ABSTRACT
Brush border myosin-I is a class I myosin with calmodulin light chains that has been identified in several vertebrate species. In chicken, it is exclusively expressed in intestinal epithelial cells where it forms spirally arrayed bridges that tether the microvillar actin bundle to the membrane. To facilitate future knockout strategies, we have isolated mouse brush border myosin-I cDNA and genomic clones. The deduced primary structure of mouse brush border myosin-I is homologous to other known brush border myosins-I. Northern blot, immunoblot, and immunolocalization studies indicate that the intestine-specific and subcellular localization profile of mouse brush border myosin-I are comparable to that determined for other brush border myosins-I. Northern analysis during embryogenesis revealed a 3.9-kb transcript first detected in 15-day embryos. This is in marked contrast to chicken, where brush border myosin-I expression begins early in embryogenesis. In situ localization in 17-day embryos indicated that RNA expression is restricted to the intestine. Protein expression is first detected in 16-day embryos with decreasing levels observed in a proximal to distal fashion. Immunolocalization in embryonic intestine revealed that brush border myosin-I is evenly distributed on both apical and basolateral membrane domains. There is also pronounced localization to a supranuclear region, presumably the Golgi apparatus. This suggests that brush border myosin-I may be targeted to the plasma membrane on Golgi-derived vesicles rather than by direct targeting to microvillar actin cores.
Subject(s)
Cloning, Molecular , Gene Expression Regulation, Developmental , Microvilli/genetics , Myosins/genetics , Sequence Analysis, DNA , Animals , Base Sequence , Blotting, Northern , Cell Membrane/ultrastructure , Chickens , Embryonic and Fetal Development/genetics , Humans , Intestines/growth & development , Intestines/physiology , Mice , Microvilli/metabolism , Molecular Sequence Data , Myosins/metabolism , RatsABSTRACT
The human intestinal cell line, Caco-2BBe, has been established as an excellent model system for analysis of the enterocyte cytoskeleton including that of the actin rich apical brush border. To facilitate its use for functional analysis of a major component of the brush border, brush border myosin-I, human cDNAs encoding the heavy chain of this class I myosin were isolated and sequenced. The identity of this myosin as human brush border myosin-I was verified based on similarity with other vertebrate sequences, as well as its expression profile at both the RNA and protein levels. Localization of the protein in human intestine along the crypt-villus axis closely resembles that previously determined for brush border myosin-I in chicken, and is quite distinct from that of myosin-Ic, another myosin-I expressed in human intestine and Caco-2BBe cells. In immature cells of the crypt, brush border myosin-I staining is low, and there is significant cytosolic and basolateral localization, while villus cells stain much more intensely, and the protein is primarily localized to the brush border. Localization of myosin-Ic is essentially the inverse of brush border myosin-I in that crypt cells exhibit higher levels of staining, while villus cells have very low levels of myosin-Ic. The expression of both myosins-I was also examined during cell-contact induced differentiation of Caco-2BBe cells where expression and changes in localization closely resemble those that accompany differentiation of enterocyte in vivo.
Subject(s)
Intestinal Mucosa/metabolism , Myosin Heavy Chains/genetics , Amino Acid Sequence , Animals , Base Sequence , Caco-2 Cells , Cell Communication , Cell Differentiation , Cloning, Molecular , DNA, Complementary , Gene Expression , Humans , Microvilli/metabolism , Molecular Sequence Data , Myosin Heavy Chains/classification , RNA , Sequence Analysis, DNA , Tissue DistributionABSTRACT
Myosins are molecular motors that move along filamentous actin. Seven classes of myosin are expressed in vertebrates: conventional myosin, or myosin-II, as well as the 6 unconventional myosin classes-I, -V, -VI, -VII, -IX, and -X. We have mapped in mouse 22 probes encompassing all known unconventional myosins and, as a result, have identified 16 potential unconventional myosin genes. These genes include 7 myosins-I, 2 myosins-V, 1 myosin-VI, 3 myosins-VII, 2 myosins-IX, and 1 myosin-X. The map location of 5 of these genes was identified in human chromosomes by fluorescence in situ hybridization.
