Slit diaphragm maintenance requires dynamic clathrin-mediated endocytosis facilitated by AP-2, Lap, Aux and Hsc70-4 in nephrocytes

Background The Slit diaphragm (SD) is the key filtration structure in human glomerular kidney that is affected in many types of renal diseases. SD proteins are known to undergo endocytosis and recycling to maintain the integrity of the filtration structure. However, the key components of this pathway remain unclear. Methods Using the Drosophila nephrocyte as a genetic screen platform, we screened most genes involved in endocytosis and cell trafficking, and identified the key components of the cell trafficking pathway required for SD protein endocytosis and recycling. Results We discovered that the SD protein endocytosis and recycling pathway contains clathrin, dynamin, AP-2 complex, like-AP180 (Lap), auxilin and Hsc70-4 (the endocytosis part) followed by Rab11 and the exocyst complex (the recycling part). Disrupting any component in this pathway led to disrupted SD on the cell surface and intracellular accumulation of mislocalized SD proteins. We also showed the first in vivo evidence of trapped SD proteins in clathrin-coated pits at the plasma membrane when this pathway is disrupted. Conclusions All genes in this SD protein endocytosis and recycling pathway, as well as SD proteins themselves, are highly conserved from flies to humans. Thus, our results suggest that the SD proteins in human kidney undergo the same endocytosis and recycling pathway to maintain the filtration structure, and mutations in any genes in this pathway could lead to abnormal SD and renal diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-021-00595-4.


Supplemental Figure Legends
Supplemental Table 1: The RNAi screen of endocytosis genes required for slit diaphragm protein endocytosis and recycling. There are usually multiple human genes for the fly homolog and here only showed the representative one. DIOPT score is calculated by the DRSC Integrative Ortholog Prediction Tool (DIOPT) at http://www.flyrnai.org/diopt. The max score between fly and human homologs is 16, and genes with a score higher than 2 could be considered as orthologs.
Supplemental Figure 1: The clathrin genes, Chc and Clc, are required for nephrocyte function. A MHC-ANF-RFP derived hemolymph ANF-RFP (red) uptake by nephrocytes. Hand-GFP labels both nephrocytes (big nucleus) and heart cells (small nucleus). Wild type (Control) flies accumulated abundant ANF-RFP. Subsequent panels show that Chc and Clc genes RNAi silencing severely reduced the ANF-RFP accumulation level. B Quantification of relative ANF-RFP fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. C Texas Red-labeled 10 kD Dextran particles uptake by nephrocytes. Particles were easily taken in and collected by wild type control nephrocytes in an ex vivo assay. Silencing of Chc and Clc genes clearly reduced levels of accumulated Dextran. D Quantification of relative Dextranlinked Texas Red fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. ***P<0.001.
Supplemental Figure 2: The clathrin genes, Chc and Clc, are required for slit diaphragm protein Sns endocytosis. A-A' Sns (red) distributed uniformly and smoothly in parallel lines in fingerprint like pattern in control (wild type) nephrocytes. Scale bar: 1µm. B-H Silencing of Chc and Clc resulted in severe disruption of Sns cell surface localization. The Sns-positive lines became more curved and shorter, and many became dots. The lines were no longer parallel, and the spacing became irregular.
Supplemental Figure 3: The Drosophila dynamin homolog, Shi, is required for nephrocyte function. A MHC-ANF-RFP derived hemolymph ANF-RFP (red) uptake by nephrocytes. Hand-GFP labels both nephrocytes (big nucleus) and heart cells (small nucleus). Wild type (Control) flies accumulated abundant ANF-RFP. Subsequent panels show that Chc and Clc genes RNAi silencing severely reduced the ANF-RFP accumulation level. B Quantification of relative ANF-RFP fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. C Texas Red-labeled 10 kD Dextran particles uptake by nephrocytes. Particles were easily taken in and collected by wild type control nephrocytes in an ex vivo assay. Silencing of Chc and Clc genes clearly reduced levels of accumulated Dextran. D Quantification of relative Dextranlinked Texas Red fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. ***P<0.001.
Supplemental Figure 4: The Clc-IR transgene can specifically decrease Clc protein level. Anti-Clc antibody fluorescent staining of Clc expression (red) in nephrocytes. A In control nephrocytes, Clc is strongly expressed and localized mainly close to plasma membrane. B In clc-silenced nephrocytes, the protein level of Clc is below the detection limit, suggesting highly effective knockdown.
Supplemental Figure 5: AP-2 complex is required for nephrocyte function. A MHC-ANF-RFP derived hemolymph ANF-RFP (red) uptake by nephrocytes. Hand-GFP labels both nephrocytes (big nucleus) and heart cells (small nucleus). Wild type (Control) flies accumulated abundant ANF-RFP. Subsequent panels show that Chc and Clc genes RNAi silencing severely reduced the ANF-RFP accumulation level. B Quantification of relative ANF-RFP fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. C Texas Red-labeled 10 kD Dextran particles uptake by nephrocytes. Particles were easily taken in and collected by wild type control nephrocytes in an ex vivo assay. Silencing of Chc and Clc genes clearly reduced levels of accumulated Dextran. Supplemental Figure 10: The Rab7-IR transgene can specifically decrease Clc protein level. Anti-Rab7 antibody fluorescent staining of Rab7 expression (green) in nephrocytes. In control nephrocytes, Rab7 is strongly expressed. In rab7-silenced nephrocytes, the protein level of Rab7 is significantly reduced, suggesting highly effective knockdown