Cytoskeletal Protein and MRNA Accumulation During Brush Border Formation in Adult Chicken Enterocytes

We have explored the development of the brush border in adult chicken enterocytes by analyzing the cytoskeletal protein and mRNA levels as enterocytes arise from crypt stem cells and differentiate as they move toward the villus. At the base of the crypt, a small population of cells contain a rudimentary terminal web and a few short microvilli with long rootlets. These microvilli appear to arise from bundles of actin filaments which nucleate on the plasma membrane. The microvilli apparently elongate via the addition of membrane supplied by vesicles that fuse with the microvillus and extend the membrane around the actin core. Actin, villin, myosin, tropomyosin and spectrin, but not myosin I (previously called 110 kD; see Mooseker and Coleman, J. Cell Biol. 108, 2395-2400, 1989) are already concentrated in the luminal cytoplasm of crypt cells, as seen by immunofluorescence. Using quantitative densitometry of cDNA-hybridized RNA blots from cells isolated from crypts, villus middle (mid), or villus tip (tip), we found a 2- to 3-fold increase in villin, calmodulin and tropomyosin steady-state mRNA levels; an increase parallel to morphological brush border development. Actin, spectrin and myosin mRNA levels did not change significantly. ELISA of total crypt, mid and tip cell lysates show that there are no significant changes in actin, myosin, spectrin, tropomyosin, myosin I, villin or alpha-actinin protein levels as the brush border develops. The G-/F-actin ratio also did not change with brush border assembly. We conclude that, although the brush border is not fully assembled in immature enterocytes, the major cytoskeletal proteins are present in their full concentration and already localized within the apical cytoplasm. Therefore brush border formation may involve reorganization of a pool of existing cytoskeletal proteins mediated by the expression or regulation of an unidentified key protein(s).