The slit diaphragm in Drosophila exhibits a bilayered, fishnet architecture

Abstract

The kidney relies on the glomerulus to filter large volumes of blood plasma, with the slit diaphragm (SD) as a key structural component of the glomerular filtration barrier. Despite its central role, the molecular architecture of the SD has remained elusive for decades. Using cryo-electron tomography on focused ion beam-milled Drosophila nephrocytes, an invertebrate podocyte model, we show that the SD exhibits a bilayered fishnet architecture. In the cryo-electron tomography map, we observe criss-crossing strands spanning the extracellular space that can be populated with Sns and Kirre, the Drosophila orthologs of nephrin and Neph1, respectively. We show that sns silencing shortens the SD lines until disappearance, linking the fishnet architecture directly to Sns. After Rab5 silencing, which causes Sns mistrafficking and ectopic formation of the SD, the fishnet pattern also appears ectopically. Elucidating the molecular SD architecture establishes a crucial link between the SD organization and its (patho)physiology.

Fig. 1. Comparative analysis of the nephrocyte slit diaphragm (SD) using light microscopy and cryo-electron tomography.

a The schematic illustrates the anatomy and structure of nephrocytes including the SD, highlighting the two key SD proteins. (b,c) STED microscopy images of nephrocytes expressing Myc-tagged Sns from a CRISPR-edited gene locus. Myc staining reveals a fingerprint-like pattern for the tagged SD protein in a tangential section (b) and a dot-like pattern in a cross-section (c). d Visualization using IMARIS software illustrates the relationship between linear and dot-like patterns based on Airyscan fluorescent microscopy of surface detail from nephrocytes stained for Myc-Sns. e–g Fluorescent microscopy of nephrocytes after co-labeling of (Myc)-Sns and Kirre shows colocalization of both SD proteins (enlarged inset). Nuclei are marked by Hoechst 33342 in blue. h A 3 nm thick computational slice of a low-magnification tomogram shows the cross-section of two nephrocytes. The cytoplasm of the cells is highlighted in light yellow, the basement membrane (BM) in gray. In the cytoplasm, several vesicles and cytoskeletal elements can be seen. Circles indicate regions containing SDs, where Sns and Kirre colocalize. i Computational slice of a tomographic reconstruction at higher magnification (1 nm pixel size) revealing the nephrocyte SD (highlighted by blue arrows and circles). The SD appears textured and not just as two lines connecting the plasma membranes. j Segmentation of the tomogram in (i), displaying the 3D architecture of the nephrocyte close to the cell surface, where SDs are found. Beige: membranes, blue: nephrocyte SDs, gray: basement membrane (BM), bright yellow: ribosomes.

Fig. 2. Cryo-ET reveals that the slit diaphragm (SD) resembles a bilayered fishnet.

a Orthogonal planes through the SD average provide an overview of the 3D geometry. b–d 1 nm thick slices through the tomographic reconstructions show the nephrocyte SD from three different perspectives: b top view revealing the fishnet pattern of the SD between the two plasma membranes (PM); c membrane view revealing two parallel lines of “pearls on a string”; and d classical view displaying two parallel electron-dense lines reflecting the bilayered architecture of the SD. (e–g) 1 nm thick slices through the 3D volume of the SD obtained after subtomogram averaging, shown from the same perspectives as in (b–d) (pixel size: 2.7 Å). The slice showing the membrane view (f) illustrates the cross-section of the fishnet pattern at the center of the fishnet, parallel to the membrane. The slice showing the classical view (g) illustrates one of the cross-sections at the center of the fishnet perpendicular to the direction of the SD line, and thus appears as two dots. The result was obtained by averaging 595 particles selected from 16 tomograms, collected on 3 lamellae from three different Drosophila larvae and three different electron microscopy grids. h–j Isosurface representation of the cryo-ET map illustrating the 3D bilayered fishnet architecture, shown from the same perspectives as in (e–g) (pixel size: 2.7 Å). Individual strands criss-cross the space between the two plasma membranes, creating the fishnet pattern. k Stereo-pair of the SD at an oblique angle showing the bilayered architecture, with each of the layers resembling a fishnet.

Fig. 3. Four possible configurations of Sns and Kirre within the slit diaphragm (SD).

a Blueprint of the densities observed by cryo-ET. In the middle of the SD, a prominent rhombus with an axis length of ~17 nm and an angle of ~110 degrees can be seen. All relevant dimensions and spacings are indicated. b Kinked arrangement of Sns and Kirre, as observed in the X-ray structure by Özkan et al. c Stretched arrangement, where Sns and Kirre interact at an angle of 180°. d Four possible molecular configurations based on the fishnet pattern of the SD. Arrows indicate potential interaction interfaces between criss-crossing Ig domains.

Fig. 4. Reduction of Sns to intermediate levels reveals shorter slit diaphragm (SD) lines, while the fishnet architecture of the SD remains.

a–c Fluorescence microscopy images acquired in Airyscan mode of nephrocytes after transient silencing of sns illustrate the reduction in the level of Sns (nephrin) protein, indicated by shortened lines of the SD. d–f Cryo-electron tomography confirmed the shorter SD lines, spanned by a fishnet pattern that is identical to the SD in wild-type nephrocytes. d 1 nm thick computational slice of a tomographic reconstruction illustrating a short SD line spanned by a fishnet-like SD. e Different region of interest of the same tomographic reconstruction as in (d), revealing the end or onset of two SD lines. f Zoom-in of the end/onset of the left line in (e). For Dot;Gal80^ts>sns-RNAi nephrocytes, six tomograms displaying SDs could be acquired on 2 lamellae from two different Drosophila larvae on one electron microscopy grid.

Fig. 5. In the absence of Sns, the fishnet architecture of the slit diaphragm (SD) is abolished.

a,b Fluorescence microscopy of nephrocytes expressing sns-RNAi persistently reveals a finely dotted pattern of Kirre in tangential sections (a) while the SD protein largely maintains an association with the cell surface, as indicated by cross-sections (b). c RT-TEM image shows detail of a nephrocyte expressing sns-RNAi. In the absence of Sns, SD formation is abrogated while electron-dense pits are formed on the surface. d A slice through the low-magnification tomographic reconstruction of a cross-section of two sns knockdown nephrocytes. No SD lines or labyrinthine channels can be identified on the cell surface. e A slice through the high-magnification tomographic reconstruction revealing electron-dense clusters close to the outer membrane of the nephrocyte. f Segmentation illustrates the 3D geometry of the clusters inside the plasma membrane of the nephrocyte (blue: clusters, beige: plasma membrane). For pros>sns-RNAi nephrocytes, 16 tomograms were acquired at the edge of the nephrocytes, where SDs would be expected to be formed. Tomograms were acquired on 4 lamellae of 3 Drosophila larvae on three different electron microscopy grids. Clusters could be found on two tomograms, no SD-like structures or channels could be seen on the other 14 tomograms.

Fig. 6. The fishnet pattern appears ectopically upon Rab5-associated disruption of sns trafficking.

a,b Fluorescence microscopy of nephrocytes after short-term expression of Rab5-RNAi for 18 h shows a partially maintained SD pattern in a tangential section (a) and ectopic Sns protein protruding in lines from the nephrocyte surface in a cross-section (b). c An RT-TEM image of two labyrinthine channels of a nephrocyte expressing Rab5-RNAi for 18 h reveals ectopic SD formation. d A slice through the high-magnification tomographic reconstruction after cryo-ET of a nephrocyte expressing Rab5-RNAi shows the SD at the cell surface in top view. Insert: The zoom-in reveals the fishnet pattern, similar to that found in the wild-type nephrocyte. e A slice through the low-magnification tomographic reconstruction, where SDs can be found translocated towards the interior of the cell. f A slice of the high-magnification tomographic reconstruction of the same region of interest as in (e). Insert: The zoom-in reveals classical views of the SDs found in the nephrocyte expressing Rab5-RNAi. For Rab5-RNAi nephrocytes, seven tomograms displaying SDs could be acquired on 2 lamellae from two different Drosophila larvae on one electron microscopy grid.