title: Progressive association of a "soluble" glycolytic enzyme with the detergent-insoluble cytoskeleton during in vitro morphogenesis of MDCK epithelial cells.
abstract: In MDCK epithelial cells, cell contact at confluency initiates a protracted process of morphogenesis during which several proteins known to bind the cytoskeleton become progressively associated with the detergent-resistant cell fraction and distributed to their characteristic polarized domains. Using extraction protocols that identify this tight cytoskeletal linkage, here we show a similar but slower, time-dependent enrichment in the detergent resistant fraction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a highly abundant glycolytic enzyme that is traditionally considered soluble. Similar enrichment did not occur for two other glycolytic enzymes, phosphoglycerate mutase or lactate dehydrogenase. Insoluble GAPDH was not homogeneously distributed in the cytoplasm but rather displayed several discrete patterns that varied within and among MDCK cells. It also localized prominently to a few nuclei in the phenotypically heterogeneous cells of late confluency cultures. Disruptors of cytoskeletal filaments were relatively ineffective in the postconfluent epithelial monolayers, although use of disrupting agents implicated actin as the cytoplasmic filament that tethers insoluble GAPDH. Catalytic activity could be demonstrated in the insoluble fraction of GAPDH from postconfluent cultures, but only after release by mechanical disruption of insoluble extracts. Treatment of postconfluent cells with agents that deplete ATP diminished the fraction of cytoskeletally associated GAPDH, and levels of insoluble GAPDH were restored with ATP repletion, suggesting that ATP levels may regulate cytoskeletal linkage and thereby local enzyme activity. We conclude that the highly abundant and ubiquitous enzyme GAPDH becomes progressively enriched in detergent stable subcellular compartments during the process of epithelial morphogenesis. The process that produces GAPDH compartments is slow, suggesting that epithelial cells just at confluency, when they are typically analyzed, have not yet maximized the organizational state that can be attained in monolayer culture.
authors.affiliation: Departments of Ophthalmology and Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: F
authors.initials: F
authors.lastname: Cao
authors.forename: N
authors.initials: N
authors.lastname: Yanagihara
authors.forename: J M
authors.initials: JM
authors.lastname: Burke
mesh_terms: D020106:Acrylamide / Q000494:pharmacology; D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D003902:Detergents; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000187:drug effects / Q000378:metabolism*; D007633:Keratins / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology; D013997:Time Factors
chemical_list: D000199:Actins; D003902:Detergents; D020106:Acrylamide; D007633:Keratins; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases