Phospholipid scramblase 1: an essential component of the nephrocyte slit diaphragm

Abstract

Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca²⁺-binding sites in Scramb1 are essential for its activity raising the possibility that Ca²⁺ signaling may control the assembly of SDs by impacting on Scramb1 activity.

Keywords: Intercellular junction; Membrane dynamics; Slit diaphragm.

Fig. 1

scramb1 expression and subcellular localization. (A-B) scramb1 is expressed in the garland nephrocytes of stage 15 Drosophila embryos (A, arrow) and of third instar larvae (B) as detected by in situ hybridization. (C-E’) Distribution of Scramb1-A-V5 driven by the indicated Gal4 lines (anti-V5 antibody) and Duf in garland nephrocytes of third instar (C-D’) and first instar (E-E’) larvae shown at a medial section (C-C’) and at higher magnifications at cortical levels (D-E’). Scramb1-A-V5 colocalizes with Duf in SDs (arrows). Asterisks in C and C’ point to cytoplasmic aggregates. (C) Nuclei were stained with DAPI (blue)

Fig. 2

scramb1 loss of function phenotype. (A-B’) Immunostaining of wild-type (A-A’) and scramb1⁴³ (B-B’) garland nephrocytes depicting the distribution of the SD proteins Duf and Pyd. In scramb1⁴³ nephrocytes, SD strands are sparse (arrow, expressing Duf and Pyd). Pyd predominantly accumulates in cortical foci devoid of Duf (arrowheads). Furthermore, Duf and Pyd colocalize in certain regions of contact between clustered nephrocytes (asterisk). (C-C’) Immunostaining of scramb1⁴³ larval nephrocytes phenotypically rescued by the expression of UAS-scramb1-A-V5 driven by pros-Gal4, to show the expression of Duf and Pyd, as indicated. SD strands cover the entire nephrocyte surface. (A, B and C) medial planes. (A’, B’ and C’) cortical planes. (D-F) TEM images of scramb1⁴³ nephrocytes. An overview of a complete nephrocyte is shown in D (n: nucleus). The highlighted region is shown at higher magnification in E. Electron-dense plaques (black arrowheads in D and E) that bridge the plasma membrane with sub-cortical lacunae are frequently observed. (F) Tangential section through the nephrocyte cortex displaying electron-dense circular structures (black arrowheads) that might correspond to the cortical electron-dense plaques observed in cross-sections. Occasional SDs are also observed (D, red arrows. See also Fig. S2 G). (G) TEM image of a scramb1⁴³ mutant nephrocyte rescued by the expression of UAS-scramb1-A-V5, displaying a normal density of SDs (red arrows). Blue arrowheads in E and G point to clathrin coated vesicles and pits. (H) Immunogold labelling of Scramb1-A-V5 (anti-V5 antibody) in a nephrocyte of the same genotype as in G, showing that gold particles associate with SDs (red arrows). Statistical analysis described in the Methods section

Fig. 3

Time-course analysis of the induction of SD formation by Scramb1-A. (A-B) Immunostaining of scramb1⁴³ or scramb1⁴³ nephrocytes rescued by the expression of UAS-scramb1-A-V5 for increasing periods of time (0, 12 and 18 h, as indicated) using the TARGET technology. See the Methods section for the complete genotype. The distribution of Scramb1-A-V5 (anti-V5 antibody), Sns, Pyd, Duf and phospho-Src64B in the cortical region are shown, as indicated. Each image corresponds to a Z-projection of several cortical planes. No SD strands are observed in nephrocytes that do not express UAS-scramb1-A-V5. Instead, abundant cortical foci containing Pyd, Sns and phospho-Src64B cover the nephrocyte surface (white arrowheads). At the 12 h window (two examples shown), Duf is visible in those foci and some acquire an elongated shape (yellow arrowheads). At 18 h, multiple short SD strands cover the surface of the nephrocytes. All images shown at the same magnification. See Fig. S3 for additional time points and medial sections. (C) TEM image of a scramb1⁴³ nephrocyte rescued by the expression of UAS-scramb1-A-V5 for 12 h. Multiple SDs sealing small labyrinthine channels are visible (red arrows). Electron-dense plaques, marked by black arrowheads in Fig. 2E, are rare

Fig. 4

Scramb1-A interacts with Pyd. (A) Co-IP of Scramb1-A-V5 and Pyd-PΔCC from a lysate of salivary glands coexpressing both proteins. The same lysate was incubated with a magnetic matrix coupled to either anti-β-galactosidase in the control (ctrl) experiment or anti-V5 (V5) antibodies. The eluates were analyzed by western blot using anti-V5 to detect Scramb1-A-V5 and anti-Pyd. Pyd-PΔCC was notably elevated in the eluate from the V5 matrix compared to the control, which shows some unspecific Pyd binding to the matrix. (B) Proximity labeling with biotin of Scramb1-A-V5 by Pyd-P-TurboID-V5. Biotinylated proteins were isolated from lysates of salivary glands expressing Scramb1-A-V5 alongside Pyd-P-TurboID-V5 or TurboID-V5 (control). The lysates (input, 10% loaded) and purified fractions (P) were analyzed by western blot using anti-V5 antibody to detect TurboID-V5, Pyd-P-TurboID-V5 and Scramb1-A-V5. Scramb1-A-V5 was biotinylated by Pyd-P-TurboID-V5 but not by the control TurboID-V5, indicating a close association between Scramb1-A and Pyd. Notice that Pyd-P-TurboID-V5 and TurboID-V5 auto-biotinylate themselves. (C) Immunostaining of pyd^ex147 nephrocytes expressing UAS-scramb1-A-V5 driven by pros-Gal4 to detect Scramb1-A-V5 (anti-V5 antibody), Sns and Duf, as indicated. Nuclei were labeled with DAPI (blue). pyd^ex147 nephrocytes lack SDs and both Sns and Duf accumulate in regions of contact between aggregated nephrocytes (arrows) whereas Scramb1-A-V5 shows a cytoplasmic distribution

Fig. 5

Scramb1-A proline-rich domain is required for protein stability and localization to SDs. (A-C’) Nephrocytes overexpressing the isoform Scramb1-B-V5 driven by sns-GCN-Gal4 in an otherwise wild-type background (+), and in nephrocytes with compromised protein degradation via the lysosome pathway (dor⁸ mutants) or the proteasome pathway (Prosα1 silencing), immunostained to reveal Scramb1-B-V5 (anti-V5 antibody) and Pyd in medial sections, as indicated. Scramb1-B-V5 is undetectable in A (+), but accumulates in cytoplasmic vesicles in dor⁸ (arrows in B-B’) and in aggresomes in Prosα1 depleted nephrocytes (arrows in C-C’). Scramb1-B-V5 does not accumulate in SDs (arrowheads). (D-D’) Silencing the proteasome subunit Prosα6 in nephrocytes expressing UAS-scramb1-B-V5 (sns-GCN-Gal4) results in the formation of aggresomes, identified by the accumulation of Ubiquitin (arrows), that also contain Scramb1-B-V5. Medial sections are shown. (E-H’’) Nephrocytes expressing Scramb2-HA or the chimera Spro-scramb2-V5 (Scramb1-A proline-rich region fused to Scramb2), driven by sns-GCN-Gal4, stained with anti-Pyd and anti-HA or anti-V5, as indicated. E-E’’ and G-G’’ depict medial sections. The boxed regions in E and G are magnified in E’-E’’ and G’-G’’ respectively. Scramb2-HA accumulates at similar levels in the plasma membrane (arrows in E’-E’’) and the subcortical region, whereas Spro-scramb2-V5 accumulates at higher levels in the plasma membrane, colocalizing with Pyd (arrows in G’-G’’). The corresponding intensity profiles for Pyd and Scramb2-HA or Spro-scramb2-V5, expressed in arbitrary units, are shown in I and J, as indicated. The plasma membrane was registered at the 1 µm position. (F-F’’) Cortical section displaying partial colocalization between Pyd and Scramb2-HA, indicated by a Pearson’s colocalization coefficient of 0.335. (H-H’’) Cortical sections showing the distribution of Pyd and Spro-scramb2-V5’, colocalizing in a fingerprint-like pattern (arrows) with a Pearson’s colocalization coefficient of 0.516. Nuclei are labeled with DAPI (blue). A-C’ shown at the same magnification

Fig. 6

Requirement of the putative Ca²⁺-binding region of Scramb1-A. (A-B) Immunostaining of scramb1⁴³ nephrocytes partially rescued by the expression of Scramb1-A^F378A-V5, containing one residue substitution within its putative Ca²⁺ binding region, driven by pros-Gal4 and shown at medial (A) and cortical (B) planes. The nephrocyte surface is partially covered by short SD strands identified by Duf accumulation, that coexist with foci containing Scramb1-A^F378A-V5 and low levels of Duf (green arrow). White arrow in A points to a region devoid of SDs. (C-D⁴) Immunostaining of scramb1⁴³ nephrocytes expressing Scramb1-A^{D372A, F378A}-V5, a variant containing two residue substitutions within its putative Ca²⁺ binding region, driven by pros-Gal4, shown at a medial (C) and a cortical view at a higher magnification (D). The highlighted region in D is also shown as single channels (D¹-D⁴), as indicated. Very few SDs are formed. Pyd, Sns and Scramb1-A^{D372A, F378A}-V5 (anti-V5 antibody) accumulate in abundant cortical foci that contain low levels of Duf. (E-E’’’) Cortical view of a first instar larval nephrocyte expressing Scramb1-A^{D372A, F378A}-V5 (pros-Gal4), immunostained as indicated. Similarly to Scramb1-A, this Ca²⁺-insensitive variant accumulates in SDs, identified by Duf and Pyd co-expression. (A, C) Nuclei were labeled with DAPI (blue). D-D⁴ shown at the same magnification

Fig. 7

Requirement of Scramb1-A palmitoylation sites. (A-B) Scheme of Scramb1-A domain composition, highlighting a cluster of conserved cysteine residues (yellow) matching the human PLSCR1 palmitoylation site and sequence alignment of the region (B). The construct UAS-Scramb1-ANP3-V5A contains mutations in residues 184, 188 and 189, in red. A conserved putative Ca2+- binding site (green) is also indicated. (C) Co-IP from salivary glands coexpressing Scramb1A-V5 and Flo2-RFP. The extract was incubated with a magnetic matrix coupled to anti-V5 or to anti-?-galactosidase as a control, and the eluates analyzed by western blot to detect Scramb1-A-V5 (anti-V5 antibody) and Flo2-RFP (anti-RFP), as indicated. Flo2-RFP was co-immunoprecipitated with Scramb1-V5. (D) Genetic interaction between scramb1 and Flo2. Three genotypes were quantitated: scramb1/scramb1 (n = 105 cells), scramb1/scramb1 (n = 87 cells) and a double mutant combination flo2/ Y; scramb1/scramb1 (n = 193 cells). Nephrocytes were immunostained for Duf and Pyd and classified into four categories, from no SD strands observed (0) to SDs covering the entire nephrocyte surface (3). Examples are shown in Fig. S7. A mutation in Flo2 normalizes the scramb1 phenotype. Asterisks show statistical significance (* P < 0.05, *** P < 0.001). (E-F’’) Subcellular localization of non-palmitoylable Scramb1-ANP3-V5 driven by pros-Gal4. Immunostainings with anti-V5 and anti-Pyd are shown in medial planes (E-E’) and in cortical planes at higher magnification (F-F’). Scramb1-ANP3-V5 colocalizes with Pyd in SDs (F-F’’) and also accumulates in nuclei, colocalizing with DAPI in blue (arrows). (G-G) scramb1 nephrocytes expressing UAS-scramb1-ANP3-V5 (pros-Gal4), immunostained to show the cortical distribution of Scramb1-ANP3-V5 (anti-V5 antibody), Sns, Duf and Pyd in foci and occasional short SD strands (yellow arrows). E-E’, F-F’’ and G-G shown at the same magnification

Fig. 8

Scramb1 interaction with membrane-remodeling proteins. (A) Wild-type nephrocyte displaying the distribution of Past1 and Cubn, as indicated. Higher magnification, single-channel data are presented at medial sections, corresponding to the boxed region, as well as at cortical levels. Past1 expression is higher in the labyrinthine channels regions, marked by Cubn expression. (B) Immunostaining of a nephrocyte expressing UAS-Scramb1-A-V5 driven by pros-Gal4, depicting the distribution of Past1, Scramb1-A-V5 (anti-V5 antibody), and Duf in high magnification images. At medial sections, Past1 is detected subcortically and at the plasma membrane, alongside Duf and Scramb1-A-V5 (arrows). In cortical sections, Past1 partially overlaps with Scramb1-A-V5, with a Pearson’s colocalization coefficient of 0.763. Arrows points to one region of overlap, as reference. (C) Quantitation of the nephrocyte phenotypes in the following genotypes and conditions: scramb1^GFP/ scramb1^GFP at 25 °C (n = 105 cells), 18 °C (n = 118 cells) and 30 °C (n = 128 cells) and in the double mutant genotypes: scramb1^GFP, Past1^110.1/scramb1^GFP, Past1^110.1 (n = 154 cells) and Amph²⁶/Amph²⁶; scramb1^GFP/scramb1^GFP (n = 92 cells) at 25 °C. Nephrocytes were classified into four categories ranging from the absence of SDs (category 0) to SDs covering the entire nephrocyte surface (category 3, examples shown in Fig. S7). The statistical significance of the observed differences compared to scramb1^GFP at 25 °C is indicated (ns: non-significant, *** P < 0.001). (D) Distribution of Amph, Cubn and Duf in wild-type nephrocytes, as indicated. Amph exhibits a cytoplasmic distribution with a preferential accumulation in the labyrinthine channels region, identified by Cubn expression (bracket). Amph is also detected in the plasma membrane, displaying partial colocalization with Duf (arrows), as depicted in high-magnification single-channel images of medial section (upper panels) and cortical sections (lower panels. Pearson’s colocalization coefficient for Amph-Duf: 0.577). (E-F’’) Medial (E) and cortical (F’-F’’) sections of Amph²⁶ mutant nephrocytes illustrating the distribution of SDs (anti-Duf and anti-Pyd), which decorate only a fraction of the nephrocyte surface (arrows), while the remaining surface exhibits low Duf levels and Pyd accumulation in foci and short rods (arrowheads). (G) Intensity profiles across medial sections of the nephrocytes shown in B (Duf, Scramb1-A-V5 and Past1) and in wild-type nephrocytes (Duf, Cubn and Amph). The plasma membrane is registered to the 0 μm position. (H) Quantitative analysis of the Amph²⁶ phenotype, depicting the fraction of cell surface containing SDs for 19 wild-type and 19 Amph²⁶ nephrocytes. (I) Scramb1-A oligomerization. Lysates from salivary glands coexpressing Scramb1-A-ProtA and Scramb1-A-V5 or only Scramb1-A-V5 (control), were incubated with a matrix conjugated to rabbit IgGs to precipitate Scramb1-A-ProtA. Lysates (input) and eluates (precipitate) were analyzed by western blot with anti-V5, which detects both Scramb1-A-V5 and Scramb1-A-ProtA. Scramb1-A-V5 co-precipitates with Scramb1-A-ProtA, indicating a capacity to oligomerize. A, D and E, nuclei are labeled with DAPI (blue)