Tau Protein Function in Living Cells

Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The injected tau protein increases tubulin polymerization and stabilizes microtubules against depolymerization. This increased polymerization does not, however, cause major changes in cell morphology or microtubule arrangement. Thus, tau protein acts in vivo primarily to induce tubulin assembly and stabilize microtubules, activities that may be necessary, but not sufficient, for neuronal morphogenesis. N 'EURONAL processes are densely filled with parallel arrays of microtubules that act as scaffolds necessary for process extension (Daniels, 1975; Yamada et al., 1976) and serve as tracks for axonal transport (Schnapp et al., 1985). Studies using clonal cell lines that extend neuritic processes in culture have shown that neurite extension involves net microtubule assembly, and not simply rearrangement of existing microtubules into growing processes (Drubin et al., 1985; Gard et al., 1985; Olmstead, 1981). Microtubule assembly during neuronal process extension appears to involve the recruitment of tubulin from preexisting monomer pools into tubulin polymers (microtubules) (Drubin et al., 1985; Gard et al., 1985; Olmsted, 1981). It has been proposed that this assembly is driven by nontubulin protein factors that accumulate during process extension. Support for this proposal comes from the observation that extracts from mature rat brain tissue and from differentiated neuroblastoma ceils contain higher levels of protein factors that promote microtubule assembly than do extracts from their respective less-differentiated counterparts (Nunez et al., 1975; Seeds and Maccioni, 1978). Three major protein factors that promote microtubule assembly in vitro have been identified in brain extracts. These include the tau proteins of 50-70 kD (Weingarten et al., 1975; Cleveland et al., 1977), microtubule-associated protein 1 (MAP1) ~ of '~330 kD, and microtubule-associated protein 2 (MAP2) of "~300 kD (for a review of MAP1 and MAP2 see Vallee et al., 1984). During neurite extension in PC12 pheochromocytoma cells, levels of tau protein correlate precisely with levels of assembled tubulin (Drubin et al., 1985). Furthermore, tau protein is bound to microtubules in PC12 cells (Drubin et al., 1986). These observations make tau pro1. Abbreviations used in this paper: MAP1 and MAP2, microtubule-associated proteins 1 and 2. tein a strong candidate for a factor that promotes microtubule assembly during process extension. Still, given the complex function that microtubules serve, it is not clear which associated proteins regulate polymer assembly and which regulate the interaction of microtubules with other components in the cell or serve some other function. Subtle questions about the importance of microtubule-associated proteins for polymer stability and dynamics have not been addressed in an in vivo context. To test the activity of tau protein in vivo we have microinjected purified tau protein into living fibroblast cells (RATI) that normally contain undetectable levels of this factor. Immunofluorescence shows that microinjected tau protein binds specifically to microtubules (Drubin et al., 1985). Two effects were demonstrated: first, tau causes a marked increase in the level of assembled tubulin. Even more dramatically tau protein increases both centrosome-nucleated and nonnucleated assembly in cells treated with low levels of microtubule depolymerizing drugs. Second, tau protein decreases the depolymerization rate of microtubules. These observations support the conclusion that tau protein promotes the assembly of microtubules that is required for neuronal process extension, and additionally stabilizes the resuiting polymers. Materials and Methods Cell Culture and Microinjections RAT1 fibroblast cells were cultured in DME supplemented with 10% calf serum (Gibco, Grand Island, NY). For microinjection experiments, cells were trypsinized and replated on poly-D-lysine coated coverslips. To compare microinjected cells with control sister cells, single cells were plated at low density ,,o15 h before microinjection. This allowed many cells time to divide once, often resulting in pairs of associated sister cells with similar morphologies. Micruinjections were carried out exactly as described by Schulze and Kirschner (1986). © The Rockefeller University Press, 0021-9525/86/12/2739/8 $1.00 The Journal of Cell Biology, Volume 103 (No. 6, Pt. 2), Dec. 1986 2739-2746 2739 on D ecem er 8, 2017 jcb.rress.org D ow nladed fom Figure 1. Localization of microinjected tau protein on RAT1 microtubules by double-label immunofluorescence. (A) Rhodamine antitubulin staining showing micmtubule networks in RATI cells fixed 40 min after one cell (arrow) was microinjected with 2 mg/ml bovine brain tau protein. (B) Fluorescein anti-tau staining showing tau localization on microtubules in the tau-micminjected cells, and absence of tan in the uninjected cells shown in A. Bar, 10 p.m.