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Pluripotent P19 embryonal carcinoma (EC) cells were differentiated along the neuronal and muscle pathways. Comparisons of class I, II, III, and IV beta tubulin isotypes in total and colchicine-stable microtubule (MT) arrays from uncommitted EC, neuronal, and muscle cells were made by immunoblotting and by indirect immunofluorescence microscopy. In undifferentiated EC cells the relative amounts of these four isotypes are the same in both the total and stable MT populations. Subcellular sorting of beta tubulin isotypes was demonstrated in both neuronal and muscle differentiated cells. During neuronal differentiation, class II beta tubulin is preferentially incorporated into the colchicine-stable MTs while class III beta tubulin is preferentially found in the colchicine-labile MTs. The subcellular sorting of class II into stable MTs correlates with the increased staining of MAP 1B, and with the expression of MAP 2C and tau. Although muscle differentiated cells express class II beta tubulin, stable MTs in these cells do not preferentially incorporate this isotype but instead show increased incorporation of class IV beta tubulin. Muscle cells do not show high levels of MAP 1B and do not express MAP 2C or tau. These results are consistent with the hypothesis that a subcellular sorting of tubulin isotypes is the result of a complex interaction between tubulin isotypes and MT-associated proteins.

Two posttranslational modifications of alpha-tubulin, acetylation and detyrosination, are associated with stable microtubule (MT) populations, including those of neuronal processes. We have used a pluripotent embryonal carcinoma cell line, P19, to investigate changes in MT isotype and stability found in MT arrays during neurogenesis. This cell line has an advantage in that both commitment- and differentiation-related events can be observed. Uncommitted P19 cells have minimal arrays of acetylated and detyrosinated MTs. Following neuronal induction with retinoic acid (RA), indirect immunofluorescence microscopy shows that the first MT modifications occur during commitment and before any morphological change is observed. RA-induced cells initially polymerize a temporarily enlarged population of MTs. Included in this population is a new array of acetylated MTs arranged in a bundle of parallel MTs. This bundle is colchicine-stable, although no MT-associated proteins (MAPs) are detectable using a battery of anti-MAP antibodies. Observation of MT arrays with patterns that are intermediate between the early bundles and short neurites suggests that the acetylated MT bundle subsequently extends to form a neurite. MAP 2 is first detected at about the time of neurite extension. However, at this early stage of differentiation, MAP 2 is not yet limited to dendritic processes. This report provides the first evidence that the stable MTs of mature neurons may be initiated during neuronal commitment.

We present the first evidence of fusion from without induced in tissue culture cells by a nonenveloped virus. Electron micrographs of two strains of rotavirus, bovine rotavirus C486 and rhesus rotavirus, show that virally mediated cell-cell fusion occurs within 1 h postinfection. Trypsin activation is necessary for rotavirus to mediate cell-cell fusion. The extent of fusion is relative to the amount of virus used, and maximum fusion occurs between pHs 6.5 and 7.5. Fusion does not require virus-induced protein synthesis, as virus from both an empty capsid preparation and from an EDTA-treated preparation, which is noninfectious, can induce fusion. Incubation of rotavirus with neutralizing and nonneutralizing monoclonal antibodies before addition to cells indicates that viral protein 4 (VP4; in the form of VP5* and VP8*) and VP7 are involved in fusion. Light and electron micrographs document this fusion, including the formation of pores or channels between adjacent fused cells. These data support direct membrane penetration as a possible route of infection. Moreover, the assay should be useful in determining the mechanisms of cell entry by rotavirus.