary walls (Cap). (E and F) TEM images of a LR-White ultrathin section of a membrane-extracted glomerulus labeled with an antibody to vimentin. Low (E) and high (F) magnification TEM images show that the microfilaments in MPs but not in FPs are labeled with anti-vimentin immunogold nanoparticles. (G and H) Low and high magnification HMDS-EM images show a membrane-extracted podocyte labeled with anti-vimentin immunogold nanoparticles (yellow enhanced particles). These images demonstrate that only the microfilaments in MP contain vimentin, and they are therefore IFs.

Figure 5.

FIB-SEM imaging of a healthy membrane-extracted glomerulus. (A) FIB-SEM overview image shows a healthy glomerulus with intact nuclei and GBM. Note the lack of cell membranes and the electron dense actin cables in FPs. (B) Zoomed-in area of the yellow box in (A) shows a capillary wall with intact GBM and FPs. Note the continuity between the FPs and the adjacent SDs (arrows). (B’ and B’’) Two high magnification images taken from the boxed areas in (B) demonstrate the connection between the FP CA and the SD areas (asterisks) via the side actin cables (SA, orange arrows). (C–E) 3D segmentation of 150 frames of the capillary loop in the white boxed area in (A). (C) Top, (D) side, and (E) bottom views of the segmented area demonstrate intact GBM and show the interdigitated FPs.

Figure 6.

FIB-SEM 3D visualization of a membrane-extracted healthy glomerulus by manual segmentation reveals the detailed architecture of the capillary wall. (A and B) One ROI for segmentation of the actin network (boxed area in Supplemental Figure 3) with the two GBM segments colored red in (B). (C and D) Top (C) and side (D) views of the 3D visualization of the GBM in (B) after manual segmentation (approximately 150 frames). (E–G) Three different views of the segmented GBM areas superimposed with an orthogonal image from the FIB-SEM image stack. (H) Manual segmentation of both the podocyte cell body and the actin cables of neighboring podocytes atop the GBM (highlighted in green and blue). The nucleus is yellow and the electron dense patches at the periphery of the cell body are pink. (I) and (J) Two different 3D visualizations of manually segmented GBM, FPs, and cell body. (K) 3D visualization of manually segmented FPs with an orthogonal image from the FIB-SEM image stack. This 3D image demonstrates that the actin cables in the adjacent FPs are interdigitated.

Figure 7.

Manual segmentation of peripheral actin cables surrounding a podocyte cell body and MPs. (A) Overview FIB-SEM single-frame image of a membrane-extracted glomerulus shows two podocyte cell bodies with electron dense patches (red arrows) at the periphery of the cell bodies. (B) One frame of the ROI (boxed in Supplemental Figure 4) shows an example of a peripheral actin patch highlighted for manual segmentation. The peripheral electron-dense patches surrounding the cell bodies is highlighted in magenta, and the GBM is highlighted in white. Two adjacent FPs are highlighted as well, in green and cyan. (C) 3D reconstruction of the segmented patches in (B) shows that they are parts of thick parallel actin cables along the longitudinal axis of the podocyte. (D, E, and F) 3D renderings of the podocyte’s peripheral actin cables and the adjacent GBM show the position of these longitudinal cables relative to the GBM. (G) A FIB-SEM slice of a membrane-extracted podocyte shows a podocyte with two MPs extending to form FPs that attach to two different capillary loops. Similar to the cell bodies, this image demonstrates that the MPs are also surrounded by peripheral electron-dense areas (red arrows). (H–K) Overview 3D visualization (H and I) and zoomed-in views (J and K) of the segmented areas in (G) show thick, branched actin cables around the MPs (red arrows). For orientation, white arrows indicate the openings of the capillaries. Scale: tickmarks in the x- and y-axes are in nm.

Figure 8.

Machine learning-mediated segmentation of FIB-SEM images of a membrane-extracted healthy mouse glomerulus reveals the architecture of the capillary wall. (A) Inverted single-frame FIB-SEM image of a membrane-extracted healthy glomerulus shows the GBM and the CA of the FPs (arrows). (B) Example of the training process for deep learning segmentation of the image in (A) shows five classes assigned to a selected training stack (15 images). (C) The probability map of FP (green) and GBM (red) channels shows the result of the deep learning classifier. (D) Front view of the 3D visualization of approximately 250 FIB-SEM segmented images showing interdigitating podocyte FPs on top of the GBM. (E and F) Bottom and flipped views of the 3D visualization in (D). GBM, yellow; FPs, silver. (G) Inverted single-frame FIB-SEM image of a membrane-extracted glomerulus shows the GBM, the CA of FPs, and the SDs. (H) Example of the training process for deep learning segmentation of the image in (G) shows six classes assigned to a selected training stack (15 images). (I) The probability map of FP (green), GBM (red), and SD (blue) channels shows the result of the deep learning classifier. (J) Front view of the 3D visualization of a podocyte shows the FP with SDs. (K and L) Bottom and flipped views of the 3D visualization in (J). GBM, yellow; FPs, silver; SDs, light blue. Scale: tickmarks in the x- and y-axes are in nm. (M) Schematic summary diagram shows the layout of the actin cables and IFs within the podocytes. (1) Although IFs (cyan lines) are restricted to the cell body and the MPs, actin cables are connected to the SDs (red lines) via the SA (green and magenta lines) that are connected to the CA within the FPs (block dots) and to the actin cables within the MPs (black lines), which in turn connect to one another and to the actin cables at the periphery of the podocyte cell body (thick magenta lines). (2) Representative single frame image of the FIB-SEM stacks demonstrates the actin cables at the periphery of the cell body (magenta). Other structures are highlighted as well: the podocyte cell nucleus (yellow) and adjacent FPs (green and cyan). (3) Representative single frame image of the FIB-SEM stacks shows the actin assembly within the FPs. SA (green arrows) connect the CA (white arrows) to the adjacent SDs (red arrows).