Myosin Vlla is a newly identified member of the myosin superfamily of actin-based motors. Recently, the

ZO-1 is a peripheral membrane protein of approximately 225 kDa located on the cytoplasmic side of all tight junctions. ZO-1 cDNA sequencing disclosed the presence of a 240-bp sequence in only some of the ZO-1 cDNAs studied. This 240-bp region encoded an inframe insertion of 80 amino acids, named motif-alpha. Expression of the predicted transcripts in normal rat and human tissues and in human epithelial cell lines (Caco-2, T84, Hep G2) was shown by reverse transcription of RNA and then DNA amplification. Immunoblot analysis showed both protein isoforms were present; however, in different cell lines, their amounts differed markedly relative to each other. Immunolocalization at light and ultrastructural levels, using antibodies generated against motif-alpha or shared sequences flanking it, indicated both forms localized indistinguishably to tight junctions. These observations demonstrate the existence and variable expression of ZO-1 isoforms and raise the question whether these isoforms contribute to tight junction diversity in different epithelia.

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Centrin, a approximately or equal to 20 kDa calcium-binding protein also known as caltractin, is a component of centrosome-associated algal flagellar roots capable of calcium-mediated contraction, and is also found in the centrosomes of vertebrate cells. Our analysis of a centrin gene from a protist, the amoeboflagellate Naegleria gruberi, reveals conserved features that distinguish centrins from calmodulin. Antibodies to bacterially expressed Naegleria centrin, which also recognize yeast Cdc31p, were employed to localize centrin immunoreactivity in selected organisms possessing specialized microtubule-organizing centers (MTOCs) or accessory structures. There is a striking morphological diversity of such structures. In the simplest associations, as found in Naegleria flagellates and vertebrates tracheal epithelium, centrin is intimately associated with the cylinder of the basal bodies. In cells with unfocused mitotic spindles, Naegleria amoebae and onion root tips, no localization of centrin was detected. In Dictyostelium discoideum and Saccharomyces cerevisiae, which lack centrioles, centrin immunoreactivity was observed as punctate cytoplasmic bodies but not associated with spindle pole MTOCs. In Paramecium multimicronucleatum, centrin immunoreactivity is localized to the infraciliary lattice, previously shown to exhibit calcium-mediated contraction. In Vorticella microstoma, known for the calcium-induced rapid contraction of its stalk, centrin immunoreactivity is localized to the contractile spasmoneme and myonemes. Similar antigens from Paramecium and Vorticella are detected by anti-centrin and anti-spasmin. The pattern of localization of centrin immunoreactivity supports the conjecture that a contractile system involving centrin, initially associated with centriolar structures, was recruited during evolution to build specialized organelles in different organisms and cell types.