Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures.
Rho GTPases are critical regulators of epithelial morphogenesis. A powerful means to investigate their function is three-dimensional (3D) cell culture, which mimics the architecture of epithelia in vivo. However, the nature of 3D culture requires specialized techniques for morphological and biochemical analyses. Here, we describe protocols for 3D culture studies with Madin-Darby Canine Kidney (MDCK) epithelial cells: establishing cultures, immunostaining, and expressing, detecting, and assaying Rho proteins. These protocols enable the regulation of epithelial morphogenesis to be explored at a detailed molecular level.
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
Lucy Erin
LE
O'Brien
Wei
W
Yu
Kitty
K
Tang
Tzuu-Shuh
TS
Jou
Mirjam M P
MM
Zegers
Keith E
KE
Mostov
D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D024042:Collagen Type I / Q000648:ultrastructure; D003560:Cysts / Q000473:pathology; D004285:Dogs; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism*; D005109:Extracellular Matrix / Q000648:ultrastructure; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D013194:Staining and Labeling; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D024042:Collagen Type I; D017228:Hepatocyte Growth Factor; D020830:rac1 GTP-Binding Protein