Podocin Participates in the Assembly of Tight Junctions between Foot Processes in Nephrotic Podocytes

Abstract

The predominant type of cellular junction between normal podocyte foot processes is the slit diaphragm. Under nephrotic conditions,however, foot process effacement leads to the loss of slit diaphragms and the new formationof tight junctions composed of the proteins coxsackievirus and adenovirus receptor (CAR) and zonula occludens 1 (ZO-1). Podocin, a protein that plays a key role in maintaining the integrity of the slit diaphragm, has also been localized to these tight junctions, but its function at this site is unknown. In this study, we confirmed that podocin colocalizes with CAR and ZO-1 at the tight junction between foot processes in nephrotic rats. Using primary cultures of rat podocytes, as well as cell lines that co-expressed podocin and CAR, we observed that podocin was recruited to sites of cell-cell contact and that it co-localized with CAR and ZO-1. Immunoprecipitation suggested that these three junctional proteins from a multi-protein complex. Consistent with this, we found that podociin facilitated the coalescence of preassembled lipid rafts containing CAR and restricted their lateral mobility, the latter likely a result of dynamic actin reorganization and subsequent tethering of CAR-podocin complexes to the cytoskeleton. In conclusion, in addition to serving as a structural protein of the slit diaphragm of normal podocytes, our data suggest that podocin may also serve as a scaffold that links tight junction proteins to the actin cytoskeleton in nephrotic foot processes.

Inverted Formin 2 Regulates Actin Dynamics by Antagonizing Rho/Diaphanous-related Formin Signaling

“…4 Podocin, a prohibitin homology domain containing protein with affinity for lipid rafts, helps recruit nephrin to the membrane and serves as a scaffolding protein for SD complex organization. [32][33][34] To understand the role of INF2 in maintaining nephrin-podocin complex trafficking, we investigated the interaction of INF2 with podocin and nephrin, as well as the lipid raft scaffolding protein caveolin-1. 35 As shown in Figure 4, A and B, in HEK 293T cells that overexpressed specific SD proteins, endogenous INF2 was co-immunoprecipitated with overexpressed hemagglutinin (HA)-podocin, but was immunoprecipitated by nephrin only in the presence of HA-podocin This finding indicates that podocin mediates an association of INF2 with nephrin.…”

Phosphorylation of Nephrin Triggers Its Internalization by Raft-Mediated Endocytosis

“…We first traced the nephrin endocytic pathway using transfected COS-7 cells, 25 because they have a favorable morphology for quantification and they internalize nephrin in a similar manner to cultured podocytes (Supplemental Figure 1). At the early phase of internalization (2 min), nephrin was taken up into fine punctate structures near the cell periphery ( Figure 1, A through C).…”

“…25 Wild-type L cells (L-wt) or L cells stably expressing podocin (L-pod) were transiently transfected with GFP-nephrin (L-wt GFP-nephrin and L-pod GFP- BASIC RESEARCH www.jasn.org nephrin, respectively), and fluorescence recovery was compared. As shown in Figure 5, A and B, in L-wt GFP-nephrin cells, fluorescence intensity reached a plateau at 70% of the prebleached level, with a recovery time of 60 to 90 s (n ϭ 6).…”

“…25,43 The homogenate was centrifuged at 1000 ϫ g for 10 min to obtain a postnuclear supernatant. The supernatant was centrifuged at 200,000 ϫ g for 30 min and resuspended in 3 ml of homogenization buffer containing 0.2% Triton X-100.…”

“…Mouse L cells, L-wt or L-pod, 25 were transiently transfected with GFP-nephrin cDNA. Forty-eight hours after transfection, cells were held in a glass-bottom dish containing 10% FCS/DMEM on the temperature-controlled stage of a Zeiss LSM PASCAL confocal microscope.…”