PMID- 10506748
OWN - NLM
STAT- MEDLINE
DCOM- 19991117
LR  - 20131121
IS  - 0886-1544 (Print)
IS  - 0886-1544 (Linking)
VI  - 44
IP  - 2
DP  - 1999 Oct
TI  - Progressive association of a "soluble" glycolytic enzyme with the 
      detergent-insoluble cytoskeleton during in vitro morphogenesis of MDCK epithelial 
      cells.
PG  - 133-42
AB  - 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.
CI  - Copyright 1999 Wiley-Liss, Inc.
FAU - Cao, F
AU  - Cao F
AD  - Departments of Ophthalmology and Cell Biology, Neurobiology & Anatomy, Medical 
      College of Wisconsin, Milwaukee, Wisconsin, USA.
FAU - Yanagihara, N
AU  - Yanagihara N
FAU - Burke, J M
AU  - Burke JM
LA  - eng
GR  - P30EY01931/EY/NEI NIH HHS/United States
GR  - R01EY10832/EY/NEI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Cell Motil Cytoskeleton
JT  - Cell motility and the cytoskeleton
JID - 8605339
RN  - 0 (Actins)
RN  - 0 (Detergents)
RN  - 20R035KLCI (Acrylamide)
RN  - 68238-35-7 (Keratins)
RN  - 8L70Q75FXE (Adenosine Triphosphate)
RN  - EC 1.2.1.- (Glyceraldehyde-3-Phosphate Dehydrogenases)
SB  - IM
MH  - Acrylamide/pharmacology
MH  - Actins/drug effects/metabolism
MH  - Adenosine Triphosphate/pharmacology
MH  - Animals
MH  - Blotting, Western
MH  - Cell Differentiation
MH  - Cell Division
MH  - Cell Line
MH  - Cytoskeleton/drug effects/*metabolism
MH  - Detergents
MH  - Glyceraldehyde-3-Phosphate Dehydrogenases/drug effects/*metabolism
MH  - Keratins/drug effects/metabolism
MH  - Microscopy, Confocal
MH  - Microtubules/drug effects/metabolism
MH  - Protein Binding
MH  - Solubility
MH  - Subcellular Fractions/enzymology
MH  - Time Factors
EDAT- 1999/10/03 00:00
MHDA- 1999/10/03 00:01
CRDT- 1999/10/03 00:00
PHST- 1999/10/03 00:00 [pubmed]
PHST- 1999/10/03 00:01 [medline]
PHST- 1999/10/03 00:00 [entrez]
AID - 10.1002/(SICI)1097-0169(199910)44:2<133::AID-CM5>3.0.CO;2-9 [pii]
AID - 10.1002/(SICI)1097-0169(199910)44:2<133::AID-CM5>3.0.CO;2-9 [doi]
PST - ppublish
SO  - Cell Motil Cytoskeleton. 1999 Oct;44(2):133-42. doi: 
      10.1002/(SICI)1097-0169(199910)44:2<133::AID-CM5>3.0.CO;2-9.