Journal of Cell Science 110, 281-294 (1997) 281 Printed in Great Britain © The Company of Biologists Limited 1997 JCS8072

Overexpression of full- or partial-length MAP4 stabilizes microtubules and alters cell growth

Hoang-Lan Nguyen1, Sripriya Chari2, Dorota Gruber3, Chia-Man Lue3, Steven J. Chapin3,* and Jeannette Chloë Bulinski3,† 1Department of Pathology, 2Integrated Program in Cellular, Molecular & Biophysical Studies, and 3Department of Anatomy & Cell Biology, Columbia University, College of Physicians & Surgeons, 630 W. 168th St, New York, NY 10032, USA *Present Address: Department of Anatomy, University of California, San Francisco, CA 94143-0452, USA †Author for correspondence (e-mail: [email protected])

SUMMARY

To investigate the in vivo functions of MAP4, a micro- equivalent efficacy demonstrated in studies of in vitro MT tubule-associated protein expressed almost ubiquitously in polymerization (Aizawa et al. (1991) J. Biol. Chem. 266, vertebrate cells, we prepared stably transfected clonal 9841-9846). L-MAP4 and L-MTB cells grew significantly mouse Ltk− cell lines expressing full-length MAP4 (L-MAP4 more slowly than control cells; this growth inhibition was cells) or its MT-binding domain (L-MTB cells). Although not due to mitotic arrest or cell death. L-MAP4 and L-MTB transfectants showed no dramatic defect in morphology, cells also exhibited greater tolerance to the MT-depoly- organellar distribution, or level of MT polymer, as merizing agent, nocodazole, but not to the MT-polymeriz- compared to naive Ltk− cells or L-MOCK cells (transfected ing agent, Taxol. Our results demonstrate that MAP4 and with vector alone), MTs in L-MAP4 and L-MTB cells its MT-binding domain are capable of MT stabilization in showed greater stability than those in control cells, as vivo, and that increasing the intracellular level of MAP4 monitored by the level of post-translationally detyrosinated affects cell growth parameters. α-tubulin and by a quantitative nocodazole-resistance assay. In vivo, the MT-binding domain of MAP4 stabilized Key words: Nocodazole, Cell cycle, Drug resistance, MTs less potently than full-length MAP4, in contrast to the Dexamethasone, Inducible expression, Stable microtubule

INTRODUCTION MTs may play an important role both in cell cycle events and in differentiative processes. Microtubules (MTs) are believed to play important roles in MT-associated proteins (MAPs) have been postulated to a variety of cellular processes, including such diverse function as in vivo regulators of the dynamics and functions of functions as mitosis, cell motility, and intracellular vesicle MTs. Based on in vitro studies, several MAPs have been classi- transport. Both in vitro and in vivo, individual MTs have fied as assembly-promoting MAPs. These include MAP2 been observed to undergo alternating periods of growth and (Murphy and Borisy, 1975) and tau (Cleveland et al., 1977), shrinkage along their lengths, a process known as dynamic proteins that are abundantly expressed in neurons, and MAP4, instability (Mitchison and Kirschner, 1984). Studies in which which is more widely expressed (Bulinski and Borisy, 1980a,b; pharmacological antagonists were used to alter MT Parysek et al., 1984; Murofushi et al., 1986). Results of previous dynamics have demonstrated that the dynamic behavior of studies of tau and MAP2, the two most extensively studied MTs is intimately related to their ability to participate in assembly-promoting MAPs, suggest that these MAPs also cellular functions such as cell division (Jordan et al., 1992, promote MT assembly in vivo. For example, in cultured cells in 1993) and cell motility (Liao et al., 1995; A. Mikhailov and which the level of MAP2 or tau was experimentally increased by G. G. Gundersen, unpublished work). The dynamic proper- transfection of MAP cDNAs (Kanai et al., 1989; Lee and Rook, ties of MTs have been shown to be regulated during both cell 1992; Bramblett et al., 1993; Ferralli et al., 1994) MTs were growth and differentiation. For example, MT dynamics shown to be increased in their stability. In cultured cells in which change during the cell cycle, exhibiting greater dynamics MAP2 and tau were decreased by anti-sense strategies (Caceres during M phase than during interphase (Saxton et al., 1984; and Kosik, 1990; Dinsmore and Solomon, 1991), cells were Zhai and Borisy, 1994). In contrast, accumulation of stable inhibited in their extension of processes, a function believed to MTs during myogenesis (Gundersen et al., 1989) and be dependent upon the stabilization of MTs (Wehland and Weber, neuronal differentiation (Wehland and Weber, 1987; Baas 1987). These results implicate MAP2 and tau as molecules and Black, 1990) suggest that MT dynamics are decreased capable of stabilizing and promoting assembly of MTs in cultured during cell differentiation. Thus, the dynamic behavior of cells, in agreement with the MAPs’ in vitro properties. 282 H.-L. Nguyen and others

MAP4 is a MT-associated protein of ~210 kDa found in all that other assembly-promoting MAPs may perform overlap- vertebrate organisms that have been examined. In vivo, MAP4 ping or redundant roles in cultured cells or in the whole animal. is distributed along most or all MTs in both proliferating and Therefore, to investigate the in vivo function of MAP4, it differentiated cells; in contrast to tau and MAP2, it is normally would be advantageous to carefully perturb the cell’s content absent from mature neurons (Bulinski and Borisy, 1980b; of MAP4 and assay resulting changes in MT behavior quanti- Parysek et al., 1984; Chapin and Bulinski, 1994). In vitro tatively. In this way, one could limit, as much as possible, alter- experiments have identified two domains within the structure ations in other MAPs or other compensatory behaviors that of MAP4, including a MT-binding domain and a ‘projection might otherwise confound the interpretation of experiments. domain’, a region of unknown function that is thought to In this paper we report the results of our investigations into project from the MT wall (Aizawa et al., 1987, 1991; Chapin MAP4’s in vivo function. Our goal was to answer several and Bulinski, 1991). The MT-binding domain of MAP4 questions: first, does MAP4 stabilize MTs or dampen MT contains a series of imperfectly repeated elements of 18 amino dynamics in vivo, as it does in vitro, and if so, how potent is acids each, separated by spacer sequences; this region is this stabilizing effect? Second, if full-length MAP4 effects MT homologous with the MT-binding domains of MAP2 and tau. stabilization in vivo, can this function be mimicked by the MT- A number of developmentally regulated MAP4 transcripts are binding domain, or are elements of the projection domain produced by alternative splicing and these encode at least three required for full stabilization of MTs in the living cell? Finally, heterogeneous forms of MAP4 that differ in the number of when MAP4 level is increased, do the cells show any pheno- repeated elements they contain within their MT-binding typic defects directly, or are slight deficits compounded so as to domains (Chapin et al., 1995). In addition to its homologous affect cell growth properties? To answer these questions defin- MT-binding domain, MAP4 shares other properties with itively, we chose an experimental system in which we could MAP2 and tau; for example, all three MAPs exhibit both a C- induce the overexpression of human MAP4 or its MT-binding terminal MT-binding and an N-terminal projection domain, domain in stably transfected, clonal rodent L cell lines. To and all show developmentally regulated, alternatively spliced determine whether MAP4 stabilizes MTs in vivo, we applied a forms that contain alterations in MT-binding domain structure quantitative assay of MT stability to characterize transfectant (Chapin and Bulinski, 1992). cell lines whose MAP4 expression was carefully controlled. Given MAP4’s capacity to augment MT assembly in vitro, Since one or a combination of subtle defects in a cell will often its in vivo distribution along MTs, and its structural similari- affect its growth, we assayed the effects of overexpressed ties to MAP2 and tau, it would be logical to hypothesize that MAP4 on growth parameters of the transfected cells. Our MAP4’s function in vivo is to promote MT assembly and to results demonstrate that increased expression of MAP4 in L cell regulate MT dynamics. However, to date, the results obtained fibroblasts increases stability of cellular MTs and alters cell from probing MAP4’s in vivo function(s) have yielded growth and response to a MT-depolymerizing drug. We also confusing information. One experimental approach used was show for the first time that the MT-binding domain of MAP4, to increase the abundance of MAP4 in living cells, in order to alone, cannot mimic the functions of full-length MAP4 in vivo. test whether MT assembly or dynamics are concomitantly altered. Barlow et al. (1994) transfected MAP4 cDNA into CHO cells and reported that these cells displayed no pheno- MATERIALS AND METHODS typic changes and no heightened MT stability. However, con- tradictory findings were obtained when Olsen et al. (1995) Materials transfected the MAP4 MT-binding portion into cultured cells, Except as noted, all chemicals were purchased from Sigma (St Louis, and Yoshida et al. (1996) used microinjection to introduce full- MO) or from Fisher Scientific (Tustin, CA). All tissue culture length MAP4 or its MT-binding domain portions into cultured materials were from Gibco Life Sciences (Gaithersburg, MD). cells; each group discovered that some MTs were stabilized as Immunochemicals were obtained from Organon Teknika (West a result. These disparate results can best be explained by con- Chester, PA). All restriction enzymes were purchased from Promega sidering that variations in the levels of MAP4 expression, the Biotech (Madison, WI) or New England Biolabs (Beverly, MA). characteristics of the host cells chosen for the experiments, Preparation of stable cell lines overexpressing MAP4 and/or the different qualitative assays used to detect MT A full-length MAP4 cDNA encoding the tau-like four-repeat isoform stability may have given rise to divergent outcomes in each of human MAP4 (isoform III; Chapin et al., 1995), contained in experiment. pGEM7Zf(+) was cleaved at a site upstream of the MAP4 insert with Another experimental strategy that was used to test MAP4 XbaI, blunt-ended with Klenow, ligated to a HindIII linker, and function in vivo was to attempt to abrogate its function and digested with HindIII. After ligating the ends of this linear construct determine the resulting phenotype. Wang et al. (1996) microin- together, the full-length MAP4 cDNA was excised with HindIII and jected cells with antibodies that inhibited binding of MAP4 to cloned into the HindIII site of pMMTV (obtained from Drs Steve Chin cellular MTs. Surprisingly, these cells showed no detectable and Ron Liem, Columbia University). After screening plasmids for phenotype, and their MT turnover appeared to be identical to proper direction of insertion, this construct, referred to as pMMTV- sham-injected cells. Similarly, no obvious phenotype and no MAP4, was used for transfection. The plasmid encoding the entire aberrant MT stability were obtained in Drosophila in which MT-binding domain of human MAP4 isoform III, called pMMTV- MTB, was generated from pMMTV-MAP4, by incomplete digestion 205 kDa MAP, thought to be a relative of MAP4 despite the with StuI (at nucleotide positions 97 and 2,025) and religation in the lack of homologous sequences, was genetically knocked out presence of an eight nucleotide XhoI linker. The resulting protein (Pereira et al., 1992). The failure to discern a phenotype in encoded by pMMTV-MTB included the entire proline-rich region, these studies suggests either that the hypothesis that MAP4 or repeat-bearing portion and C terminus, but lacked sequences encoding its relatives function in regulating MT stability is incorrect, or the projection domain of MAP4 (amino acid residues 33-675). MAP4 stabilizes microtubules in vivo 283

Mouse Ltk− cells, originally obtained from Dr Peter Gunning with a silicon-intensified tube camera (MTI Corp., Michigan City, (Children’s Hospital, University of Sydney, Australia) were grown in IN), and the intensities of the bands in each lane were determined Dulbecco’s MEM (DMEM) containing 10% calf serum for all exper- using IMAGE-1 software (Universal Imaging Corp., West Chester, iments; induction of transfected constructs was performed by adding PA), Lotus1-2-3 Release 5 spreadsheet software (Lotus Development DMEM containing 1 µM dexamethasone (dex), for a 24 hour interval, Corp., Cambridge, MA), and SigmaPlot graphing software (Jandel except as noted. Ltk− cells grown to about one-half confluence in 100 Scientific, San Rafael, CA). Each clonal cell line’s MAP4 or MT- mm culture dishes were transfected by the calcium phosphate binding domain content was determined in three separate experiments, procedure (Graham and van der Eb, 1973), with 20 µg pMMTV- and the average for each cell line was expressed as picomoles of MAP4 or pMMTV-MTB DNA, along with 2 µg pSV2-neo DNA as MAP4 or MT-binding domain per mg of total protein. a selectable marker. To prepare ‘mock-transfected’ cells, referred to as L-MOCK lines, pMMTV lacking a cDNA insert was substituted in Immunofluorescent staining of transfected cells the transfection procedure. Some cells were analyzed by immunoflu- Cells cultured on coverslips were fixed in methanol and immunos- orescence 48 or 72 hours after transfection, for a rapid assessment of tained as described by Gundersen et al. (1989), except that primary the success of the transfection procedure. Stably transfected cell lines antibodies used were a polyclonal, primate-specific MAP4 antibody used for all experiments described here were derived by placing the (Chapin and Bulinski, 1991), a guinea pig antibody directed against antibiotic, G418, in the culture medium from days 2-16 following human MAP4 MT-binding domain (see above), a β-tubulin antibody transfection. For each transfection, a small number of colonies (≤30) (a generous gift from Dr James Lessard, University of Cincinnati, grew up on each 100 mm culture plate, and at least 20 independent Cincinnati, OH), or antibodies specific for detyrosinated or tyrosi- G418-resistant colonies were isolated from two or three plates, using nated tubulin (Gundersen et al., 1989). Staining of other cellular com- cloning cylinders (Bellco, Inc., Vineland, NJ). Subcultures from each ponents utilized antibodies directed against γ-actin (Otey et al., 1986), G418-resistant colony were treated with dex for 24 hours, and the Golgi apparatus (monoclonal antibody against mannose-6- analyzed for expression of human MAP4 by western blotting and phosphate, generously donated by Dr Chris Gabel, Pfizer, Inc. Groton, immunofluorescence (see procedural details below). Those colonies CT), and mitochondria (monoclonal anti-bovine cytochrome oxidase showing the highest level of inducible expression of human MAP4 subunit IV; Molecular Probes, Inc., Eugene, OR). Microscopy of were subcloned and several clones were chosen for analysis. In clonal immunostained cells was performed as previously described L-MOCK lines, the presence of pMMTV was assayed by Southern (Gundersen et al., 1989), except that images were captured with a blot (Sambrook et al., 1989). To avoid plasmid loss or development Micromax cooled CCD camera (Princeton Instruments, Trenton, NJ) of undesirable heterogeneity, cells were used within ten passages of with a Kodak KAF1400 chip (Kodak, Rochester, NY). Each picture cloning; the level of heterogeneity was assayed by immunofluor- was exposed for 4 seconds and processed with the MetaMorph system escence in each experiment. For each plasmid transfected, stable, (Universal Imaging Corp. West Chester, PA). All images were clonal lines derived from at least two different colonies isolated in the imported into Adobe Photoshop 3.0 (Adobe Systems, Inc., Mountain original G418 selection were compared, in order to rule out pheno- View, CA) and converted into 8-bit format. Pixels were scaled iden- typic changes that might otherwise be ascribed to point of insertion tically in each image, such that the lightest and the darkest pixels in or number of copies of plasmid within the genome. On average, we the 256 shades of gray were represented. The images displayed in the found that at least 70% of colonies exhibiting resistance to G418 figures were printed on a Kodak dye sublimation printer (Speed possessed at least modest inducible expression of human MAP4. Graphics, New York, NY). Immunoassay of levels of MAP4 and MT-binding domain in Assays of MT stability transfected cells Monomer, polymer, and total tubulin in all cell lines were determined Western blots were performed as described previously, using primate- as described by Gundersen et al. (1987), using bovine brain tubulin specific rabbit MAP4 antibodies (Chapin and Bulinski, 1991). For to generate a standard curve. In control experiments, analysis of cells some experiments, such as the sensitive detection of MAP4’s MT- whose MTs had been completely depolymerized by treatment with 10 binding domain in MTB cells, antibodies were prepared in guinea pigs µM nocodazole for 16 hours gave a value of 0% polymer, while cells by Pocono Farms (Canadensis, PA), using a bacterially expressed whose MTs were completely stabilized by treatment with 1 µM Taxol MAP4 MT-binding domain portion as immunogen. Briefly, this for 4 hours gave a value of 100% polymer using this partitioning protein, termed JS3, was prepared by inserting a BglII-SmaI fragment technique. into the BamHI site of pET3a, transforming BL21/DE3 bacteria, and Nocodazole resistance of MTs was assayed as follows: L-MAP4, inducing their expression with IPTG (Studier et al., 1990). Prepara- L-MTB, L-MOCK, and naive mouse Ltk− cells were cultured on glass tion of 98% pure samples of JS3 was accomplished by boiling the coverslips until they were approx. an eighth confluent, treated with extract of sonicated bacteria for 20 minutes in a buffer containing 0.1 dex for 24 hours and treated with 10 µM nocodazole in culture M Pipes, pH 6.9, 1 mM dithiothreitol, 1 mM EGTA, 1 mM MgCl2, medium for 0-120 minutes at 37¡C. Coverslips were extracted in a 0.5 M NaCl, and 1 µg/ml each of chymostatin, leupeptin, antipain, solution of 0.2 mg/ml saponin in 0.1 M Pipes, pH 6.9, 1 mM EGTA and pepstatin, and centrifuging the sample at 48,000 g to separate and 1 mM MgSO4, for 2 minutes at 37¡C, fixed in methanol for 5 heat-stable JS3 protein in the supernatant from heat-labile bacterial minutes at −20¡C, and then stained with anti-tubulin antibody. Cells proteins in the pellet. Guinea pigs were injected with 50 µg JS3 in were scored to determine the number of MTs that remained after each of four injections, using either soluble JS3 protein or JS3 protein nocodazole treatment, exactly as previously described (Khawaja et al., excised from polyacrylamide gel bands. 1988). Briefly, a MT was scored as fluorescence of length greater than For quantifying expression of human MAP4, L-MTB and L-MAP4 width, as resolvable under ×63 magnification. One coverslip of each clonal cell lines were cultured until approx. half confluent and induced cell type was not treated with nocodazole and was fixed in methanol with dex for 24 hours Immunoblots stained with anti-JS3 or anti- without prior saponin extraction (saponin treatment is known to MAP4 antibodies, respectively, were quantified using standard curves extract MAP4 from MTs; Bulinski and Bossler, 1994); this coverslip generated by blotting either JS3 (for quantifying expression of MAP4 was stained with antibodies to MAP4, in order to assess the unifor- MT-binding domain in L-MTB cell lines) or full-length bacterially mity of MAP4 expression. expressed MAP4 (for quantifying expression in L-MAP4 cell lines). Full-length MAP4 was prepared as described above for JS3, except Analysis of cell growth and phenotype that a cDNA encoding full-length MAP4 was inserted between NdeI Cell growth was assayed by plating 2×104 cells of each cell type and BamHI sites in the pET vector. Images of each blot were captured (passaged from half-confluent cultures) into each of 16 tissue culture 284 H.-L. Nguyen and others plates (60 mm diameter). Dex was added 24 hours later (t=0), and the of our experimental system was that we express exogenous MAP4-expressing or control cells were allowed to grow for 8 days. MAP4 that could be detected with antibodies and would Dex-containing medium was changed every second day. At each time behave similarly to the endogenous MAP4 in the cultured cells. point, cells in duplicate cultures were released from the substratum To this end, we chose to express human MAP4 in a rodent cell with Viokase protease solution, counted in a haemocytometer, and line, since this would allow us to conveniently assay trans- assayed for viability by trypan blue exclusion, and for mitotic index fected MAP4 with available primate-specific MAP4 anti- with either a Bac-Light stain kit (Molecular Probes, Eugene, OR), tubulin immunofluorescence, or phase microscopy at ×40 magnifica- bodies, and obviate the need to add an epitope tag or other tion. For each cell type, linear regression analysis was performed on modification to the MAP4 molecule expressed. cell counts of duplicate cultures from at least two separate experiments The second requirement of our system was that the cells in order to calculate cell doubling time. Cells were also monitored for under study comprise a uniform population of cells that would their ability to grow in culture medium containing the MT-antagonis- exhibit high levels of expression of the transfected MAP4. This tic agents, nocodazole and Taxol. For these experiments, cells were would allow us to perform analyses of MAP4 behavior or plated and induced with dex as described for cell growth experiments, function, without concern that our results were affected by then they were treated at t=2 days with the specified concentration of variations that might exist among individual cells. To prepare drug. A lethal dose of a drug was defined as a concentration sufficient cells that would meet this requirement, we stably transfected to kill >98% cells following a four day treatment. an inducible plasmid, pMMTV-MAP4, into mouse Ltk− cells, Assay of bromodeoxyuridine incorporation and isolated homogeneous, clonal cell lines that efficiently and Cells were plated identically at a density of 6×103 cells/cm2 and uniformly expressed the desired MAP4 constructs. Because the cultured on coverslips. At approx. an eighth confluence they were MMTV plasmid contains a glucocorticoid-inducible promoter, induced with dex for 24 hours Bromodeoxyuridine (BrdU) was added we were able to use dexamethasone (dex), a synthetic hormone to each culture at a concentration of 9 µM, along with 1 µM that has no deleterious effects on the cells, to induce expression − deoxythymidine, 5 µM deoxycytidine (dCyt), and 5 µM 5-fluorouracil of MAP4. The mouse Ltk cell line was an ideal cell line for (5-FU), as specified (Dolbeare and Selden, 1994) in order to ensure a our purposes, since Ltk− cells are well-studied, readily trans- BrdU substitution level which allows efficient binding of anti-BrdU fected cells that had been used in functional studies of other antibodies. The 5-FU was added to inhibit endogenous thymidine MT-binding proteins (Kanai et al., 1989; Nakata and Hirokawa, synthesis (Dolbeare and Selden, 1994), and dCyt to prevent the inhi- 1995); they are also highly responsive to dex (Hall et al., 1983). bition of DNA synthesis by BrdU (Meuth and Greene, 1974). After the A final requirement of our overexpression system was that our desired periods of incubation in BrdU, coverslips were rinsed briefly transfected constructs encode the most appropriate isoform of in Earle’s balanced salt solution (EBSS) at 37ºC, fixed in methanol at −20ºC, and post-fixed for 90 seconds in methanol/ acetic acid, 3:1 MAP4; that is, the form most likely to be amenable to a direct (v/v). Coverslips were denatured for 30 minutes in 2 M HCl, rinsed test of our hypotheses of MAP4 functions. Our lab. had previ- briefly in EBSS, and immediately inverted over 50 µl of TBS (pH 7.5), ously found that several isoforms of MAP4, derived from alter- containing 0.1% mouse anti-BrdU (monoclonal antibody BU-33, natively spliced transcripts, are differentially expressed in Sigma Immuno Chemicals, St Louis, MO) and 1% normal goat serum, various tissues and developmental stages (Chapin et al., 1995). for 30 minutes. After rinsing in TBS, secondary antibody staining was Each of these isoforms is unique in the number of imperfectly performed with 1% rhodamine-labeled goat anti-mouse IgG and 1% repeated sequences it possesses within its MT-binding domain. normal goat serum in TBS. Coverslips were mounted on glass slides, Although proliferating cells whose MTs are dynamic express a in Fluoromount-G mounting medium (Southern Biotechnology, Birm- MAP4 isoform bearing five repeats, differentiated cells and ingham, AL), containing 0.1% 4,6-diaminidino-2-phenylindole dihy- tissues whose MTs are more stable express a four-repeat isoform drochloride (DAPI). The number of cells that incorporated BrdU was (called the tau-like MAP4 isoform, or form III; Chapin et al., scored as a percentage of the number of DAPI-stained cells in each field. For each experiment, at least 400 cells were scored per sample 1995). In rodents, expression of MAP4 form III, the four-repeat coverslip. Two of the three experiments were scored double blindly, form of tau, and an analogously structured four-repeat form of and a Student’s t-test was performed to assess the significance of the MAP2c all correlate with increased states of differentiation, and differences observed. DAPI staining was viewed as previously each has been hypothesized to possess a heightened capacity to described (Gundersen et al., 1989), except using a Fluor 40 lens and a stabilize MTs, relative to other forms of the same MAP (Chapin UV-2 filter cube (Nikon Instruments, Garden City, NY). et al., 1995; Goedert and Jakes, 1990; Doll et al., 1993, respec- tively). Accordingly, we chose to transfect cells with cDNAs encoding MAP4 form III, since we considered this four-repeat RESULTS MAP4 species to be the form most likely to confer a MT stabi- lization phenotype on cells into which it was transfected. A technique commonly used to ascertain the in vivo function With our experimental system we also chose to investigate of a protein is to express the protein in cultured cells and to whether the MT-binding domain of MAP4 could mimic the analyze the cells for phenotypic changes that result from function of the entire molecule in vivo. Aizawa et al. (1991) increased function of the protein. To probe the function(s) of had reported the results of in vitro experiments that demon- MAP4 under physiological conditions, we focused on two strated that the MT-binding domain of MAP4 was fully capable straightforward questions: first, does MAP4 induce a dis- of promoting MT assembly. In fact, on a mole per mole basis, cernible phenotype in cells in which it is overexpressed? the MT-binding domain studied by Aizawa et al. (1991) Second, does MAP4 stabilize MTs in vivo, as it does in vitro? exhibited equivalent activity in in vitro MT polymerization and We designed an experimental system that satisfied several binding assays. Therefore, we tested whether this result could requirements and, thus, was amenable to answering these be extrapolated to the in vivo situation. We transfected Ltk− questions. Since MAP4 is expressed and codistributed with cells with a construct encoding the MT-binding domain of MTs in all vertebrate cells examined thus far, one requirement MAP4 form III (MTB) in order to test whether sequences MAP4 stabilizes microtubules in vivo 285

Table 1. Overexpression of MAP4 and MT-binding domain in stably transfected cell lines L-MAP4 L-MAP4 L- MTB L-MTB #13-2 #18-1 #3-6 #8-4 HeLa MAP4, % of total protein (w/w) 0.67±0.12 0.37±0.15 1.73±0.22 0.31±0.06 0.1* Ratio, MAP4: tubulin (mol/mol) 0.15±0.04 0.14±0.04 0.49±0.26 0.13±0.08 0.02* Overexpression, relative to total protein (w/w)† 7× 4× 17× 3× 1× Overexpression, relative to tubulin (mol/mol)† 8× 7× 25× 7× 1×

*Values for HeLa were reported by Bulinski and Borisy (1979). †MAP4 expression is normalized to HeLa, a cell line in which MAP4 and tubulin expression have been characterized extensively. Immunoassays to quantifiy expression of tubulin and human MAP4 in each cell line are described in Materials and Methods. Note that endogenous murine MAP4 is not assayed here, since it is not reactive with primate-specific MAP4 antibodies. Standard deviation is reported for each value. contained in the C-terminal one-third of the MAP4 molecule, synthesis in L-MAP4 cells. As shown in the western blot in equivalent to the region shown to substitute for full-length Fig. 2 and the micrographs in Fig. 3, human MAP4 could not MAP4 in vitro (Aizawa et al., 1991) could functionally mimic be detected in cells that were not induced by dex (Fig. 2, 0 hour full-length MAP4 in vivo. lane). Within 3 hours of dex treatment (Fig. 2, 3 hour lane), expression of human MAP4 was detectable, and its abundance Generation of stable L-cell lines inducibly increased to a plateau level between 12 and 24 hours after dex overexpressing MAP4 addition (Fig. 2, compare 12 hour and 24 hour lanes). − We transfected mouse Ltk cells with MMTV plasmids con- Similarly, by immunofluorescence human MAP4 was taining a cDNA insert encoding either full-length MAP4 or its detectable at 6 hours after induction with dex (Fig. 3b) and was MT-binding domain, or with empty MMTV plasmid (we refer much more brightly stained in cells induced with dex for 24 to these cells as L-MAP4, L-MTB, and L-MOCK, respec- hours (Fig. 3c). We performed all experiments with L-MAP4 tively). We isolated several colonies from each, following transfectants between 24 hours and 7 days after dex induction, antibiotic treatment selective for a co-transfected plasmid, since MAP4 levels were constant during this interval (Fig. 2, pSV2neo. We characterized the transfectant cell colonies, compare lanes 24 hour, 2D, 4D, and 6D), and bright immuno- assessing inducibility of pMMTV expression by dex, fluorescence was not changed in appearance during this time abundance of MAP4 expression, and intracellular distribution period (data not shown). Fig. 2 documents MAP4 level in one of induced MAP4. Assays of all L-MAP4 and L-MTB colonies L-MAP4 clonal line; identical results were obtained for all L- resistant to the antibiotic, G418, revealed that 70-90% of these MAP4 and L-MTB cell lines (data not shown). colonies expressed MAP4 or MT-binding domain that was We quantified the expression level of full-length MAP4 and readily detectable by immunofluorescence and western MT-binding domain in the transfectant cell lines, as shown in blotting. Of these, 10% were chosen as high level expressers; Table 1. We performed all experiments with clonal cell lines these were cloned to yield L-MAP4 and L-MTB cell lines that selected from at least two independent colonies derived from exhibited a uniform, high level of expression of MAP4 when the original G418 antibiotic selection. Thus, we studied one induced with dex (Table 1). Fig. 1 shows immunofluorescence line each of cells expressing low and high levels of each MAP4 patterns typical of clonal lines in the presence (a-f) and absence species; these lines would be expected to differ in plasmid copy (g-i) of dex. Note that bright immunofluorescence with MAP4 number and genomic integration points. Several other inde- antibodies was observed in dex-induced L-MAP4 and L-MTB pendent clonal L-MAP4 and L-MTB lines that exhibited cells, and these MAP4 forms colocalized with anti-tubulin similar behavior were analyzed less extensively. staining of the same cells (compare a and b with d and e, respectively). Fig. 1 also demonstrates the uniformity of Transfected human MAP4 does not alter the expression level of MAP4 or MT-binding domain obtained in appearance of cells or their MT arrays each line. In the presence of dex, >95% of L-MAP4 and L- One complication that often arises when overexpression of a MTB showed bright anti-MAP4 staining, with an additional protein is used to test in vivo function is the tendency of proteins population (<1%) showing even brighter staining, and the present at high concentration to exhibit nonspecific interactions. remaining minority of cells showing dim or barely detectable In our system, we were initially concerned that grossly overex- immunofluorescence with MAP4 antibodies. In Fig. 1, a poly- pressed MAP4 or MT-binding domain might interact non- clonal antibody raised to full-length human MAP4 was used to specifically with low affinity binding sites within the cell, stain both the full-length MAP4 and the MT-binding domain yielding ‘false’ phenotypes. However, in all cell lines we in transfected cells. Perceptibly dimmer or less distinct staining analyzed, regardless of the level of MAP4 or MT-binding was observed in the L-MTB transfectants, compared to the L- domain that was expressed, all exogenous MAP4 or MT-binding MAP4 transfectants (compare Fig. 1a and b). This decreased domain appeared to be localized to the MTs (Fig. 3). Thus, the brightness is due to the fact that, in comparison to full-length intracellular distribution of MAP4 and MT-binding domain MAP4, the MT-binding domain is a smaller molecule that provided evidence that each was behaving like the endogenous contains fewer epitopes reactive with the polyclonal MAP4 MAP4, rather than exhibiting nonspecific interactions favored by antibody. Nonetheless, immunofluorescence patterns demon- unusually high concentrations of MAP4 in the cell. strate that MAP4’s MT-binding domain, like full-length MAP4, We performed a careful analysis of the cell morphology and is bound to cellular MTs in transfectants. intracellular organelle distribution of L-MAP4 and L-MTB cells, We next examined the time course of induction of MAP4 in order to determine if the transfectants displayed morphological 286 H.-L. Nguyen and others phenotypes that could be detected upon gross examination. In par- that had not been cultured in dex, and it also allowed the MTs ticular, L-MTB cells expressing high levels of MT-binding domain and other organelles to be visualized more easily (Fig. 3). Other might effectively compete with endogenous full-length MAP4. than their flatter morphology, however, cells overexpressing We hoped that, in highly expressing L-MTB lines, we might MAP4 did not appear altered in their morphology. L-MAP4, L- observe ‘dominant negative’ phenotypic abnormalities, because MTB, and L-MOCK cells all exhibited a range of morphologies the overexpressed MT-binding domain might lack regulatory typical of fibroblasts in culture (Figs 1 and 3). sequences or projection domain sites of interaction by which Since increased expression of MAP4 did not affect overall cell molecules or organelles would normally be tethered to the MTs. morphology, we next examined the distribution of a number of Thus, we assayed multiple L-MAP4 and L-MTB cell lines, different organelles within L-MAP4 and L-MTB cells induced examining both cell morphology and organelle distribution. to overexpress MAP4, comparing these to dex-treated L-MOCK A striking feature of all of our transfected cell lines, including or Ltk− cells. The distribution of MTs was indistinguishable from L-MAP4, L-MTB, and L-MOCK lines, was the altered, flat mor- that of control cells. No MT aggregates, bundles, or circumfer- phology that each adopted upon induction with dex. Since flat- ential whorls of MTs characteristic of cells transfected with other tening was characteristic of L-MOCK cells as well as L-MAP4 MAPs (e.g. Kanai et al., 1989; Lewis et al., 1989; Lee and Rook, and L-MTB cells, we concluded that this morphological change 1992; Ferralli et al., 1994) were noted. The distribution of actin, was not attributable to overexpressed MAP. This flattened mor- the Golgi apparatus, and mitochondria also appeared similar in phology caused the dex-induced cells to appear larger than cells transfected and control cells (data not shown).

Fig. 1. Mouse L cell lines inducibly expressing MAP4 constructs. Immunofluorescence of mouse L-cell transfectants in the presence (a-f) and absence (g-i) of dex, the synthetic hormone used to induce expression from the MMTV plasmid. Cells shown are clonal cell lines stably transfected with pMMTV with insert encoding full-length MAP4 (L-MAP4; a,d,g) or MT-binding domain (L-MTB; b,e,h), or with MMTV plasmid lacking insert (L-MOCK; c,f,i). Cells were stained with antibodies reactive with either primate MAP4 (a-c,g,h) or anti-tubulin (d-f, i). Bar, 10 µm. MAP4 stabilizes microtubules in vivo 287

MAP4 contributes to MT stability in vivo. The first approach we used was to determine whether MAP4-transfected cells possessed more stable MTs than control cells. As a measure of stable MTs, we analyzed the amount of detyrosinated (Glu) tubulin in L-MAP4, L-MTB, and L-MOCK cells. Glu tubulin is formed as the result of a post-translational event in which an enzyme, tubulin carboxypeptidase, removes the carboxyl terminal tyrosine residue of α-tubulin, and exposes the adjacent glutamate residue. Glu tubulin has been demonstrated to be enriched in stable MT populations (Gundersen et al., 1987; Khawaja et al., 1988). As displayed in the western blot in Fig. 4, both L-MAP4 and L-MTB cells had significantly higher levels of Glu tubulin when induced with dex than did the control L-MOCK cells. This result suggests one of two possibilities; either that cells overexpressing full-length MAP4 or MT-binding domain possess MTs that are more stable than those of the control cells, Fig. 2. Time-course of dex-induction of MAP4 synthesis. Extracts or that L-MAP4 and L-MTB cells contain a larger sub-popu- were prepared from log phase HeLa cells (left panel) or from L cell lation of stable MTs in their MT pools than do L-MOCK cells. stable transfectants expressing MAP4 (L-MAP4) at 0, 3, 6, 12, and 24 hours, and at 2, 4 and 6 days (D) after the addition of dex. A 40 The correlation between increased expression of Glu tubulin µg sample of total extract protein was electrophoresed and and high level expression of MAP4 and MT-binding domain in electroblotted in each lane. The blots were immunostained with the transfected cells (Fig. 4) suggest that it is the exogenous primate-specific anti-MAP4 antibodies, as described in Materials and MAP4 and MT-binding domain expressed in the cells that is Methods. The bars at left show the positions of electrophoretic responsible for the elevated levels of Glu tubulin observed. standards of molecular mass (kDa). MTs in cells overexpressing MAP4 are more resistant to depolymerization by nocodazole MAP4-overexpressing cells have elevated levels of We next examined the hypothesis that overexpressed MAP4 stable microtubules alters MT stability or polymerization. First, we measured the The transfected cell lines we prepared and characterized steady-state level of total tubulin, MT monomer, and polymer allowed us to address the question of whether overexpressed by extracting cells in MT-stabilizing buffer. As shown in Table

Fig. 3. Dex-induced MAP4 is localized on microtubules. L- MAP4 cells were induced with dex for 0 hour (a,d), 6 hours (b,e), and 24 hours (c,f), fixed, and double-stained with primate- specific anti-MAP4 (a,b,c) and anti-tubulin (d,e,f) antibodies. Note that the presence of human MAP4 can be detected as early as 6 hours after dex treatment, and expression is visibly increased following a 24 hour induction with dex. Notice that all MAP4 expressed from the transfected plasmid appeared to be colocalized with MTs. Bar, 10 µm. 288 H.-L. Nguyen and others

Fig. 4. MAP4-transfected cells possess elevated levels of detyrosinated (Glu) tubulin. Electropherograms of protein extracts prepared from L-MOCK (a,b), L- MTB (c,d), and L-MAP4 (e,f) cell lines, without (a,c,e) or with (b,d,f) treatment with dex for 24 hours. (A) Coomassie- stained polyacrylamide gel of protein extracts (a-f). (B) Electropherograms were blotted onto nitrocellulose and immunostained with (a-f) primate-specific anti- MAP4 antibodies; (a′-f′) antibody specific for tyrosinated α-tubulin; (a′′ f′′) antibody specific for detyrosinated (Glu) α-tubulin. Note that cells overexpressing MAP4 or its MT-binding domain contained significantly higher levels of Glu tubulin than control L-MOCK cells. Protein loadings were 40 µg in each lane of A, and lanes a-f of B; and 20 µg in lanes a′-f′ and a′′-f′′ of B. The bars at left show the positions of electrophoretic standards of molecular mass (kDa).

2, overexpression of full-length MAP4 or MTB had a dose- nocodazole depolymerization have been observed; however, dependent effect on the cellular content of tubulin; however, it these are usually bundled aggregates of several MTs that do did not affect the proportion of tubulin that was present as not resemble the individual MTs seen in non-transfected cells polymer in any of the cell lines. On average, 74.3±6.7% of (Kanai et al., 1989; Lee and Rook, 1992; Ferralli et al., 1994; cellular tubulin was present as polymer in all cell lines tested. Olsen et al., 1995). Therefore, the significant but lesser degree From these measurements we conclude that no significant of MT stabilization we observed in cells stably transfected with change in MT assembly had occurred in the presence of over- MAP4 constructs suggested that MAP4 increased the stability expressed MAP4 or MT-binding domain. of individual MTs, but did not contribute to reorganizing the For a quantitative assessment of the effects of MAP4 over- MTs into bundles. expresssion on stability of MTs, we used a standard nocoda- The nocodazole resistance assay we used to analyze the zole depolymerization assay similar to one we had used previ- MTs in MAP4-transfected cells proved to be both highly ously to assay stable and labile populations of MTs in another reproducible and amenable to quantification. From four indi- cell type (Khawaja et al., 1988). In these experiments, L- vidual experiments, which are graphically represented in MAP4, L-MTB, and L-MOCK cells were grown and induced Fig. 6A, we quantified MT stability in L-MAP4, L-MTB and with dex, and the MT-depolymerizing agent, nocodazole, was L-MOCK cells. The results demonstrate that MTs in L- added at a concentration (10 mM) that rapidly depolymerized MAP4 or L-MTB cells exhibited greater stability than MTs all labile cellular MTs, while working more slowly on stable in L-MOCK cells. MTs. Fig. 5 shows a striking difference in the abundance of MTs that resisted nocodazole depolymerization in each cell Full-length MAP4 is a more effective microtubule type. For example, following a 20 minute treatment with noco- stabilizing agent than MT-binding domain dazole, more MTs remained in L-MAP4 and L-MTB cells than The demonstration that MAP4 and its MT-binding domain in control cells (Fig. 5b,e,h). However, after nocodazole were both capable of in vivo stabilization of MTs (Figs 5 and treatment for longer periods, very few MTs remained in all the 6A) allowed us to ask the following question: Does the MAP4 cell lines, suggesting that MAP4 overexpression enhanced the MT-binding domain, which constitutes only about one-third of stability of cellular MTs to some degree, but did not give rise the entire MAP4 molecule, substitute effectively for the full- to extremely stable polymers or MT bundles that were refrac- length molecule in vivo? In vitro, the sequences encoded in the tory to nocodazole (Fig. 5c,f,i). In some cells transiently over- MTB construct constitute a region that, analogous to the MT- expressing other MAPs, MTs that are completely resistant to binding regions of other MAPs, has been shown to mimic the

Table 2. Steady-state level of microtubule polymer in stably transfected cell lines L-MAP4 L-MAP4 L- MTB L-MTB #13-2 #18-1 #3-6 #8-4 L-MOCK HeLa Tubulin, % of total protein 4.65±0.92 2.85±0.64 4.42±0.49 2.66±1.41 2.63±0.66 5.0* Tubulin in polymer, % 66.84±3.47 69.32±8.91 77.14±4.38 78.76±4.69 79.38±8.80 76.5

*Values for HeLa were reported by Bulinski and Borisy (1979). Quantification of tubulin and determination of fraction in monomer and polymer were performed as described in Materials and Methods. MAP4 stabilizes microtubules in vivo 289

Fig. 5. Overexpression of MAP4 stabilizes cellular microtubules. L-MAP4 (a-c), L-MTB (d-f), and L-MOCK (g-i) cells were treated with nocodazole (10 µM) for 0 minute (a,d,g), 20 minutes (b,e,h), and 60 minutes (c,f,i) before being extracted with detergent, fixed, and stained with anti-tubulin antibodies in order to visualize those MTs that resisted depolymerization by nocodazole. Notice that L- MAP4 and L-MTB cells have significantly more MTs remaining after the 20 minute treatment than L-MOCK cells; after the 60 minute treatment, virtually all of the MTs have been depolymerized in all cell lines. MTs in Ltk− cells showed nocodazole sensitivity that was indistinguishable from those in L-MOCK cells (data not shown). Bar, 10 µm. whole molecule in its capacity to interact with MTs in vitro shown in Fig. 6B, demonstrate that, relative to control cells, (Aizawa et al., 1991). Therefore, we applied the nocodazole- MAP4 was, mol/mol, a more potent stabilizer of MTs in vivo resistance assay of MT stability to assess the potency with than was the MAP4 MT-binding domain. This result is which full-length MAP4 and its MT-binding domain stabilized somewhat surprising, since the MTB cDNA encodes the only MTs in vivo. In Fig. 6A, a quantitative analysis of MT stability site of MT binding in the MAP4 molecule (Aizawa et al., 1991; in L cell transfectants, one can see that expression of MAP4 Chapin and Bulinski, 1991), and the region of bovine MAP4 yielded a significant enhancement of MT stability, and the MT that is most analogous to the human MT-binding domain stability in L-MTB cells was nearly as great as in L-MAP4 molecule expressed in L-MTB cells was previously shown to cells. However, because we analyzed cell lines with disparate contribute as effectively as full-length MAP4 in enhancing the levels of expression of each MAP4 species, the degree of MT rate and final level of MT polymerization in vitro (Aizawa et stabilization brought about by MAP4 and the MAP4 MT- al., 1991). binding domain might be expected to be a function of the level of each molecule expressed. To address this possibility, we Growth properties of L-cell transfectants quantified the extent of MT stabilization in clonal cell lines Even minor perturbations of MT dynamics have been with high and low levels of expression of the MAP4 constructs, reported to interfere with cell growth and progression through and quantified the MT stabilization observed as a function of cell division. For example, low concentrations of the MT-sta- the molar level of MAP4 or MT-binding domain. The results, bilizing drug, Taxol, which are sufficient to alter MT 290 H.-L. Nguyen and others

Table 3. Cell growth properties of stably transfected cell lines L-MAP4 L-MAP4 L-MTB L-MTB #13-2 #18-1 #3-6 #8-4 L-MOCK Ltk− Doubling time, hours 43.2 55.7 45.6 41.8 31.7 28.0 (r2=0.934) (r2=0.974) (r2=0.989) (r2=0.972) (r2=0.980) r2=0.997) Mitotic index, % 3.7±1.1 4.7±0.25 4.76±0.74 3.55±1.03 3.3±0.89 3.9±0.72 Lethal [nocodazole], nM* 75 nM N.D. 60 nM N.D. 40 nM 35 nM Lethal [Taxol], nM* 300 nM N.D. 300 nM N.D. 300 nM 300 nM

Cell growth properties were assayed as described in Materials and Methods. At least two cell growth experiments were performed for each cell line. *>98% of cells killed by a 4 day treatment (see Materials and Methods). N.D., not determined.

dynamics (A. Mikhailov and G. G. Gundersen, unpublished might arise from a delay in progression through the G1 phase of work) but are insufficient to affect the cellular amount of MT the cell cycle. In addition, in the rat, the MAP4 form we trans- polymer, have been reported to alter mitotic progression fected into Ltk− cells (form III), is only expressed in differenti- and/or cell growth (Jordan et al., 1992, 1993). Accordingly, ated cells that are resting in the G0 phase of the cell cycle we tested the hypothesis that stabilizing MTs with overex- (Chapin et al., 1995). It is possible that the presence of excess pressed MAP4 would inhibit cell growth or mitosis. We MAP4 form III or its MT-binding domain would be sufficient to compared the growth rates of L-MAP4 and L-MTB cell lines prolong G1 transit or promote arrest of cells in G0. Therefore, tk− to those of L-MOCK and L control cells. These data, we tested for abnormalities in the G1 phase of the cell cycle by presented in Table 3, clearly demonstrate that overexpression measuring the kinetics of entry into S-phase in the transfected of either MAP4 or its MT-binding domain lengthens cell cell lines. L-MAP4 cells incorporated bromodeoxyuridine doubling time. Since MAP4 and the MT-binding domain (BrdU) into their genomic DNA at a rate that was markedly could slow the growth rate of cells by killing them or similar to that of L-MOCK cells; in contrast, the L-MTB cells arresting them in mitosis, we measured cell viability and entered S-phase at a much slower rate (Fig. 7). The vast majority mitotic index. The results of these experiments (Table 3) of the L-MAP4 and L-MOCK cells incorporated the BrdU label show that overexpressed MAP4 and MT-binding domain during a 72 hour incubation, indicating that they had entered at exert effects on cell growth, without being deleterious to the least an initial round of DNA replication. L-MTB cells, however, cells’ viability, or arresting cells in mitosis. displayed a much slower rate of BrdU incorporation (P<0.05); Variation in cell cycle time of MAP4-overexpressing cells this effect was observed in lines expressing either high levels (L-

120 A 100

100 B

L-MAP4 80 80

60 60 L-MTB L-MAP4

40 40

% cells with >10 MTs L-MOCK 20 20 min nocodazole treatment % of cells with >10 MTs after 20 L-MTB 0

0 0 10203040506070 0 50 100 150 200 250 300 350 400 Time of nocodazole treatment (min) MAP4 fraction of total cellular protein (pmol/mg)

Fig. 6. Quantitative measurement of MT stability in MAP4-expressing cells. (A) L-MAP4, L-MTB, and L-MOCK cells were induced with dex for 24 hours, treated with nocodazole (10 mM) for 0, 10, 20, and 60 minutes, and then scored for the presence of more than 10 MTs per cell, in order to assess quantitatively the resistance of MTs to drug depolymerization in each cell type. For each time point, at least 200 cells from each cell line were scored. Results from four experiments on a single cell line of each type (L-MAP4-#13-2 and L-MTB-#3-6, respectively) are shown here. (B) Two different clones of L-MAP4 (#13-2 and #18-1) and L-MTB cells (#3-6 and #8-1), each expressing high and low levels of their respective MAP4 products (see Table 1), were induced with dex for 24 hours and treated with 10 mM nocodazole for 20 minutes. The proportion of cells in which more than 10 MTs remained was then plotted as a function of the level of MAP4 expressed. Each point on the graph represents one clone of L-MAP4 or L-MTB; each data point is the result of at least two separate experiments. For each experiment, at least 200 cells of each clonal cell line were scored. Results here demonstrate that, on a molar basis, full-length MAP4 is a more potent stabilizer of MTs than the MT-binding domain alone. MAP4 stabilizes microtubules in vivo 291

100 1991), by dampening the parameters of dynamic instability (Itoh and Hotani, 1994). Molecular analysis of MAP4 structure 80 established that the C-terminal one-third of the MAP4 molecule contains a basic, proline-rich segment that lies just

60 N-terminal to a set of three to five imperfectly repeated elements; this is followed, in turn, by an ~100 amino acid C- terminal segment possessing a nearly neutral pI (Aizawa et al., 40 1990; West et al., 1991; Chapin and Bulinski, 1991; Chapin et al., 1995). Examination of proteolytically generated fragments 20

% BrdU-labeled cells of MAP4, as well as recombinant MAP4 fragments purified L-MAP4 from Escherichia coli, showed that the C-terminal one-third of 0 L-MTB L-MOCK MAP4 comprises the MAP’s only MT-binding domain (Aizawa et al., 1991; Chapin and Bulinski, 1991). In fact, in a

0 122436486072 thorough dissection of the MAP4 molecule, the C-terminal MT-binding domain, or even the proline-rich segment by itself, Time of BrdU treatment (hr) was shown to be as potent as the entire MAP4 molecule, Fig. 7. Expression of MT-binding domain delays entry into S-phase. mol/mol, in promoting in vitro MT assembly (Aizawa et al., L-MAP4 (#13-2), L-MTB (#3-6), and L-MOCK cells were induced 1991). We and others embarked on the study of MAP4’s in vivo for 24 hours with dex, and were treated with bromodeoxyuridine function in order to elucidate the in vivo correlates of MAP4’s (BrdU) for 0-72 hours. Cells were scored to determine the proportion in vitro behavior, and to analyze the function(s) of each portion of cells that displayed anti-BrdU staining (see Materials and Methods, for details). Each data point is the average of 3 independent of the MAP4 molecule within the living cell. experiments ± s.e.m. Student’s t-test was performed, pairwise, Initial studies of MAP4 function in vivo proved to be incon- comparing L-MAP4, L-MTB, and L-MOCK. Significant differences clusive, however. Three studies employing MAP4 overexpres- were observed (P<.05) in comparisons of L-MTB with L-MAP4 and sion and one using MAP4 depletion techniques reported results L-MTB with L-MOCK, respectively that appeared to be difficult to reconcile with one another. For example, both Barlow et al. (1994) and Olsen et al. (1995) transfected cultured cells to bring about MAP4 overexpression. MTB#3-6; Fig. 7) and, to a lesser extent, in lines expressing low Surprisingly, Barlow et al. reported that MTs in Chinese levels (L-MTB#8-4; data not shown) of MT-binding domain. hamster ovary cells were unchanged in either stability or level These results suggest that the delay in entry into S-phase of polymer in the presence of a significantly increased MAP4 observed in L-MTB cells is dose-dependent and is independent level, while Olsen et al. reported that MTs were increased in of either the degree of MT stability or the degree of growth inhi- their stability in Chinese hamster ovary and BHK cells trans- bition conferred by the MT-binding domain. fected under similar conditions. Olsen et al. (1995) also trans- Because greater MT stability in the presence of MAP4 over- fected cultured cells with various chimeric proteins containing expression affected cell growth parameters, we tested the hypoth- an epitope tag and a green fluorescent protein moiety attached esis that greater MT stability might affect the responses of cells to full-length MAP4 or various fragments. Analysis of these to drugs that interact with MTs. For example, increased MT stably or transiently transfected cells established that stability due to MAP4 overexpression might have antagonistic expression of most or all of the MT-binding domain of MAP4 effects on the cells’ response to the MT-destabilizing drug, noco- brought about an increase in MT stability, as assayed qualita- dazole, or synergistic effects on the cells’ response to the MT-sta- tively by resistance to nocodazole depolymerization. Yoshida bilizing drug, Taxol. We assayed the threshold concentrations of et al. (1996) increased total MAP4 level in PtK2 cells by the drugs, nocodazole and Taxol, that killed >98% of the cells of microinjecting a recombinant MAP4 MT-binding domain; each transfectant cell line, as shown in Table 3. Overexpression these cells also exhibited stabilized MTs, as well as some of MAP4 had a modest effect on the cells’ tolerance of the MT changes in their actin cytoskeleton. In contrast to the study of antagonistic drug, nocodazole, while overexpression of the MT- Barlow et al. (1994), the studies of Olsen et al. (1995) and binding domain had a less significant effect. In contrast, we could Yoshida et al. (1996) suggested that MAP4 does, indeed, not detect any change in the cells’ tolerance to Taxol. Nonethe- possess the capacity to stabilize MTs in vivo. The latter two less, to our knowledge, this is the first instance in which altered studies also provided in vivo evidence that a fragment of MAP4 expression of a protein known to stabilize MTs has been found could substitute for full-length MAP4 in stabilizing MTs. It is to affect a long-term response to nocodazole. The ‘protection’ possible that the apparently disparate results obtained in these against nocodazole afforded by increased expression of MAP4 studies stemmed from differences in experimental methods. may have pharmacological relevance: altered expression of Wang et al. (1996) approached the study of MAP4 function MAP4 or other MAPs might constitute a molecular mechanism from the opposite direction, attempting to deplete functional to explain the variation in the response of different cell types or MAP4 from proliferating cells by an antibody microinjection tumors to chemotherapeutic MT-depolymerizing drugs. strategy. In these cells, which were thought to contain less than 20% of the usual complement of MAP4, no phenotype was observed, no striking changes in nocodazole stability were DISCUSSION seen, and no changes in MT turnover were measurable. This study raised at least three possibilities. The first possibility is Previous studies have demonstrated that MAP4 promotes MT that MAP4 actually does not contribute significantly to MT assembly and stabilizes MTs in vitro (Aizawa et al., 1987, assembly or stabilization in vivo. Second, MAP4 does con- 292 H.-L. Nguyen and others tribute to MT stability, but other MAPs can provide MT stabi- and to alter their dynamic parameters (A. Mikhailov and G. G. lization normally conferred by MAP4, allowing the cell to Gundersen, unpublished work) have been shown to exert compensate for diminished amounts of MAP4. The presence profound effects on MT-dependent functions such as mitosis in the cells of other MAPs with potentially overlapping or and cell motility (Jordan et al., 1993; Liao et al., 1995). Indeed, redundant functions is a drawback inherent in the MAP4 of the sequelae of MAP4 overexpression that we observed, depletion approach taken by Wang et al. (1996); in MAP4 over- perhaps the most exciting was that the cells acquired a expression studies, compensation by other molecules is less tolerance to low levels of nocodazole in their growth medium. likely. A third possible explanation for the results of Wang et This result suggested that the modest perturbation in MT al. is that there was a phenotype that resulted from MAP4- stability brought about by altered MAP4 expression was suffi- depletion, but under their experimental system and with the cient to change the efficacy of at least one MT-depolymerizing assays utilized, the phenotype was not detectable. The drug. somewhat ambiguous results obtained previously in these two A striking finding from our studies is that the MT-binding systems suggested the need to perform further studies. domain of human MAP4, which includes the entire proline- Accordingly, we carried out experiments on Ltk− cells that rich region, the imperfectly repeated elements, and the C we induced to overexpress human MAP4. Our experimental terminus, was not as effective in stabilizing MTs, mol/mol, as system, in which we compared clonal cell lines with high or the full-length MAP4 molecule (Fig. 6B). Our results are not low levels of MAP4 expression to control cells, allowed us to inconsistent with recent studies in which introduction of determine exactly the quantity of MAP4 present and the several versions of the MAP4 MT-binding domain, alone, sta- degree of MT stabilization conferred on MTs by a particular bilized MTs against nocodazole treatment (Olsen et al., 1995; amount of MAP4. Our finding, that MAP4 stabilized MTs Yoshida et al., 1996), since in the latter studies no measure of against depolymerization by nocodazole, demonstrates that, in the effective stoichiometry of the MT-binding domain activity the cellular milieu containing other MT-associated proteins was available. However, our data do appear to be inconsistent and MT modulators, MAP4 carries out at least one activity with in vitro data showing that the most analogous portion of previously attributed to it from similarly quantitative in vitro the bovine MAP4 MT-binding domain substituted fully for studies performed with purified molecules. Our experiments full-length MAP4 in promoting MT assembly in vitro (Aizawa differed from those performed recently in several other labo- et al., 1991). Differences abound between the in vitro system ratories, since we expressed a different isoform of MAP4, the of Aizawa et al. (1991), in which recombinant, purified (i.e. tau-like four-repeat form characteristic of differentiated cells heat-treated) MAP4 was assayed with pure tubulin, and our in (isoform III; Chapin et al., 1995), and found that four-repeat vivo system, in which MAP4 or its MT-binding domain was MAP4 was capable of in vivo MT stabilization. Preliminary synthesized and utilized in a more nearly physiological envi- results suggest that in our L cell overexpression system other ronment known to contain other MT-binding proteins and MAP4 forms, such as the five-repeat form used in the exper- regulatory molecules such as protein kinases. Taking these dif- iments of Barlow et al. (1994) and Olsen et al. (1995) also ferences into account, we would attribute the observed dis- stabilize MTs, at least to some degree (H.-L. Nguyen and J. crepancies in results to in vitro purification artifacts, in vivo C. Bulinski, data not shown). We have not yet determined the regulation by phosphorylation, or in vivo modulation of MT- efficacy with which different MAP4 forms that possess five, binding domain activity by sequences within MAP4’s N- four, or even three repeats in their MT-binding domains terminal projection domain. stabilize MTs in vivo. The underlying goal of this study was to elucidate all in vivo Although we found a measurable effect of MAP4 overex- functions of MAP4, whether or not these functions were related pression on MT stability, we also found, in agreement with to the MAP’s capacity to stabilize MTs. We did not observe any Barlow et al. (1994), that MAP4 overexpression did not change obvious morphological phenotype in the cells overexpressing the steady-state level of MT polymer (see Table 2). This result MAP4. We decided to analyze cell growth in the hope of suggests that MAP4 stabilizes MTs to an extent similar to very detecting a phenotype, for two reasons: First, earlier studies low concentrations of the drug Taxol (Jordan et al., 1993). (Crossin and Carney, 1981; Wang and Rozengurt, 1983; Jordan Although this degree of MT stabilization may appear at first et al., 1993; Hwang and Ding, 1995) pointed to an interplay glance to be less impressive than the extensive stabilization that between MT stability and cell growth or metabolic activation. was previously found to give rise to extensive bundled arrays Second, we reasoned that slight defects in cellular functions, of MTs (e.g. Kanai et al., 1989; Lewis et al., 1989; Jordan et which might not produce an observable phenotype, might be al., 1993), we would argue that this lower level of MT stabi- compounded into a detectable inhibition of cell growth. Our lization, and the dampened MT dynamics it generally corre- findings, that overexpression of MAP4 or its MT-binding sponds to, are actually of great physiological significance. In domain lengthened the recipient cells’ doubling time, and that cells overexpressing MAP4, the degrees of MT stability we slower cell growth was not readily attributable to mitotic abnor- observed from measurement of either nocodazole stability or malities or to cell death (Table 3), raise the possibility that in increased post-translational detyrosination of α-tubulin were normal physiological settings MAP4 itself, or MTs stabilized comparable to levels of stability characteristic of differentiat- by MAP4, may play a role in the regulation of cell growth. The ing cells, either in myogenesis (Gundersen et al., 1989) or neu- suggestion that MAP4 influences the cell cycle is compatible ritogenesis (Wehland and Weber; 1987; Baas and Black, 1990). with a previous demonstration that antisense inhibition of Furthermore, slight alterations in MT stability have been inves- MAP2 expression in stably transfected embryonal carcinoma tigated in studies of the MT-stabilizing drug, Taxol. Levels of cells inhibited neuronal differentiation (Dinsmore and Taxol that are insufficient to alter MT polymer level (Jordan et Solomon, 1991), and that anti-sense inhibition of a muscle- al., 1993; Liao et al., 1995), but are sufficient to stabilize MTs specific MAP4 RNA species in C2C12 myogenic cells led to a MAP4 stabilizes microtubules in vivo 293 perturbation in myogenic differentiation (Mangan and Olmsted, lines we have generated, which inducibly overexpress different 1996). In each of these studies, the diminution in assembly- levels and domains of MAP4, will undoubtedly allow us to promoting MAP may have resulted in failure of the cells to address further questions about MAP4’s functional interactions undergo normal, differentiation-induced withdrawal from the and dynamics in vivo. cell cycle. Further investigation will be required in order to determine if MAPs play a role at the interface between the cell The authors thank Drs Gregg Gundersen, Ron Liem, Kathy Faire, cycle and morphological differentiation. Gary Borisy, and Eckhard and Eva Mandelkow for stimulating scien- The precise mechanism by which MAP4 or its MT-binding tific discussions concerning the results presented in this paper. This domain affects the cell cycle is not known. The fact that Taxol, research was supported by American Cancer Society Grants (#NP- 192C and CB#168D) to J.C.B.; H.L.N. was an NIH predoctoral another MT stabilizing agent that has no structural similarity trainee (#T32 AG00189). S. Chari was supported by CTR Grant to MAP4, also depresses cellular growth kinetics, suggests #3747 during a portion of this work. that MT stabilization may directly slow cell growth. MTs undergo breakdown and changes in their dynamic behavior during the transition from the G2 portion of interphase to the REFERENCES mitotic (M) phase of the cell cycle (Saxton et al., 1984; Verde et al., 1990; Belmont et al., 1990). Overexpressed MAP4, Aizawa, H., Murofushi, H., Kotani, S., Hisanaga, S.-I., Hirokawa, N. and analogous to Taxol, may stabilize MTs sufficiently that the Sakai, H. (1987). Limited chymotryptic digestion of bovine adrenal cell’s normal disassembly of interphase MT’s is hampered and 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly. J. Biol. Chem. 262, 3782- entry into M is delayed. The G2-M transition in MT dynamics 3787. is accompanied by phosphorylation of MAP4 or other MAPs, Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai, H., and Suzuki, which limits their capacity to stabilize MTs (Vandré et al., K. (1990). Molecular cloning of a ubiquitously distributed microtubule- 1991; Tombes et al., 1991; Shiina et al., 1992; Andersen et al., associated protein with Mr 190,000. J. Biol. Chem. 265, 13849-13855. 1994; Ookata et al., 1994; Masson and Kreis, 1995). A Aizawa, H., Emori, Y., Mori, A., Murofushi, H., Sakai, H. and Suzuki, K. (1991). Functional analysis of the domain structure of microtubule- possible consequence of overexpressing MAP4 is that, if the associated protein 4 (MAPU). J. Biol. Chem. 266, 9841-9846. enzymes that control the phosphorylation states of MAP4 are Andersen, S. S., Bendia, B., Dominguez, J. E., Sawyer, A. and Karsenti, E. present in a limited quantity, they may be unable to regulate (1994). Effect on microtubule dynamics of XMAP230, a microtubule- greater amounts of MAP4 in a timely manner. Thus, we would associated protein present in Xenopus laevis eggs and dividing cells [published erratum appears in J. Cell Biol. 128 (1995) following p. 988]. J. predict that one facet of the cell cycle that would be retarded Cell Biol. 127, 1289-1299. in L-MAP4 and L-MTB cells would be the G2-M transition. Baas, P. W. and Black, M. M. (1990). Individual microtubules in the axon Further experiments will be needed to pinpoint the regulation consist of domains that differ in both composition and stability. J. Cell Biol. of overexpressed MAP4 or MT-binding domain at the G2-M 111, 495-509. transition. Barlow, S., Gonzalez-Garay, M. L., West, R. R., Olmsted, J. B. and Cabral, F. (1994). Stable expression of heterologous microtubule-associated proteins Our efforts to examine the behavior of L-MAP4 and L- (MAPs) in Chinese hamster ovary cells: Evidence for differing roles of MTB cells at another cell cycle checkpoint, the G1-S tran- MAPs in MT organization. J. Cell Biol. 126, 1017-1030. sition, yielded a provocative result: although L-MAP4 cells Belmont, L. D., Hyman, A. A., Sawin, K. E. and Mitchison, T. J. (1990). exhibited a slight delay in their entry into S-phase, as deter- Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62, 579-589. mined by monitoring incorporation of BrdU, L-MTB cells Bramblett, G. T., Goedert, M., Jakes, R., Merrick, S. E., Trojanowski, J. Q. exhibited a more significant delay (Fig. 7). This result and Lee, V. M.-Y. (1993). Abnormal tau phosphorylation at Ser396 in suggested that transit through the G1-S checkpoint was Alzheimer’s disease recapitulates development and contributes to reduced inhibited by the overexpressed MAP4 MT-binding domain. microtubule binding. Neuron 10, 1089-1099. This inhibition was not correlated with MT stability, since L- Bulinski, J. C. and Borisy, G. G. (1980a). Microtubule-associated proteins from cultured HeLa cells: Analysis of molecular properties and effects on MTB lines with either high or low (data not shown) levels of microtubule polymerization. J. Biol. Chem. 255, 11570-11576. expression were delayed in their entry into S-phase more sig- Bulinski, J. C. and Borisy, G. G. (1980b). Widespread distribution of a nificantly than L-MAP4 lines, while all L-MTB cell lines 210,000 mol wt microtubule-associated protein in cells and tissues of displayed MT stability that was, at most, equivalent to L- primates. J. Cell Biol. 87, 802-808. Bulinski, J. C. and Bossler, A. (1994). Purification and characterization of MAP4 lines (Fig. 6). The MT-binding domain molecule is not ensconsin, a novel microtubule stabilizing protein. J. Cell Sci. 107, 2839- toxic to cells, nor does it slow overall cell growth as signifi- 2849. cantly as the full-length MAP4 molecule (Table 3). Therefore, Caceres, A. and Kosik, K. S. (1990). Inhibition of neurite polarity by antisense this result raises the intriguing possibility that the MAP4 pro- oligonucleotides in primary cerebellar neurons. Nature 343, 461-463. jection domain, which is missing from the MT-binding Chapin, S. and Bulinski, J. C. (1991). Non-neuronal 210 kDa microtubule- associated protein (MAP4) contains a domain homologous to the domain expressed in L-MTB cells, has activities or binding microtubule-binding domains of MAP2 and tau. J. Cell Sci. 98, 27-36. partners that are necessary for the MAP’s normal function. It Chapin, S. and Bulinski, J. C. (1992). Microtubule stabilization by assembly is possible that the presence of MAP4 MT-binding domain promoting microtubule-associated proteins: A repeat performance. Cell alone competes with the full-length molecule and results in Motil. Cytoskel. 4, 236-243. Chapin, S. and Bulinski, J. C. (1994). Cellular microtubules heterogeneous in ‘dominant negative’ effects on the MT cytoskeleton. A search their content of MAP4 (210 Kd MAP). Cell Motil. Cytoskel. 27, 133-149. for functional interactions of the MAP4 projection domain is Chapin, S., Lue, C.-M., Yu, M. T. and Bulinski, J. C. (1995). Differential currently underway. expression of alternatively spliced forms of MAP4: A repertoire of The results of our current studies yield exciting information structurally different microtubule-binding domains. Biochemistry 34, 2289- about MAP4’ s function in vivo, and provide support for the 2301. Cleveland, D. W., Hwo, S.-Y. and Kirschner, M. W. (1977). Physical and hypothesis that each of the MAP’s domains plays an important chemical properties of purified tau factor and the role of tau in microtubule tk− role in regulating MT stability and cell growth. The L cell assembly. J. Mol. Biol. 116, 227-247. 294 H.-L. Nguyen and others

Crossin K. L. and Carney, D. H. (1981). Microtubule stabilization by Taxol Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N. and inhibits initiation of DNA synthesis by thrombin and by epidermal growth Sakai, H. (1986). Purification and characterization of a 190-kD microtubule- factor. Cell 27, 341-350. associated protein from bovine adrenal cortex. J. Cell Biol. 103, 1911-1919. Dinsmore, J. H. and Solomon F. (1991). Inhibition of MAP2 expression Murphy, D. B. and Borisy, G. G. (1975). Association of high molecular affects both morphological and cell division phenotypes of neuronal weight proteins with microtubules and their role in microtubule assembly in differentiation. Cell 64, 817-826. vitro. Proc. Nat. Acad. Sci. USA 72, 2696-2700. Dolbeare, F. and Selden, J. R. (1994). Immunochemical quantitation of Nakata, T. and Hirokawa, N. (1995). Point mutation of adenosine bromodeoxyuridine: Application to cell-cycle kinetics. Meth. Cell Biol. 41, triphosphate-binding motif generated rigor kinesin that selectively blocks 297-316. anterograde lysosome membrane transport. J. Cell Biol. 131, 1039-1053. Doll, T., Meichsner, M., Riederer, B. M., Honegger, P. and Matus, A. (1993). Olsen, K. R., McIntosh, J. R. and Olmsted, J. B. (1995). Analysis of MAP4 An isoform of microtubule-associated protein 2 (MAP2) containing four function in living cells using green fluorescent protein (GFP) chimeras. J. repeats of the tubulin-binding motif. J. Cell Sci. 106, 633-640. Cell Biol. 130, 639-650. Ferralli, J., Doll, T. and Matus, A. (1994). Sequence analysis of MAP2 Ookata, K., Hisanaga, S., Bulinski, J. C., Murofushi, H., Aizawa, H., Itoh, function in living cells. J. Cell Sci. 107, 3115-3125. T., Hotani, H., Okumura, E., Tachibana, K. and Kishimoto, T. (1994). Goedert, M. and Jakes, R. (1990). Expression of separate isoforms of tau Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets protein: correlation with the tau pattern in brain and effects on tubulin p34cdc2 kinase to microtubules and is a potential regulator of M-phase polymerization. EMBO J. 9, 4225-4230. microtubule dynamics. J. Cell Biol. 128, 849-862. Graham, F. L. and van der Eb, A. J. (1973). A new technique for the assay of Otey, C. A., Kalnoski, M. H., Lessard, J. L. and Bulinski, J. C. (1986). infectivity of human adenovirus DNA. Virology 52, 456-461. Immunolocalization of the gamma isoform of nonmuscle actin in cultured Gundersen, G. G., Khawaja, S. and Bulinski, J. C. (1987). Post- cells. J. Cell Biol. 102, 1726-1737. polymerization detyrosination of alpha tubulin: a mechanism for subcellular Parysek, L. M., Asnes, C. F. and Olmsted, J. B. (1984). MAP4: Occurrence in differentiation of microtubules. J. Cell Biol. 105, 251-264. mouse tissues. J. Cell Biol. 99, 1309-1315. Gundersen, G. G., Khawaja, S. and Bulinski, J. C. (1989). Generation of a Pereira, A., Doshen, E., Tanaka, E. and Goldstein, L. S. B. (1992). Genetic stable, posttranslationally modified microtubule array is an early event in analysis of a Drosophila microtubule-associated protein. J. Cell Biol. 116, myogenic differentiation. J. Cell Biol. 109, 2275-2288. 377-383. Hall, C. V., Jacob, P. E., Ringold, G. M. and Lee, F. (1983). Expression and Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A regulation of Escherichia coli LacZ gene fusions in mammalian cells. J. Mol. Laboratory Manual. 2nd edn. New York: Cold Spring Harbor Laboratory Appl. Gen. 2, 101-109. Press. Hwang, S. and Ding, A. (1995) Activation of NF-kappa B in murine Saxton, W. M., Stemple, D. L., Leslie, R. J., Salmon, E. D., Zavortink, M. macrophages by Taxol. Cancer Biochem. Biophys. 14, 465-472. and McIntosh, J. R. (1984). Tubulin dynamics in cultured mammalian cells. Itoh, T. J. and Hotani, H. (1994). Microtubule-stabilizing activity of J. Cell Biol. 99, 2175-2186. microtubule-associated proteins (MAPs) is due to increase in frequency of Shiina, N., Moriguichi, T., Ohta, K., Gotoh, Y. and Nishida E. (1992). rescue in dynamic instability: shortening length decreases with binding of Regulation of a major microtubule-associated protein by MPF and MAP MAPs onto microtubules. Cell Struct. Funct. 19, 279-290. kinase. EMBO J. 11, 3977-3984. Jordan, M. A., Thrower, D. and Wilson, L. (1992). Effects of vinblastine, Studier, F. W., Rosenberg, A. W., Dunn, J. J. and Dubendorff, J. W. (1990). podophyllotoxin, and nocodazole on mitotic spindles: Implications for the Use of T7 RNA polymerase to direct expression of cloned genes. Meth. role of microtubule dynamics in mitosis. J. Cell Sci. 102, 401-416. Enzymol. 185, 60-89. Jordan, M. A., Toso, R. J., Thrower, D. and Wilson, L. (1993). Mechanism of Tombes, R. M., Peloquin, J. G. and Borisy, G. G. (1991). Specific association mitotic block and inhibition of cell proliferation by Taxol at low of an M-phase kinase with isolated mitotic spindles and identification of two concentrations. Proc. Nat. Acad. Sci. USA 90, 9552-9556. of its substrates as MAP4 and MAP1B. Cell Regul. 2, 861-874. Kanai, Y., Takemura, R., Oshima, T., Mori, H., Ihara, Y., Yanangisawa, M., Vandré, D. D., Centonze, V. E., Peloquin, J., Tombes, R. M. and Borisy, G. Masaki, T. and Hirokawa, N. (1989). Expression of multiple isoforms and G. (1991). Proteins of the mammalian mitotic spindle: microtubule bundle formation in fibroblasts transfected with a single tau phosphorylation/dephosphorylation of MAP4 during mitosis. J. Cell Sci. 98, cDNA. J. Cell Biol. 109, 1173-1184. 577-588. Khawaja, S., Gundersen, G. G. and Bulinski, J. C. (1988). Enhanced Verde, F., Labbé, J. C., Dorée, M. and Karsenti, E. (1990). Regulation of stability of microtubules enriched in detyrosinated tubulin is not a direct microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus function of detyrosination level. J. Cell Biol. 106, 141-149. eggs. Nature 343, 233-238. Liao, G., Nagasaki, T. and Gundersen, G. G. (1995). Low concentrations of Wang, Z.-W. and Rozengurt, E. (1983). Interplay of cyclic AMP and nocodazole interfere with fibroblast locomotion without significantly microtubules in modulating the initiation of DNA synthesis in 3T3 cells. J. affecting microtubule level: Implications for the role of dynamic Cell Biol. 96, 1743-1750. microtubules in cell locomotion. J. Cell Sci. 108, 3473-3483. Wang, X. M., Peloquin, J. G., Zhai, Y., Bulinski, J. C. and Borisy, G. G. Lee, G. and Rook, S. L. (1992). Expression of tau protein in non-neuronal (1996). Removal of MAP4 from microtubules in vivo produces no cells: microtubule binding and stabilization. J. Cell Sci. 102, 227-237. discernible phenotype at the cellular level. J. Cell Biol. 132, 349-358. Lewis, S. A., Ivanov, I. F., Lee, G. H. and Cowan, N. J. (1989). Organization Wehland, J. and Weber, K. (1987). Turnover of the carboxy-terminal tyrosine of microtubules in dendrites and axons is determined by a short hydrophobic of alpha-tubulin and means of reaching elevated levels of detyrosination in zipper in microtubule-associated proteins MAP2 and tau [correction appears living cells. J. Cell Sci. 88, 185-203. in Nature (1990) 345, 674]. Nature 342, 498-505. West, R. R., Tenbarge, K. M. and Olmsted, J. B. (1991). A model for Mangan, M. E. and Olmsted, J. B. (1996). A muscle-specific variant of microtubule-associated protein 4 structure. J. Biol. Chem. 266, 21886-21896. microtubule-associated protein 4 (MAP4) is required in myogenesis. Yoshida, T., Imanaka-Yoshida, K., Murofushi, H., Tanaka, J., Ito, H. and Development 122, 771-781. Inagaki, M. (1996). Microinjection of intact MAP-4 and fragments induces Masson, D. and Kreis, T. E. (1995). Binding of E-MAP-115 to microtubules is changes of the cytoskeleton in PtK2 cells. Cell Motil. Cytoskel. 33, 252-262. regulated by cell cycle-dependent phosphorylation. J. Cell Biol. 131, 1015- Zhai, Y. and Borisy G. G. (1994). Quantitative determination of the proportion 1024. of microtubule polymer present during the mitosis-interphase transition. J. Meuth, M. and Greene, H. (1974). Induction of deoxydytidineless state in Cell Sci. 107, 881-890. cultured mammalian cells by bromodeoxyuridine. Cell 2, 109-112. Mitchison, T. J. and Kirschner, M. W. (1984). Dynamic instability in microtubule growth. Nature 312, 237-242. (Received 23 July 1996 Ð Accepted 1 November 1996)