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

Zhanzheng Zhao,Zhe Han,Luyao Wang,Pei Wen,Joyce Van De Leemput

doi:10.1186/s13578-021-00595-4

Zhanzheng Zhao, Zhe Han + Show 3 more

Open Access

https://doi.org/10.1186/s13578-021-00595-4

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Abstract

BackgroundThe 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.MethodsUsing 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.ResultsWe 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.ConclusionsAll 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.

Highlights

The glomerular filtration barrier, which contains the fenestrated endothelium of the glomerular capillaries, the glomerular basement membranes and podocytes, Nephrin, a transmembrane protein encoded by the NPHS1 gene [15], plays an essential role in assembly of the slit diaphragm structure and functionsWang et al Cell Biosci (2021) 11:83 as a signaling platform regulating the podocyte actin cytoskeleton, membrane trafficking and calcium mechano-signaling [17]
The CLIC/GEEC pathway, which is the route for uptake of glycosylphosphatidylinositol-anchored proteins (GPI-APs) and fluid phase, is conserved and the only clathrin-independent endocytosis (CIE) route that has been molecularly characterized and confirmed in Drosophila [7, 9, 10]
We found that silencing Drosophila homologs, aux and Hsc70-4 in the nephrocytes showed highly similar phenotypes to those of Clathrin (Chc or Clathrin light chain (Clc))-silenced nephrocytes, including a significant reduction in their filtering/absorption function (Additional file 1: Figure S8), and disrupted location of slit diaphragm proteins (Pyd and Sns) (Fig. 4c–d′′, Additional file 1: Figure S9)

Summary

Introduction

The glomerular filtration barrier, which contains the fenestrated endothelium of the glomerular capillaries, the glomerular basement membranes and podocytes, Nephrin, a transmembrane protein encoded by the NPHS1 gene [15], plays an essential role in assembly of the slit diaphragm structure and functionsWang et al Cell Biosci (2021) 11:83 as a signaling platform regulating the podocyte actin cytoskeleton, membrane trafficking and calcium mechano-signaling [17]. The glomerular filtration barrier, which contains the fenestrated endothelium of the glomerular capillaries, the glomerular basement membranes and podocytes, Nephrin, a transmembrane protein encoded by the NPHS1 gene [15], plays an essential role in assembly of the slit diaphragm structure and functions. Perturbations in nephrin protein level and localization have been found in podocytes of patients with membranous glomerulonephritis (GN), minimal change GN and diabetic nephropathy [4, 5], suggesting regulation of nephrin is critical for slit diaphragm function. The above studies were carried out in cultured cells, while the results have provided possible mechanisms in the regulation of nephrin endocytosis, it remained unclear whether nephrin and other slit diaphragm proteins are regulated in podocytes in vivo.

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