An in vitro model for the analysis of intestinal brush border assembly. II. Changes in expression and localization of brush border proteins during cell contact-induced brush border assembly in Caco-2BBe cells

Abstract

In the companion paper (M. D. Peterson and M. S. Mooseker (1993). J. Cell Sci. 105, 445–460) we describe a method for modeling brush border assembly in the Caco-2BBe clones. In this study we have examined the molecular changes accompanying cell contact-induced brush border assembly. A subset of brush border proteins was tracked throughout brush border assembly by immunoblotting and by immunofluorescent localization using laser scanning confocal microscopy. Actin, fodrin, villin and presumptive unconventional myosin immunogens were distributed at the periphery of depolarized cells. All proteins partitioned primarily with the membrane fraction upon differential sedimentation of depolarized cell lysates; the fractionation patterns were comparable to those of confluent cells. After a monolayer had formed, each protein showed a redistribution to the apical domain in a discrete sequence. Actin and villin began to shift apically at 2 d, while fodrin and the unconventional myosin immunogens did not redistribute until 3 d. Enterocyte-like localization was observed by 5 d for all proteins. Sucrase-isomaltase was not reliably detectable until 9 d by immunofluorescence, after brush border assembly was complete. Quantitative immunoblot analysis of total cell extracts demonstrated an average 10-fold increase in villin levels, while fodrin levels appeared to remain unchanged. Three putative unconventional myosin immunogens of 140 kDa, 130 kDa, and 110 kDa have been detected previously in the C2BBe cells with a head-specific monoclonal antibody to avian brush border myosin I (M. D. Peterson and M. S. Mooseker (1992) J. Cell Sci. 102, 581–600). Each of these immunogens displayed distinct expression patterns during brush border assembly. The 140 kDa species decreased by half, while the 130 kDa immunogen(s) did not change in any consistent fashion. The 110 kDa protein, presumed to be human brush border myosin I, rose on average 8-fold. A ribonuclease protection assay was also performed using a probe for human brush border myosin I. Equal amounts of total RNA from depolarized and confluent cells were assayed; the level of protected product was approximately 9-fold greater in the confluent cells. The expression patterns of the brush border proteins, coupled with the correlation to the ultrastructural features during brush border assembly in C2BBe cells, show that differentiation of the C2BBe cells closely resembles the changes that occur during human fetal intestinal differentiation.