Summary This review discusses the possible role of a-tubulin detyrosination, a reversible post-translational modification that occurs at the protein's C-terminus, in cellular morphogenesis. Higher eukaryotic cells possess a cyclic post-translationalmechanism by which dynamic microtubules are differentiated from their more stable counterparts; a tubulin-specificcarboxypeptidase detyrosinates tubulin protomers within microtubules, while the reverse reaction, tyrosination, is performed on the soluble protomer by a second tubulin-specific enzyme, tubulin tyrosine ligase. In general, the turnover of microtubules in undifferentiated, proliferating cells is so rapid that the microtubules accumulate very little detyrosinated tubulin; that is, they are enriched in tyrosinated tubulin. However, an early event common to at least three well-studied morphogenetic events myogenesis, neuritogenesis, and directed cell motility is the elaboration of a polarized array of stable microtubules that become enriched in detyrosinated tubulin. The formation of this specialized array of microtubules in specific locations in cells undergoing morphogenesissuggests that it plays an important role in generating cellular asymmetries.

Introd uction During the past two decades, the results of pharmacological and genetic experiments have demonstrated a vital role for microtubules in a variety of morphogenetic events. In the same pcriod. biochemical cxperiments havc provided a detailed knowledge of the in vitro properties of the microtubule subunit protein, tubulin, and its interactions with other proteins (reviewed by McKeithan and Rosenbaum(l)), and cell biological approaches havc furnished information on the d namics and behavior of microtubules in living cells(2-JI The existing knowledge of microtubules has made it possible to formulatc and bcgin to test specific hypotheses of how microtubules function during the dramatic alterations in cell shape that accompany differentiation and motility. In this review, we examine recent evidence suggesting that the dynamic properties of microtubules are coupled to post-translational

modifications of the substituent tubulin subunits, and we suggest that this biochemical differentiation of microtubules may be important in control of cell shape. Microtubules are self-assembled in the cytoplasm from heterodimcric tubulin protomers. The in vivo assembly process is best understood in cultured fibroblastic cells, in which an extensive array of microtubules fills the cytoplasm. Although the tubulin protomers in these cells are long-lived proteins (tl >1 day), the microtubules formed from them are remarkably dynamic (tl l2 -5 niin(')). Moreover, in fibroblasts, one end of each microtubule is attached to the centrosomc, a microtubule-organizing center adjacent to the nucleus(7), and the other (growing) end of each is oriented toward the cell periphery. While the location of the centrosome specifies the location of one end of each microtubulc, the mechanisms that specify the location of the other (dynamic) end of each microtubule and consequently, the shape of the three-dimensional array of microtubules, are unknown at present. In other cell types. e.g., epithelial cells, many of the microtubules are not nucleated at the centrosome('); in this case, the problem of how the microtubule array is organizcd is a more complcx one, sincc thc location of both ends of each microtubule must be determined. Although understanding how the microtubule array is specified is of obvious importance, it is also vital to understand the function or functions carried out by microtubules once they have adopted their particular configurations. It is reasonable to expect that biochemical alterations in microtubules would modulate their function during cellular morphogenesis. For example, functionally distinct tubulin forms could bc generated by the expression of distinct tubulin genes(9). or by posttranslational modification(''). Similarly, changes in the cellular complement of microtubule-associated proteins(") could serve to modify the composition and function of microtubules. Of these liochemical alterations, only post-translational modification is a rapid and reversible mechanism to generate covalently different tubulin molecules; thus, the presence of posttranslationally modified tubulin subunits suggests a logical mechanism by which to alter microtubule function during the early stages of differentiative events, particularly prior to the elaboration of a stable 'endpoint' morphology. We discuss in this rcview several morphogenetic events involving tubulin post-translational modification. In migration of oriented fibroblasts, neurite outgrowth, and myogenesis, increases have been noted in the degree of post-translational modification of w-tubulin; spatially restricted subsets of microtubules that become enriched in modified tubulin have also been observed during these morphogenetic events. Post-translationally modified microtubules exhibit a striking stability compared to the bulk of the microtubules present. Here, we suggest that stablc, posttranslationally modified microtubules may play a vital

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Glu-Tyr I

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Pig. 1. Tyro5ination and detyrosination of n-tubulin. Pictured in this cartoon are a ncwly translatcd n-tubulin polypcptidc (left), which then undergoes removal and readdition of its C-terminal tyrosine residue (right) by tubulin carboxypeptidase and tuhulin tyrosine ligasc, respectively.

role in generating microtubule-based asymmetry during alterations in cell morphology and polarity.

in TTL activity and level of Tyr tubulin have been correlated with a number of developmental events(21.25.26), suggesting that these post-translational modifications may play a role in the dramatic cytoskeletal alterations that occur when cells change shape.

Tyrosination of a-Tubulin - A Unique PostTranslational Modification The post-translational modification of tubulin that has been studied in greatest detail, tyrosination/detyrosinaTyrosinated and Detyrosinated n-Tubulin Define tion, consists of the removal and subsequent readdition Distinct Microtubule Subsets in vivo of a tyrosine residue from the C-terminus of a-tubulin, To test the involvement of tyrosination/detyrosination one of the two polypeptides comprising the heteroof alpha tubulin in cellular morphogenesis, we prepared dimeric tubulin protomer. Fig. 1shows that the primary peptide antibodies specifically reactive either with Tyr modification that occurs after a-tubulin is translated is or with Glu tubulin (thefe antibodies bein called Tyr detyrosination, which is carried out by a tubulin-specific antibody and Glu antibody, respectively@ ). The two carboxypeptidase (TCP(*.l2)).Detyrosinated actubulin antibodies were found to label distinct populations of (called Glu tubulin, because of the glutamic acid microtubules in undifferentiated cultured cells. In each residue exposed at the C-tcrminus after detyrosination) of a wide variety of cultured cell types, most interphasc is then subject to a second modification. the covalent microtubules were brightly labeled with the Tyr readdition of C-terminal tyrosine by tubulin tyrosine antibody, while only a small number wcre labeled with ligase (TTL(l3,l'j), in an ATP-dependent reaction, thus the G ~ antibody; u some microtubules were labeled by regenerating tyrosinated tubulin (called Tyr tubulin both antibodies (Fig. 2). Immunoelectron mibecause oi thc tyrosine residue at its C-terminus). croscopy(28)demonstrated that all cellular microtubules Other features of tyrosination/detyrosination relcontained both forms of tubulin: yet, the level of the evant to the current studies are not shown in Fi 1. two forms in different microtubules varied widely. The First, most a-tubulin genes (6 out of7 in mammals8')), data obtained from electron microscopy suggest that cncode Tyr tubulin; the seventh gene, whose primary microtubules dctectably labeled with only one antibody translation product is a Glu tubulin form. has also bccn by immunofluorescence actually contain a low level of shown to be a substrate for tyrosination in ~ z v o ( ' ~ ) . the other species. The immunoelcctron microscopic Thus, all a-tubulins in mammals are subject to this data also showed that the level of Glu or Tyr tubulin did reversible post-translational modification. In addition, not vary appreciably along the length of an individual these modifications are nearly ubiquitous within the microtubule. Because of the special properties of animal kingdom: TTL activity has been detected in microtubules enriched in Glu tubulin (see below), we phylogenetically diverse organisms(17), including a have adopted operational definitions of microtubules variety of invertebrates (with the exception of based on the imrnunofluorescence labeling they exhibit yeast( ')). We and others have demonstrated the with Glu- and Tyr-specific antibodies: microtubules existence of both Tyr and Glu tubulin in protozoa("), containing sufficient Glu tubulin to be detectable with Cuenorlzahditid""), Xenopud21), and many higher Glu antibody are called Glu microtubules (even if they vertebrates(22). Second, both Tyr and G l ~ itubulin also display some Tyr antibody immunofluorescence), species are known to be capable of assembling into while microtubules detcctably labeled with Tyr, but not microtubules in ~ , i l r o and ( ~ ~in) viva(=). Finally, changes Glu, antibody are termed Tyr microtubules. To date, microtubules unstained with both Glu and Tyr Abbreviations used: Glu, detyrosinated; TCP. tubulin carboxypeptidasc; antibodies have not been reported in cells of higher TTL. tubulin tyrosine ligase; Tyr. tyrosinated.

8

+

Glu

Fig. 2. Double immunofluorescence localization of tyrosinated (Tyr; left panel) and detyrosinated (Glu; right panel) n-tubulin in an African green monkey kidney cell (TC-7 line). Microtubules detectable only with tht. Tyr antibody (arrowheads) or only with the Glu antibody (arroWb) are indicated. Bar. I0 micrometers. This figurc is reproduced from Gundersen el al. C" by permission of Cell Press; immunostaiiiiiig methods used have been described previous~y~~",

animals, suggesting that the operationally defined Glu and Tyr microtubule subsets together comprise the entire cellular array. While Glu and Tyr tubulin are concentrated in distinct microtubule subsets in undiffcrentiated cultured cells during interphase, wc have found that the two tubulin forms are more homogeneously distributed during mitosis(29).Tyr tubulin is the more prevalent form during all stages of mitosis, and little or no difference in the level of the two forms was noted on western blots of cells at different stages of Quantitative immunoelectron microscopy of metaphase spindles has shown that all classes of spindle mici-otubules (i.e., kinetochore, pole-to-pole, and astral) contain similar amounts of G ~ Lantibody I immunoreactivity. and that all metaphase microtubules contain lower levels of Glu antibody reactivit than any of the microtubules found in interphase cellsY'").Nonetheless, immunofluorescence studies of cells at other stages of mitosis suggest that microtubule subpopulations differing in their Glu/Tyr tubulin composition do exist in the spindle, just as they do in interphase cells. For example, Glu antibody labeling is limited to the half-spindles of early anaphase cells, while in late anaphase and telophase, Glu antibody labeling of the interzonal class of microtubules becomes as intense as that in the halfspindle(29).Results on the distribution of Glu and Tyr tubulin during mitosis are consistent with other studies that have dcmonstrated that most mitotic spindle microtubules are highly dynamic (e.g., see ref. 3 ) ; yet. these results also suggest that at certain stages of mitosis, these rapid dynamics are modified for a subset of microtubules. Detyrosinated Tubulin is Enriched in Stable Microtubules in vivo To determine the mechanism(s) by which cells are

capable of generating distinct cellular distributions of tubulin molecules that differ only in the presence or absence of a single amino acid, cultured cells were treated with agents (e.g.. nocodazole, cold) that depolymcrized microtubules, then the drug was washed away or the cells were re-warmed to permit the repolymerization of microtubules('"). In these experiments, Tyr microtubules reappeared rapidly, while Glu microtubules appeared only after an extended interval. From this result-it was postulated that the regrown Tyr microtubulcs arise from a pool of soluble protomcr containing only Tyr tubulin, and that Glu microtubules arise by the time-dependent action of TCP on the newly-formed Tyr microtubules. That Glu microtubules arise in a time-dependent fashion was subsequently shown by artificially stabilizing the normally dynamic microtubules in cultured cells with taxol or azidc. With both microtubule-stabilizing treatments, an increase in detyrosination of cellular microtubules was measured by immunofluorescence and western blotting(3"). These and similar experiments established that detyrosination in cultured cells is a post-polymepizution modificution; that is, that the two populations of microtubules arise by a cyclic mechanism. as shown in Fig. 3 . In this cycle. microtubules are polymerized from tubulin protomcrs in which the a-tubulin is in the Tyr form. Tyr microtubules are then acted upon by TCP in a timedependent manner; consequently, all long-lived microtubules become substantially detyrosinated, whereas microtubules turning over rapidly never achieve a significant level of Glu tubulin before they depolymerize. When microtubules enriched in dctyrosinatcd tubulin (Glu microtubulcs) eventually dcpolymerize. the resulting Glu protomers are efficiently retyrosinated, thus completing the cycle. This cyclic mechanism is consistent with previous in vitm studies of TTL and TCP, in which TTL was determined to act preferentially on protomeric tubulin("*"~ while TCP acted almost

exclusively on microtubule polymer(33934). By partitioning the tyrosine-addition and -removal reactions between separate cytoplasmic pools of tubulin, i.e., between protomeric and polymeric tubulin, a homogeneous population of subunit tubulin for polymerization is created and a heterogeneous population of microtubules is generated. This partitioning of enzymatic activities may also avoid the possibility of a futile cycle. In summary, the coupling of the tyrosination/detyrosination cycle with the rapid dynamics of microtubules in vtvo generate5 microtubules that are chemically marked, depending on their stability. Further aspects of the cycle of post-translational modification shown in Fig. 3 were demonstrated experimentally. For example, the enzymatic retyrosination of Glu tubulin protomerq by TTL was studied by microinjection of Glu tubulin into cultured cells(24). Similarly, the partitioning of Tyr and Glu tubulin between polymeric and protomeric pools was examined by extracting cultured cells under conditions that solubilized the protomeric tubulin, but left the polymeric tubulin i n ~ o l u b l e ( ~ ~Only ~ ' ~ )Tyr . a-tubulin was detectable in the extractable protomer, while both Tyr and Glu tubulin were present in the polymeric fraction. These data suggest that only three of the four species shown in Fig. 3 (Tyr protomer, and Tyr and Glu microtubules) are maintained at significant steady-state levels in vivo,and support the contention that the most likely role of the tubulin tyrosination/detyrosination cycle is to generate functionally distinct microtubules. Yet another aspect of the tyrosination/detyrosination cycle that has been experimentally examined is the heightened stability of Glu-enriched microtubules. Glu microtubules have been shown in many cell types to be more resistant than Tyr microtubules to depolymerization by microtubule-antagonistic drugs or to dilution in extracted-cell preparations('"-"). That Glu microtubules possess greater longevity in vivo has also been demonstrated in experiments in which hapten-derivatized tubulin was microinjected and allowed to incorporate into cellular m i c r ~ t u b u l e s ~ ~In~fact, ~ " ) .this approach was exploited in order to measure the degree carboxypeptidase

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of stability, or longevity, of Glu microtubules; a significant population of Glu microtubules persisted for as long as 16 h (almost the full length of interphase)("). It is noteworthy that in all systems examined thus far, Glu microtubules exhibit enhanced stability relative to other cellular microtubules. Thus, the Glu antibody is a novel and useful probe of microtubule stability, since prior treatment of the cells is unnecessary to assess the populations of stable and dynamic microtubules. The other two methods for determining microtubule stability - monitoring incorporation of microinjected derivatized tubulin or quantification of drug stability both require that the experimenter perturb the cells in order to measure stability of microtubules that were present at the start of the measurement. However, by examining the distribution of Glu and Tyr tubulin with specific antibodies, one can readily assess the distribution of stable and dynamic microtubules in populations of unperturbed cells; indeed, Glu antibody has corroborated other methods in demonstratin stable I.i microtubule subsets in a variety of cell types(# Although detyrosination is enhanced on stable microtubules. available experimental evidence indicates that detyrosination is a result of, rather than a cause of, microtubule stability. Enzymatic detyrosination of Tyr microtubules in cytoskeletons prepared from permeabilized cells is insufficient to increase thcir stability(37).Analogous results were obtained in vivo by inhibiting TTL with microinjected antibodies; the resultant Glu microtubules in the injected cells did not differ in their dynamics from their Tyr counterparts in uninjected cells(''). These results, obtained with experimentally produced Glu microtubules in living cells and in lysed cell models, are in agreement with data obtained from in v i m comparisons of the stability of Glu and Tyr microtubules; to date, preparations of tubulin enriched in either Glu or Tyr forms have shown similar pol merization and depolymerization characteristic~('~~~~.'). These results strongly suggest that detyrosination of microtubules is insufficient to generate the enhanced stability that is characteristic of Glu microtubulcs in vivo. The presence of elevated Glu tubulin in cellular microtubules also seems to be unnecessary for their stabilization in vivo. Some cultured fibroblasts have no Glu microtubules detectable by immunofluorescence. although the1 have been shown to exhibit stable microtubules( 'I. Similarly, we have found that the marginal band of toad erythrocytes, an unusually stable array of microtubules, contains no detectable Glu tubulin(22).That Glu tubulin is neither necessary nor sufficient for microtubule stabilization suggests that the role of microtubule detyrosination is of a completely different nature (see below).

Glu

Fig. 3. Cyclic model for the tyrosination-detyrosination of n-tubulin. This figure is reproduced from Gundersen et al.("')),with permission from the Rockefeller Press.

Stable Detyrosinated Microtubules in Cellular Morphogenesis Although a wide variety of types of cultured cells

contain microtubules heterogeneous both in their dynamics and their longevity, neither the distribution nor the orientation of these microtubule arrays in asynchronous populations of proliferating cells hinted at their function. Therefore. we began to examine cell types that could be triggered to undergo particular morphogenetic transformations in which microtubules were suspected of contributing to the observed change in cell shape. First. we examined cells migrating with known polarity in a wounded monolayer system. In this model system, a swath of cells is scraped from a confluent monolayer of fibroblasts, in the process inducing the cells adjacent to the 'wound' to migrate into the empty space. In these experiments, cells immediately adjacent to the wound acquired an extensive array of Glu microtubules oriented toward the direction of impending migration(38). The oriented array appeared rapidly, that is, within an hour in most cells and at about the same time that the centrosome reoriented toward the wound edge, a feature of motile fibroblasts and endothelial cells observed previously("s). but preceded actual translocation of the cells. As expected, the Glu microtubule array oriented toward the cclls' migration was enhanced in its resistance to depolymerizing drugs, confirming the TYr

correlation of Glu tubulin enrichment and enhanced stability. While the role of the Glu microtubules in the cells migrating into the wound is unclear, their orientation is certainly suggestive: perhaps these microtubules function in the transport of new membrane components to their site of insertion near the leading edge of the fibroblaht. Newly synthesized viral proteins have been shown to be inserted preferentially at the leading edge of motile cells, and this vectorial insertion does not occur in the abscnce of m i c r o t ~ b u l c s ( ~thercfore, ~); it is possible that Glu microtubules serve as specialized tracks for the transport of membrane components to insertion sites at the plasma membrane. Vectorial insertion of new membrane at the leading edge of motile cells has been proposed as an important facet of cell motility'"), although some recent experiments have qiuestioned the universality of this hypothesis'47.4 ) We investigated Glu and Tyr microtubules in PC-12 cells, since the outgrowth of neuritm in these cells has been a classic model system for studying the mechanism by which microtubules function to bring about the formation of a neuronal process in response to ncrve growth factor (Fig. 4). With this homogeneous cell

Glu

Fig. 4. Immunofluorcscencc localization of tyrosinated (Tyr; left pancl) and detyrosinated (Glu; right pancl) cu-tubulin during neurite outgrowth. Single arrowhead indicates a developing neurite in which both Tyr and Glu microtubules are apparent: double arrowhead indicates a neurite with only Tyr microtubules. PC-12 pheochromocytoma cells were grown in DMEM supplemented with S % fetal bovine serum and 100'2 horse serum (GIBCO, Grand Island, NY); SOngml-' nerve growth factor (Collaborative Research, Boston MA) was added to the rnediurn 24h prior t o fixation and irnmunostaining of Tyr arid Glu tuhulin, which were performed by protocols described previously("). Bar, 10 pm.

population, we also found that the appearance of Glu microtubules accompanied the morphogenetic change: in this case we were also able to measure an increase in thc lcvcl of Glu tubulin during the time course of differentiation, and these results were corroborated by others(”). In PC-12 cells, we noted that detyrosination of individual microtubules was occurring and, as in the case of the motile fibroblasts, that this was signalled locally, rather than occurring throughout the cytoplasm. One can see from Fig. 4 that each asymmetric forming neuritc responds individually in generating Glu microtubules. In general, the longer the neurite, the greater the likelihood that it contained Glu microtubules. In PC-12 cells, as in other cell types, the Glu microtubules were stable ones, as determined by their resistance to depolymerizing drugs. The enhanced stability of Glu microtubules in neuronal processes has been convincing1 demonstrated by others (e.g., see Baas and Black j . A third morphogenetic event we have investigated is the differentiation of L6 rat muscle cells. The L6 rat myoblasts offer the advantages of an inducible and highly synchronous differentiation system that undergoes well-characterized changes in cytoskeletal proteins of the microfilament class. We found that Glu microtubules and an increased level of detyrosinated

(4

tubulin arose early in the myogenic program, at a time when the mononucleated myoblasts wcrc aligning and elongating prior to ccll fusion(”). Surprisingly, these Glu microtubules were elaborated prior to the accumulation of detectable muscle-specific myosin, making this a very early event. indeed, in the myogenic program. Finally, we determined that the Glu microtubules were stabilized relative to microtubules present in undifferentiated myoblasts. Myogenesis, thus. is a third example of a morphogenetic program leading to asymmetry in cell shape that includes the elaboration of a polarized array of Glu microtubules of heightened stability. Conclusions and Perspectives The three morphogenetic events we have examined point to a general mechanism by which microtubule stabilization is involved in morphogenetic events (Fig. 5 ) : In precursor cells, microtubules are dynamic, and are radially arrayed due to their nucleation at the centrosome. At a very early stage in the morphogenetic process, microtubules arc stabilizcd selectivcly, with the criterion for stabilization being their particular cellular location. Because the basal state of microtubules is one of dynamic instability, microtubules that

External signal 4

B

C

Pig. 5. Stable, detyrosinated microtubules in cellular morphogenesis. This model depicts the iovolveinent of microtubules in cellular morphogenesis, in which: (1) a spherical, undifferentiated cell possesses only dynamic microtubules (---): (2) An external signal generates microtubule stabilization sites (hatched area): which (3) selectively stabilize microtubules 1-( nearby; and (4) The stabilized microtubules become post-translationally modified (thick dark line; -). and may be further stabilized. Depending on the location of the original external signal and the microtubule stabilization sites, a number of outcomes are possible, and are illustrated by the examples in A-C. A. a polarized motile fibroblast with an oriented array of stable. detyrosinated microtubulcs; R . a neural cell whosencuritic process, extending from the cell body. contains many stable microtubules, but whose cell body and growth cone regions may contain dynamic microtubules, and C, an elongated muscle cell. with bipolar morphology and numerous stable microtubules oriented parallel to the elongation axis. For the sakz of clarity. only the stable microtubules are shown in each of the differentiated cells (A-C); dynamic microtubules are known to co-exist with stablc ones, at least in motile fibroblasts(3s) and in neurons(50). Tn each cell the stippled circle represents the nucleus. This model is an extension of one originally preseiited by Kirschner and Mitchi~on(~”.

are not selected for stabilization are rapidly replaced by those that have been stabilized(“). The extent of replacement of dynamic microtubules with stable microtubules would be expected to depend on a number of factors but would probably be most strongly influenced by the number of stabilization sites. If there are many stabilization sites, dynamic microtubules may be completely replaced by stable microtubules at the end of the morphogenetic process. This may be the situation in cells with extremely stable arrays of microtubules; e.g., the axoneme of sperm. Alternatively, if there is only a limited number of stabilization sites, dynamic microtubules will coexist with stable microtubules. This seems to be the inore common situation: it is observed in many types of proliferating cultured cells, such as in fibroblasts and epithelial cells, and even in cells such as neurons, that have increased their levels of stable microtubules during a differentiative event(”). A number of other factors, such as microtubule bundling, may also affect the stability and the final array of microtubules. If the model shown in Fig. 5 is correct, what role exists for the post-polymerization detyrosination of microtubules? We have previously proposed that the primary role of detyrosination is to mark the stable microtubules biochemically, enabling the cell to distin uish stable microtubules from dynamic According to this idea, the complex ones (‘335). information contained in the dynamic properties of the cellular microtubule array is converted to a rimple biochemical form with only one parameter, namely the level of Glu tubulin in a microtubule. If the level of Glu tubulin in a microtubule is acting as a signal of microtubule stability, cellular components competent to interpret this signal must exist. Specific proteins that interact with Glu tubulin could serve to further stabilize microtubules or they might utilize Glu microtubules specifically for transport processes. To date no proteins of this type have been identified. With respect to a putative signalling function for Glu tubulin, it is important to point out that tubulin undergoes three other post-translational modifications; namely, a ~ e t y l a t i o n ( ~phosphorylation(”), ~), and glutamylation(”). A t least in the cases of acetylation and phosphorylation, the modifications appear to occur on the microtubules that have been stabilized; thus. these modifications may have a function similar to that hypothesized for detyrosination, i.e., in apprising cellular effectors that stable microtubules have been generated. The multiplicity of post-translational modifications of tubulin may explain why some cells do not generate Glu microtubules even though they have stable microtubules; instead of having an effector system that recognizes Glu tubulin, they may have one that is responsive to one of the other modified forms. The multiplicity may also increase the functional repertoire of the cell so that more than one effector system can utilize the stable array of microtubules. Indeed, stable inicrotubules containing both Glu and

acctylated tubulin are a common feature of many cultured cell lines(5s). A number of issues remain unresolved and form the basis for ongoing research. First, how stable are the Glu microtubules in cells that are undergoing morphogenesis? Tn all three morphogenetic events examined, the Glu microtubules are stable, as determined by their drug stability and as predicted from work with undifferentiated cells. Still, the degree of stability of the Glu inicrotubules in differentiated cells is unknown. It is possible that different degrees of microtubule stability are effected as the cell’s final morphology is established. It will be interesting to measure the longevity of stable Glu microtubules in these cells (using the derivatized-tubulin method). since previous studies have only examined this question in cells progressing through the cell cycle. The second question is, how arc stable microtubules generated? Any mechanism must explaiii both the restricted formation of stable inicrotubules in ‘meaningful’ locations in the cell and the selective nature of the stabilization (that is, i he location-specific stabilization of only a subset of the total cellular microtubules). Experimental evidence such as the relative insensitivity of stable microtubules to end-mediated depolymerization either by drugs or dilution(”7).and their inabilitv to incorporate tubulin subunits at their distal ends(””“‘) suggests that stable microtubules have modified distal ends in v i w . Neither the post-translational modification itself, nor the binding of microtubule-associated proteins along the length of the microtubule is likely to be the primary event in generating microtubules with these peculiar ends. A GTP-cap mechanism is also unlikely, since the GTP cap model(”) postulates that microtubules are stabilized by the incorporation of protomers at their ends. It is also worth noting that the relative stability imparted by a cap of GTP-containing subunits, which has been hypothesized as a mechanism to permit growth of microtubules at steady-state levels of polymeric tubulin. is unlikely to be important for the hours-long stability of Glu microtubules in viva. One possibility is that end stabilization is accomplished by microtubule-capping proteins that act specifically to prevent addition or loss of tubulin subunits from microtubules. A structural cap has been discovered at the plus end of microtubules at the distal tip of cilia and flagella("^"); in Chlumydomonas, these flagellar capping structures are immunoreactive with an antibody that recognizes kinetochores, which are plus-end capping structures present during mitosisi57).Nonetheless, to date there is no evidence for a plus-end microtubule capping protein, structure or collar that might contribute to the stability of the Glu microtubules that have been described in proliferating or differentiating cells. A final remaining question is: how are the stable Glu microtubules utilized by the cell? Are there specialized microtubule-associated proteins or motor proteins that interact exclusively, or more efficiently, with Glu

b y brain extract. Separation of a carboxypeptidase lrom tuhiilm tyrosine ligase. M o l . Cell. Biochem. 19. 17-22. 13 RAYRIN, D. A N D FI.AVIN, M,(1977). Enzyme which adds tyrosine to (he nchain of lubulin. Biochemistry 16. 2189-2194. 14 SCHROFDER, H. C . , WEHLAND, J. AND W E B t K , K. (1985). Purification of brain tul>ulin:tyrosine ligasc by biochemical and immunological methods. J. C'~,U 100. 276-281. 15 VILLASAN'IE, A , . W A N G , I).. DOBNER. P., DOLWI,P., COWAN, W. J . (1986). Six mouse a-tubulin mKNAa encode ixotypes: testis-specific expression of two sister genes. Mol. Cell B i d 6, 2409.241 9, 16 Gu, W.. Ltwis. S. A. A.UU COWAN, h.1. (1988). Generation of antisera that discriminate among mammalian ru-tubulins: introduction of specialized isotypes Note Added In Proof. In a recent paper, Alfa and into cultured cell? results i n theit- coasscmbly without disruption of normal Hyams (Cell Motzl. Cytuskel. 18, 86-93 [1991]) microtubule function. J . C d l Riol. 106, 2011-2022. attempted to detect Glu tubulin in Schizosaccharo17 PRESTON. S., DEAUIN, G. G . , HANSOX., R. D. AND GORDON. M. W-. (1979). The phylogenetic distribution of tubulin:tyrosinc ligasc. .I. ikfol. E i d . 13, inyces ponibe. They used Glu antibodies reactive with 233-244. 3.poinbe tubulin enzymatically detyrosinated in vitro. 18 KOBAYASHI. T. AND FLAVIN,M. (1981). Tubulin tyrosylation in By immunofluorescence or western blotting. no Glu invertebrates. Cornp. Biochem. P!iysiol. 69B. 387-392. 19 STEI~ER, J . . WYLEK, T. AND S ~ L B E CT. K ,(1984). Partial purification and tubulin was detected in vivo, even when the cells were characterization of microtubular protein from Trypanosornn brucei. J. Biol. previously treated with microtubule stabilizing agents. Cliern. 259, 496-4602. These results suggest that the tyrosination/ 20 G A H ~ UH. S . J.. GKALILWK, G . ~ N CIUMTR, D F. (1983). Activity patrern of aminoacyl-tRNA synthetascs, tRNA mcthylases, arginyltran?feraseq and detyrosiiiation modification may not extend to yeast, tubu1in:tyrosine ligasc dui-ing dcvelopmcnt and aging of Cuenorhnhditiy perhaps, as the authors point out, because yeast have elegans. Eur. J . Biochem. 131. 231-233. no requirement for a differentiated microtubule array. 21 PKLSTON. S . . DEANIN. G . G , ,HANSON, R. D. ANJ G O K D OM. ~ , W . (1981). Tubu1in:tprosine ligase in oocytes and embryos of Xenopus laevis. Dev. Biol. 81, 36-42. 22 GIJUDERSEN, G. G. A U D BTJr.INSK1. .I. C . (1986). Microtubule arrays in Acknowledgements differentiated cells contain elevated levels of a ~ost-tranblationallymodifid form of tubulin. Eur. J. Call R i d . 42, 288-294. The authors thank Drs Daniel Webster, Nidia Modesti, 23 RAYBIN. D . AND FLAVIN. M.(1977). Modification of tubulin by tyrosylation Sadiqa Khawaja, and Steve Chapin for helpful in cells and extracts and its cffect on asscmbly in vifro. .I. Cell B i d . 73.492-504. 24 WEBSTLK. D. R.. ~ ~ U N D ~ K ~G.C G.. N , BULINSKI, J. C. AND BoRrsu, G . G. discussions. and Dr Andrew Chang for assistance with (1987). Assembly and turnover of dctyrosinated tubuliii in i,ivo. .I. Crl/ Riol. Fig. 4. We also thank D r John Kilmartin for providing 105, 265-276. us with monoclonal anti-Tyr tubulin antibody used for 25 D ~ A N I G. N , G . W. C. A ~ GORDON. D M. W. (1977). Tyrosyltubulin ligase activity in brain. skelctal muscle, and liver of the developing chick. DFI'. R i d . many of these studies. Research described was 57,230-233. supported by grants from the N.I.H. (CA 39755), and 26 RODRICAEZ, J . A . AND BORISP,G . G. (1978). Modification of the Cterminus of brain tubulin during development. Biochem. Biophyi. Res. the Muscular Dystrophy Association. Commun. 83. 579-586. 27 GUNOEKYEN, ti. G.M. H. . ~ N DBULLNSKI, J. C. (1984). Distinct populations of microtubules: tyrosinated and nontyrosinatcd wtubulin are distrihuted differently irr vivo. Cell 38. 779-7S9. References 28 G ~ U L N(3. S , G . G . , G L N D L R ~ R.. L NNLYULNS. . F., CORNELISSEN, .I. C. A N D 1 MCKBITHAN. T. W. AND ROSENB~UM. .I. L. (19S4). Thc biochemistry of D!~~KABANDLK. M. (1986). Ultrastructural colocalization of tyrosinatcd and microtubulcs. In Crll and r l . l u r b MaiZi+, Vol. 5 (ed. J.W. Shay), pp 255-288. detyrosinated e-tubuliii in intcrphasc and mitotic cells. J . C d l Biul. 103. Plenum, NY. 1883-1893. 2 INOI'E, S. (1982). The role of qelf-assembly in the ge ne ra hn of biological 29 GuKDeKsEN. G . G . AND BULINSKI. J. C. (1986). Distribution oftgrosinatcd form. In Dcwh~prnentulOrder: Its Origin nnd Kegulntion. Fortierh Symposium and nontyrosinated n-tubulin in the mitotic spindlc. .I. Cell Riol. 102. of the Society for 1)evelopmental Biologists (eds. S. Subtelny and P.B. Green), 1118-1126. pp. 35-76. Alan K. Liss. Inc., NY. G. G. S. AND U U L I N ~ K JI ,. C. (1987). Post-polymerization 3 S.AXTOh, W. M.. SILMPLL, D. L., LLSIJE,R. J., SALMON, E . D., ZAVORTINK, 30 GC"DERSEN, detyrosinalion of a-tubulin: a mechanism for subcellular differentiation of A M MCINIOSH, J . R. (1984). Tubuliii dynamics in cultured nian~malian microtubulea. J. Cell B i d . 106, 251-264. cells. J. C'eN Biol. 99. 2175-2186. 31 ARCE.C. A.. R ~ D R I G L EJ.Z A . . . BARRA, FI. S. A N D C A P U I LR . ~. , (1978). 4 SOLIYS,B. J. AKD BOKISY, G. G. (1985). Polymerization of tubulin in vii.o. Capability of tubulin and microtubules to incorporate and to releaie tyrosine direct evidence for assembly onto microtubule a i d s and from ccntrosonien. .I. and phenylalanine and thc effcct of incorporation of these amino acids on Cell Bzol. 100. 1682-1689. tubulin asqcmbly. J. A'eurochem. 31, 205-210. 5 SCHULZE. E. AND KIRSCHNBR, M. (1986). Microtubule dynamics i n interphase 32 WCIILAND:J . A N D WEHLK,K. (1987). Tubulin-tyrosine ligasc has a binding CCIIS. .I. Cell ~ i o i 102, . imo-in31 site on /%tubulin: A two domain structure of the enzymc. J. Cell Riol. 104, 6 SCHIJIL7F, E.. ASU, D., BULINSKI. J. c. 4 N D KIKSCHNPR. bf. (1987). Post1059-1067. translational modificatiuna and microtubule atability. J. Celell B i d . 105. 33 ARCL.C . A. AND BARXA. H. S . (1985). Release of C-terminal tyrosine from 2167-2178. tubulin and microtubules at steady state. Biochrm J. 226, 311-317. 7 BORNENS.M. A ~ DKAKSENTI. E. (1984). Thc centrononie. I n M~wzhrcrne 34 KUMAR,x. AND FLAVIN.M. (1Y81). Preferential action of a brain Strurrirre and Fu~unr.lion(ed. F,.E. Bittar) pp. 100-171. John Wiley and Sons, detyrosinolating carboxypeptidase on polymerized tubulin. .I. B i d . Chem. 256, NY. 7678-7680. 8 B K ~M.-H., . KRU, 'r. E. A N D KAKS~NTI. E. (1987). Control of microtubule 35 GCNICRSEU. G. G.. KIIAU~AJA, s. A N D BULINSKI, J . C. (1987). Microtubules nucleation and stability in Madin-Darby canine kidney cclls: the occurrence of enriched in detyroiinated tiihulin in vivo are leis dynamic than those enriched in nonccntrosomal, stable detyrosinated microtubulcs. .I. Cell B i d . 105, tyrosinated tubulin. In The Cytoskeiefon and Ceil Diflerenfintioit nrzd 1283-1296. Development (eds. J. hrechaga and R. Maccioni). pp. 75-81. ICSU Prcss. 9 Sur i IVAN, K . F. (19x8). Structure and utilization of tuhulin kotypes. Annu. Miami. REV.Cell Biol. 4. 687-716. 36 KRELS,T . E. (1987). Microtubulei containg delyroiinated tubulin are less 10 GRECR, K. AKD KOSENBAUM, J. L. (1989). Pout-translational modifications dynamic. EMBO J. 6, 2597-2606. of tubulm. In CtN Movenrerit, vol. 2 (eds. F.D. Warner, P. Satir, and 1.K. 37 KHAWAJA, S. G. G. A N D RTXINSKI. J . C. (1988). Enhanced stability of tiibbons), pp. 47-66. Alan R. Liss. NY. microtubules enriched in detyrosinaled tubulin is no1 a direct function of 11 OLMSTED, J. B. (1986). Microtubule-associated proteins. Annu. Rev. Cell detyrosination level. J. Cdl Biol. 106. 141-150. Biol. 2. 321-357. 12 A R G A R A N A . C. E.H. S. A N D CAPuno. R . (1978). Release of ["Cltyrosinc 38 GUNDERSEN. G . G. AND BULINSKI. J . C. (1988). Selective stabilization of

tubulin'? Is the multiplicity of tubulin post-translational modifications a reflection of a redundancy in signalling a single function, or a reflection of a number of different signals, each of which activates a separate function? These are important questions that need to be answered before we can formulate more accurate molecular mechanisms to explain the role of microtubules in cellular morphogenesis.

niicrotubules oriented toward the direction of cell migration. Proc. ,Vat1 Acad. Sci. US.4 85. 5946-5950. 39 GUNDERSEY, G. G., KH.~WNA, S. AND BULIUSRI, J. C . (1989). Generation of a stable. post-translationally inodificd microtubule array is an early event In myogcnic diffcrentiation. .7. Cell Biol. 109. 2275-2288. 40 WEBSTF,R, D. R..GIINIJFRSFN. fi.G . , UULINSKI, J . C . AND UOR15Y. (j.G . (1987). Differential turnover of tyrosinated and detyrosinated microtubules. Prvr. h a r l 11ca.d. Sci. il.jSA) 84, 9040-YO44. 41 WFBsrEI