Exocyst Genes Are Essential for Recycling Membrane Proteins and Maintaining Slit Diaphragm in Drosophila Nephrocytes
Authors: Wen Pei , Zhang Fujian , Fu Yulong , Zhu Jun-yi , Han Zhe 
Published: April 2020

Significance Statement
Mutations in genes encoding the exocyst protein complex have been identified in patients with kidney disease, but the underlying mechanism of renal pathology is unclear. The authors demonstrated in Drosophila nephrocytes—counterparts to mammalian podocytes—that an intact exocyst complex is essential for normal function and structure. Silencing of various exocyst genes disrupted slit diaphragm structure and reduced infoldings of the nephrocyte plasma membrane (lacuna channels), the main sites of protein reabsorption. The authors also discovered abnormal electron-dense “exocyst rods,” structures formed by lacuna membrane proteins and slit diaphragm proteins that are not appropriately recycled because of deficiency of exocyst proteins. These findings are consistent with the exocyst complex playing a key role in maintenance of slit diaphragm and lacuna channel structural integrity via completion of the endosome recycling pathway.

Background
Studies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because Drosophila nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue.

Methods
We silenced genes encoding exocyst complex proteins specifically in Drosophila nephrocytes and studied the effects on protein reabsorption by lacuna channels and filtration by the slit diaphragm. We performed nephrocyte functional assays, carried out super-resolution confocal microscopy of slit diaphragm proteins, and used transmission electron microscopy to analyze ultrastructural changes. We also examined the colocalization of slit diaphragm proteins with exocyst protein Sec15 and with endocytosis and recycling regulators Rab5, Rab7, and Rab11.

Results
Silencing exocyst genes in nephrocytes led to profound changes in structure and function. Abolition of cellular accumulation of hemolymph proteins with dramatically reduced lacuna channel membrane invaginations offered a strong indication of reabsorption defects. Moreover, the slit diaphragm’s highly organized surface structure—essential for filtration—was disrupted, and key proteins were mislocalized. Ultrastructural analysis revealed that exocyst gene silencing led to the striking appearance of novel electron-dense structures that we named “exocyst rods,” which likely represent accumulated membrane proteins following defective exocytosis or recycling. The slit diaphragm proteins partially colocalized with Sec15, Rab5, and Rab11.

Conclusions
Our findings suggest that the slit diaphragm of Drosophila nephrocytes requires balanced endocytosis and recycling to maintain its structural integrity and that impairment of the exocyst complex leads to disruption of the slit diaphragm and nephrocyte malfunction. This model may help identify therapeutic targets for treating kidney diseases featuring molecular defects in vesicle endocytosis, exocytosis, and recycling.