Identification of a putative network of actin-associated cytoskeletal proteins in glomerular podocytes defined by co-purified mRNAs.
The glomerular podocyte is a highly specialized and polarized kidney cell type that contains major processes and foot processes that extend from the cell body. Foot processes from adjacent podocytes form interdigitations with those of adjacent cells, thereby creating an essential intercellular junctional domain of the renal filtration barrier known as the slit diaphragm. Interesting parallels have been drawn between the slit diaphragm and other sites of cell-cell contact by polarized cells. Notably mutations in several genes encoding proteins localized to the foot processes can lead to proteinuria and kidney failure. Mutations in the Wilm's tumor gene (WT1) can also lead to kidney disease and one isoform of WT1, WT1(+KTS), has been proposed to regulate gene expression post-transcriptionally. We originally sought to identify mRNAs associated with WT1(+KTS) through an RNA immunoprecipitation and microarray approach, hypothesizing that the proteins encoded by these mRNAs might be important for podocyte morphology and function. We identified a subset of mRNAs that were remarkably enriched for transcripts encoding actin-binding proteins and other cytoskeletal proteins including several that are localized at or near the slit diaphragm. Interestingly, these mRNAs included those of alpha-actinin-4 and non-muscle myosin IIA that are mutated in genetic forms of kidney disease. However, isolation of the mRNAs occurred independently of the expression of WT1, suggesting that the identified mRNAs were serendipitously co-purified on the basis of co-association in a common subcellular fraction. Mass spectroscopy revealed that other components of the actin cytoskeleton co-purified with these mRNAs, namely actin, tubulin, and elongation factor 1alpha. We propose that these mRNAs encode a number of proteins that comprise a highly specialized protein interactome underlying the slit diaphragm. Collectively, these gene products and their interactions may prove to be important for the structural integrity of the actin cytoskeleton in podocytes as well as other polarized cell types.
Nabet
Behnam
B
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Tsai
Arthur
A
Tobias
John W
JW
Carstens
Russ P
RP
D000199:Actins / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D016162:Genes, Wilms Tumor; D006801:Humans; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism*; D013058:Mass Spectrometry; D009154:Mutation; D050199:Podocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D003598:Cytoskeletal Proteins; D012333:RNA, Messenger