Overexpression of Myo1e promotes albumin endocytosis by mouse glomerular podocytes mediated by Dynamin

Abstract

Background As a fundamental process internalizing molecules from the plasma membrane, endocytosis plays a crucial role in podocyte biology. Our previous study has identified that overexpression of Myole may enhance podocyte endocytosis. However, its potential mechanism has been not well understand. Thus, we aimed to analyze whether albumin endocytosis by mouse glomerular podocytes is dependent on Myo1e expression. Also, we aimed to elucidate whether the underlying mechanism is mediated by Dynamin. Methods Firstly, mouse podocyte cells (MPC5) were treated with different concentrations of FITC-bovine serum albumin (BSA). The fluorescence intensity and cell viability were detected by flow cytometry and MTT assays, respectively. Afterwards, the optimal concentration of FITC-BSA was determined. Secondly, MPC5 cells were treated with Myole overexpression or knockdown. Cell morphology was observed under microscope. Immunofluorescence assay was used to determine the expression of F-actin. The protein expression of nephrin and podocin was detected by western blot. Flow cytometry was used to detect MPC5 cell apoptosis with annexin V. Finally, MPC5 cells were treated with Myole overexpression and/or Dynasore (a GTPase inhibitor of Dynamin). The fluorescence intensity was detected using flow cytometry assay. Results MPC5 endocytosis BSA was elevated with a concentration-dependent manner. MTT results showed that MPC5 cell viability was inhibited with a concentration-dependent manner. Myo1e overexpression promoted podocyte endocytic FITC-BSA, which was contrary to its knockdown. Under microscope, after inhibition of Myo1e, podocyte foot process fusion was observed. Myo1e overexpression promoted the expression of cytoskeleton F-actin and podocyte-specific molecules (nephrin and podocin) in podocyte endocytic FITC-BSA. Furthermore, we found that Myo1e promoted the apoptosis of podocytes. Dynasore attenuated the increase in endocytosis of FITC-BSA induced by Myo1e overexpression, suggesting that podocytes might mediate albumin endocytosis via Myo1e-Dynamin-Albumin. Conclusion Our findings revealed that overexpression of Myo1e promotes albumin endocytosis in mouse glomerular podocyte endocytic albumin mediated by Dynamin.

Podocyte Endocytosis in Regulating the Glomerular Filtration Barrier

“…Depletion of Myo1E inhibits transferrin endocytosis and extends the lifetimes of clathrin and dynamin on the invaginated vesicles ( 86 , 88 ). Overexpression of Myo1E in the mouse podocytes promotes the expression of F-actin and increases albumin endocytosis mediated by dynamin ( 90 ). Mutations in MYO1E (A159P and Y695X) were reported causing autosomal recessive steroid-resistant FSGS further illustrating the importance of this gene ( 67 ).…”

“…Accumulation of blue-labeled albumin endocytic vesicles was observed in the podocytes in a rat model of puromycin aminonucleoside-induced nephrotic syndrome ( 122 ). Furthermore, overexpression of Myo1e may also enhance dynamin-mediated albumin endocytosis in mouse podocytes ( 90 ). Ang II enhances the migration of albumin-containing vesicles from the glomerular capillary to the apical aspect of the podocyte, which is eventually released into the urinary space.…”

A critical role of the podocyte cytoskeleton in the pathogenesis of glomerular proteinuria and autoimmune podocytopathies

“…Dynamin and actin are colocalized in many cell structures, and GTP enzyme activity is essential for many actin‐driven processes, such as the formation of growth cones, the cortical actin cytoskeleton, and podosomes. Dynamin can directly interact with actin, and after forming the dynamin loop, gelsolin can be replaced from the barbed end of actin filaments to promote actin polymerization 50,51,60–63 . Further studies showed a key role for dynamin‐actin interactions in the regulation of the podocyte actin cytoskeleton suggesting that dynamin is required to maintain the glomerular filtration barrier 64,65 .…”

“…Dynamin can directly interact with actin, and after forming the dynamin loop, gelsolin can be replaced from the barbed end of actin filaments to promote actin polymerization. 50,51,[60][61][62][63] Further studies showed a key role for dynamin-actin interactions in the regulation of the podocyte actin cytoskeleton suggesting that dynamin is required to maintain the glomerular filtration barrier. 64,65 Dynamin also interacts with cortactin, which provides a biochemical link with actin filament assembly.…”