Additional file 1 of Slit diaphragm maintenance requires dynamic clathrin-mediated endocytosis facilitated by AP-2, Lap, Aux and Hsc70-4 in nephrocytes
Creators: name: Wang, Luyao
name: Wen, Pei
name: van de Leemput, Joyce
name: Zhao, Zhanzheng
name: Han, Zhe
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file1: Table S1. The RNAi screen of endocytosis genes required for slit diaphragm protein endocytosis and recycling. Figure S1. 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 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. C Quantification of relative ANF-RFP fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. D Quantification of relative Dextran-linked Texas Red fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. ***P&lt;0.001. Figure S2. 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-C’ 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. Figure S3. 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 panel shows that shi gene 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 shi clearly reduced levels of accumulated Dextran. D Quantification of relative Dextran-linked Texas Red fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. ***P&lt;0.001. Figure S4. The Clc-IR transgene can specifically decrease Clc protein level. Anti-Clc antibody fluorescent staining of Clc expression (red) in nephrocytes. Left panel, in control nephrocytes, Clc is strongly expressed and localized mainly close to plasma membrane. Right panel, in clc-silenced nephrocytes, the protein level of Clc is below the detection limit, suggesting highly effective knockdown. Figure S5. 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 AP-2α, AP-2μ, AP-2Ω and AP-1-2β 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 AP-2α, AP-2μ, AP-2Ω and AP-1-2β genes clearly reduced levels of accumulated Dextran. D Quantification of relative Dextran-linked Texas Red fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. ***P&lt;0.001. Figure S6. AP-2 complex is required for Sns protein proper localization. A-A’ Sns (red) distributed uniformly and smoothly in parallel lines in finger-print like pattern in control (wild type) nephrocytes. Scale bar: 1μm. B-D Silencing of AP-1-2β, AP-2α, AP-2μ and AP-2σ resulted in severe disruption of Sns cell surface localization. Figure S7. AP-1 complex is not required for Pyd protein proper localization. A-A″ Sns (red) distributed uniformly and smoothly in parallel lines in finger-print like pattern in control (wild type) nephrocytes, with DAPI (blue nuclear stain). Scale bar: 1μm. B-D″ Silencing of AP-1γ, AP-1σ and AP-1μ showed similar pattern of Pyd cell surface localization as the control. Figure S8. The adaptor protein Lap and uncoating proteins, Aux and Hsc70-4, 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 Lap, Aux and Hsc70-4 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 Lap, Aux and Hsc70-4 genes clearly reduced levels of accumulated Dextran. D Quantification of relative Dextran-linked Texas Red fluorescence in nephrocytes expressing the indicated gene silencing RNAi construct. ***P&lt;0.001. Figure S9. The adaptor protein Lap and uncoating proteins, Aux and Hsc70-4, are required for Sns protein proper localization. A-A’ Sns (red) distributed uniformly and smoothly in parallel lines in fingerprint like pattern in control (wild type) nephrocytes. Scale bar: 1μm. B-D’ Silencing of Lap, Aux, Hsc70-4 resulted in severe disruption of Sns cell surface localization. Figure S10. The Rab7-IR transgene can specifically decrease Rab7 protein level. Anti-Rab7 antibody fluorescent staining of Rab7 expression (green) in nephrocytes, with DAPI (blue) nuclear stain. In control nephrocytes, Rab7 is strongly expressed. In rab7-silenced nephrocytes, the protein level of Rab7 is significantly reduced, suggesting highly effective knockdown.