Process formation of podocytes: morphogenetic activity of microtubules and regulation by protein serine/threonine phosphatase PP2A

Abstract

Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One-and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after ok-adaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.

Process formation of the renal glomerular podocyte: Is there common molecular machinery for processes of podocytes and neurons?

“…Taking advantage of the conditionally immortalized cell line (Mundel et al, 1997a; kindly provided by Dr Mundel, Albert Einstein College of Medicine, NY, USA), we have also shown the contribution of PP2A-driven dephosphorylation in podocytes. Like in neurons (Chiou & Westhead, 1992), application of okadaic acid at nanomolar concentrations, which preferentially inhibits PP2A and, in turn, elevates the level of protein Ser/ Thr phosphorylation, resulted in the total abolition of the formation of podocyte process, accompanied by an increase in protein Ser/Thr phosphorylation (Kobayashi et al, 2001b) and a decrease in MT stability detected by post-translational modification of tubulin (Gao et al, 2004). Conversely, inhibition of various Ser /Thr kinases enhanced process formation of podocytes ( Fig.…”

“…In previous review articles, we have discussed the machinery of morphogenesis driven by MT, microtubuleassociated proteins (MAP) and MT-based motor proteins Kobayashi, 2002). As shown by experiments in vivo (Andrews, 1977) and in vitro (Kobayashi et al, 2001b), the development and maintenance of podocyte processes depend on the assembly of MT. The dynamics and assembly of MT are controlled by MAP; a variety of MAP has been studied most intensively in neurons, whereas podocytes have been reported to express MAP that are ubiquitously distributed (Kobayashi et al, 2000).…”

Increase of Integrin-Linked Kinase Activity in Cultured Podocytes upon Stimulation with Plasma from Patients with Recurrent FSGS

“…A conditionally immortalized mouse podocyte cell line (MPC cells) was cultivated as reported previously (12). In this study, we induced differentiation of MPC cells by cultivating in cell culture dishes or glass coverslips coated with laminin‐1 (products of murine EHS tumor cells; Koken Inc., Tokyo, Japan) at 37°C, in culture medium supplemented with 5% FCS but no γ‐interferon (nonpermissive condition) for at least 14 days before use.…”

“…In this study, we induced differentiation of MPC cells by cultivating in cell culture dishes or glass coverslips coated with laminin‐1 (products of murine EHS tumor cells; Koken Inc., Tokyo, Japan) at 37°C, in culture medium supplemented with 5% FCS but no γ‐interferon (nonpermissive condition) for at least 14 days before use. Culture in the presence of lamiin‐1 and reduced FCS concentration is effective to promote MPC process formation (12).…”

Networks that Link Cytoskeletal Regulators and Diaphragm Proteins Underpin Filtration Function inDrosophilaNephrocytes

“…The actin cytoskeleton is a key component that regulates podocyte shape and function. While microtubules (MTs) and intermediate filaments (IFs) provide the major structural support in the podocyte cell body and primary processes (Kobayashi et al, 1998; Kobayashi et al, 2001), dense arrays of actin microfilaments are present in the foot processes (Zhang et al, 2013). In several nephrotic syndromes this differential distribution of cytoskeletal components is lost, leading to loss of SD integrity, foot process effacement and proteinuria (Allison, 2015; Gheissari et al, 2012; Mathieson, 2012; Sanai et al, 2000).…”