Journal of Cell Science 102, 227-237 (1992) 227 Printed in Great Britain © The Company of Biologists Limited 1992 Expression of tau protein in non-neuronal cells: microtubule binding and stabilization GLORIA LEE and SUSAN L. ROOK Program in Neuroscience, Harvard Medical School and Center for Neurologic Diseases, Department of Medicine (Division of Neurology), Brigham and Women's Hospital, Boston, MA 02115, USA Summary The microtubule-associated protein tau is a developmen- tubule contacts or for proper protein folding in the cell. tally regulated family of neuronal phosphoproteins that In our expression system, the bundling of cellular promotes the assembly and stabilization of micro- microtubules occurs only in transfections using four- tubules. The carboxy-terminal half of the protein repeat tau constructs; any four-repeat construct capable contains three copies of an imperfectly repeated se- of binding is also able to induce bundling. Our data quence; this region has been found to bind microtubules suggest that the presence of bundles is correlated with in vitro. In addition, a fourth copy of the repeat has been enhanced microtubule stability; factors that increase found in adult-specific forms of tau protein. To examine stability such as higher levels of tau protein expression or the structure and function of tau protein in vivo, we have the presence of the fourth repeat, increase the fraction of transiently expressed fetal and adult forms of tau protein transfected cells showing bundles. Finally, the presence and tau protein fragments in tissue culture cells. of tau protein in the cell allows all interphase micro- Biochemical analysis reveals full-length products with tubules to become acetylated, a post-translational modi- heterogeneity in post-translational modification syn- fication usually reserved for a subset of stable cellular thesized in the cells. Immunofluorescent staining of microtubules. transfected cells shows that, under our conditions, sequences on both sides of the repeat region are required for in vivo microtubule co-localization. These additional Key words: transient transfections, microtubule bundling, regions may be required either for enhancing micro- microtubule stability, acetylated tubulin. Introduction The earliest purification of tau protein revealed a family of related phosphoproteins (Cleveland et al., Microtubules are ubiquitous structures in eukaryotic 1977). It is now clear that some of this heterogeneity is cells and perform a variety of cellular functions. The generated by alternative splicing (Himmler, 1989). In ability of microtubules to achieve such functional human, cDNA clones for six tau isoforms and their diversity has been thought to be partially provided developmental expression have been described through the assistance of various associated proteins. (Goedert et al., 1988; Goedert et al., 1989a,b). Human Tau protein is a microtubule-associated protein found adult-specific forms are distinguished by the presence of primarily in neuronal tissue. It promotes microtubule one or more of exons2, 3 or 10 (Goedert et al., 1989a,b; assembly in vitro (reviewed by Olmsted, 1986) and exon number as assigned by Himmler (1989) for bovine stabilizes cellular microtubules when microinjected into gene). The most striking feature of the primary cells (Drubin and Kirschner, 1986). Expression of tau structure of tau protein as predicted from cDNA clones protein has been correlated with the extension of is the presence of three evenly spaced, imperfectly neurites during the differentiation of pheochromo- repeated copies of an 18 amino acid sequence in the cytoma cells (Drubin et al. 1985). Moreover, the carboxy-terminal half of the protein. One or more presence of tau antisense in primary neuronal cell copies of the repeat are able to bind to microtubules in culture appears to block the development and mainten- vitro (Butner and Kirschner, 1991; Himmler et al., ance of axon-like processes, suggesting a role for tau 1989; Lee et al., 1989). Not surprisingly, increasing the protein in the establishment of neuronal cell polarity number of copies of the repeat increased the efficiency (Caceres and Kosik, 1990; Caceres et al., 1991). In situ, of microtubule binding (Butner and Kirschner, 1991; tau protein is associated with axons (Binder et al., 1985; Lee et al., 1989). Synthetic one-repeat peptides also Kowall and Kosik, 1987; Brion et al., 1988; Trojanowski promote microtubule assembly; however, the required et al., 1989). stoichiometry indicates that the peptides are much less 228 G. Lee and S. L. Rook potent than intact protein (Ennulat et al., 1989; Joly et SV40 early promoter, filled in, then ligated to an Ncol linker a!., 1989). The high molecular weight microtubule- in order to introduce an Ncol site at this location. The sequence of the new multiple cloning site in this vector, associated protein MAP2 shares about 50% amino acid + homology with tau protein at the carboxy-terminal end named pECEN , was checked by DNA sequencing. 220 residues and also contains three repeats (Lewis et Most three-repeat human tau inserts were prepared by polymerase chain reaction (PCR) using pl9tau plasmid DNA al., 1988). As expected, a fragment of MAP2 from this (Lee et al., 1989) as template. PCR primers contained area coassembles with microtubules in vitro (Lewis et restriction sites that enabled insertion into pECEN+. pEn was al., 1988). prepared by cutting and recircularizing pEnl23c. In some The adult-specific exon 10 encodes a 31 amino acid plasmids, translation was terminated by a stop codon in the sequence that contains a fourth copy of the microtubule primer; in other plasmids, translation was terminated by the binding repeat unit. The inclusion of this fourth repeat stop codons in the vector. Table 1 lists all plasmids used in this increases the activity of tau protein in in vitro study; tau sequences expressed and new sequences contribu- microtubule assembly assays (Goedert and Jakes, ted by primers, linkers or vectors are shown. The longest 1990). The expression of a four-repeat tau protein in sequence contributed in this manner was 11 residues and its presence did not correlate with any functional property of the vivo results in the bundling of cellular microtubules expressed proteins. (Kanai et al., 1989). pEnl234c was constructed from pEnl23c by replacing the In this study, we examined the in vivo structure and tau cDNA carboxy-terminal 500 bp with the analogous 593 bp function of fetal and adult tau protein. Transiently fragment from an adult human tau four-repeat cDNA clone expressed tau protein and tau protein fragments were (Mori et al., 1989) kindly provided by Dr. H. Mori (Tokyo characterized using Western blot and immunoprecipi- Metropolitan Institute of Gerontology, Tokyo, Japan). The tation protocols. The ability of the expressed protein to resultant cDNA is a full-length four-repeat tau cDNA associate with cellular microtubules and to induce identical to a clone previously identified by Goedert et al. microtubule bundling were determined by immunoflu- (1989a). pE1234c(487) and pE1234(487) were constructed orescence. Our results indicate that, under our con- from pE123c(487) by similar replacements. pEnl23(4c) was ditions, sequences adequate for in vitro binding are not prepared by cutting and recircularizing pEnl234c. The remaining human four-repeat tau inserts were prepared by adequate for in vivo co-localization to microtubules. PCR from pEnl234c. Polymerase chain reaction primers used Furthermore, the presence of adult-specific tau protein to prepare inserts for insertion into pECEN+ were identical to can induce microtubule bundling; microtubule stability those used for the three-repeat human tau inserts and, is also increased. Bundling and stability may be related, therefore, the starts and stops of expressed sequences are since no evidence for cross-linking of microtubules by identical. tau protein was found. Lastly, the presence of tau Because most constructs were made using polymerase chain protein correlated with the acetylation of all cellular reaction technology, we acknowledge the small probability of microtubules, indicating enhanced stability for all a base change occurring during synthesis. However, most of microtubules. the DNA fragments prepared were fairly small and the biochemical characterizaton of expressed protein (see below) would detect the inadvertant production of a termination codon. Also, all of the constructs overlapped, so that if a single amino acid replacement were to alter dramatically the Materials and methods properties of the expressed protein, an inconsistency would be noted. Construction of expression plasmids The eukaryotic expression vector pECE (Ellis et al., 1986) Transfections was modified in the following manner to introduce a unique CHO or 3T3 (NIH) cells were grown in oMEM sup- Ncol cloning site into its multiple cloning site. First, the sole plemented with 10% fetal bovine serum (Hyclone, Logan, Ncol site in pECE was removed by Ncol cleavage followed by UT) and seeded onto 12 mm coverslips in 24-well plates at a fill-in and re-circularization. The plasmid was then cut with density of approximately 0.2 x 104 /well. CHO cells were Bglll, the first site in the polylinker site downstream from the transfected by calcium phosphate precipitation using standard Table 1. Eukaryotic expression plasmids NH2-terminal sequences Tau sequence COOH-terminal sequences Plasmids from primers, linkers or vector (residue no.) from primers, linkers or vector