- Email: [email protected]
Functions of tubulin isoforms
Functions
of tubulin
Douglas The Johns Hopkins
University
School
isoforms
B. Murphy of Medicine,
The biological significance of tubulin isotypes lies in different chemical and physical environments. the origin and distribution of several new tubulin ways for studying their assembly and function
Current
Opinion
in Cell
Biology
Baltimore,
Maryland,
USA
in their ability to function Recent papers document isotypes and suggest new in specialized cells.
1991,
3:43-51
p-tubulins that are only used for mitosis in the plasmodium of Pbysarum, asexual sporulation in Aspergillus, etythropoiesis in vertebrates, and spermatocyte differentiation in Drosophila have already been identified [ 3,401.
Introduction Microtubules perform diverse and essential functions in all eukaryotic cells, and during recent years it has become clear that the u- and P-tubulin subunits of microtubules, together with certain microtubule-associated proteins (MAPS), are as diverse and complex as the functions of microtubules themselves.
In articles that appeared during the past year, Oka et al. [5*] convincingly demonstrated the existence of several immunologically distinct tubulin isoforms in the asters and central spindles of dividing sea urchin eggs (Fig. 11, and Denoulet et al. [6*] showed that distinct tubulin isoforms are preferentially associated with either the soluble or cytoskeletal fractions comprising the slow components of axonal transport in fat sciatic nerve.
Depending on the context of their expression, microtubules may be either dynamic and labile or stable and inert, and also serve as substrates for a wide assortment of MAPS, cross-linking proteins and cytoplasmic microtubule motors. Although the diversity in the physical properties of microtubules can in principle be explained by changes in the intracellular environment and microtubule-interacting proteins, early observations led to the hypothesis that the tubulin subunits themselves might occur in different biochemical isoforms, each associated with unique properties for performing certain functions in the cell [ 1,2]. To cite just a few examples,
It is now well established that both a- and P-tubulin represent families of structurally and biochemically distinguishable isofon-ns that have their origin in multiple diverse tubulin genes and in several speciIic forms of posttranslational modifications. For P-tubulin, the number of genes ranges from one in yeast and two in Aspergil1u.s and Chhmydomonas to four in Drcxophih and as many
Fig. 1. lmmunofluorescence staining of dividing echinoderm eggs reveals segregation of tubulin isoform in the mitotic, spindle. Eggs of the sand dollar, C. japonicas, labeled with the following isotype-specific antibodies: (a) YLl/2, an antibody that labels microtubules, the asubunits of which contain tyrosine at their carboxy-terminal ends, labels astral microtubules near the spindle poles; (b) Class II antibody, D2D6, which recognizes specific a-tubulin isoforms, labels primarily the central spindle; (c) Class Ill antibody, DMIB, specific for the carboxyl terminus of a vertebrate P-tubulin isotype, labels peripheral microtubules of the spindle asters. Published with permission IS*]. Abbreviations MAP-microtubule-associated @
Current
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protein; Ltd
ISSN
MT-microtubule. 0955-0674
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. as six in vertebrates [ 31. The a-tubulins exhibit a similar degree of genetic complexity but have n& been studied as extensively. A striking feature of the genetic isotypes of tubulin is that many are expressed in specific tissues and cell types [ 7,8,9]. Even within single cells, tubulin isotypes may be non-uniformly distributed; for example, a class III p-tubulin &type has been found to be partially excluded from the microtubule cytoskeleton in the neurites of differentiating PC12 cells [,lO*,llm]. One of the present challenges is to distinguish whether isotypes perform spec&z functions, as Cleveland and Cowan first suggested [12,13], or whether they are redundant in their functions, as Ratf first proposed [14], their evolution resulting from selection that controls the time and place of tubulin expression during development. As this review of the literature covering the last two years will point out, the evidence is complex but appears to support both of these hypotheses.
Molecular and genetic approaches: testing for the requirements and properties of isotypes ‘Wo molecular and genetic approaches have been employed to examine the requirement for tubulin isotypes in viva: analysis of organisms with mutated or replaced tubulin genes, and introduction of exotic tubulins into tissue culture cells by transfection with tubulin DNA sequences. The deletion or mutation of tubulin genes in a genetically amenable organism puts forward the following question: because cells contain multiple isotypes, does ablation of one isotype disrupt a specific cell process? In Drosopbiila and Gzenorbabditis, mutation of tubulin genes has been shown to have striking and specific effects. As discussed later in more detail, mutations in a single B-tubulin isotype in LIros@ka arrest spermatocyte development and result in male sterility [ 141. In a recent analysis of mutations in Gzenorbabdie Savage et ul [ W] discovered another tissue-specific p-tubulin gene designated m-7. The microtubules in this organism contain 11 protolilaments, the single known exception being the microtubules in six touch-receptor neurons that contain 15 protofilaments. Mutations in m-7 resulted in touch-insensitive animals that lack the 15 protofilament microtubules in their touch-receptor neurons. This is one of the most dramatic examples relating tubulin heterogeneity to the formation of a set of microtubules that is both st.ructuraUy distinct and is located in a specific cell type. III contrast to this extreme example, however, Sawada and Cabral [16] recently showed that P-tubulin isotypes are used interchangeably in vertebrate tissue culture cells-a result that has been observed previously in at least two other organisms. In lower organisms such as yeasts and Aspeqilluswhich contain, respectively, two a- and two g-tubulin genes, the
replacement of one tubulin gene with another has no observable effect on cell function [ 31. Recently, Morns and May and their collaborators [ 17’,18*] have focused their attention on Aspergiks, where one of two P-tubulin isotypes is normally expressed only in structures associated with spore formation. As in the case of a-tub&n gene substitutions in yeast, the replacement of the general P-tubulin (benA) with the conidiation-specific one (tubC) and vice wrsu (the experiment has now been carried out in both directions in A.speygik.x) yielded no observable phenotype, and the tubulin pool size and a-tubulin$tubulin ratio were normal in the modified cells. Given that the two P-tubulins show a 17% divergence in their ammo acid sequences, and the fact that Rspergi1Iu.s contams several distinct cell types and exhibits a number of microtubule-dependent processes, the lack of an observable phenotype is puzzling. As May suggests, perhaps differences in cell function would have been observed under different environmental conditions, or perhaps the rationale for the existence of two j3-tubulin genes is not in having different sequences available, but in order to line-tune the time and place of tubulin expression. These ideas have their merits, but could the results mean that it is the ap-tubulin dimer or even the a-subunit itself that is the critical parameter that needs to be examined? It follows that it would be interesting to determine whether aand fi-tubulin form isotype-specific dimer pairs. If such a relationship is confirmed, the key experiment would be to replace both the g- and corresponding a-subunits. Other recent reports also point to this possibility, as described later. An alternative strategy has been to introduce exotic tubulin DNA sequences into cells by transfection. The rationale here is that an introduced isotype with distinct (and possibly inappropriate) biochemical properties would disrupt some cell function or possibly produce a distinct intracellular distribution of microtubules. Although a number of different isotypes and cell types have been employed, the results thus far are not definitive [3]. The introduced isotypes co-mingle with the endogenous isotypes and become incorporated in all of the microtubules, and cellular functions are not disrupted. A common problem encountered with this method has been that the introduced tubulin is expressed only at the level of a few per cent of the endogenous tub&n. Acting on the belief that low amounts of protein might bias the interpretation of the results (and indeed there is evidence that this is the case), investigators are using more efficient cell expression systems. In a recent study, Sisodia et al. [19*] used a method of gene amplification to overexpress chicken class IV @ubulin in Chinese hamster ovary (CHO) cells. Although transcription of class N mFWA was increased three-fourfold relative to that of the endogenous tubulin isolypes, the class N isotype never accumulated to greater than 10% of the total tubulin protein. The RNA and tubulin protein were synthesized at a rapid rate, but the newly synthesized P-tubulin never accumulated, suggesting that it was continuously and rapidly degraded.
Functions
Part of the di5culty in obtaining large amounts of expressed protein may lie in the autoregulatory mechanism controlling tubulin expression, but this does not totally account for the problem since, during the time the chicken class N sequences were being overexpressed, there was a compensating reduction in the transcription of the endogenous class N g-tubulin message. Sisodia et al concluded that tubulin synthesis might be a&ted by other isotype-specilic regulatory mechanisms that have not yet been identilied. If overexpression of P-tubulin did occur, there is the possibility that in the absence of compensating amounts of endogenous cc-tubulin, excess subunits would poison the cell as Burke et aC [ 20*], Katz et al. [ 21.1, and Weinstein and Solomon [22*] have shown during tubulin overexpression in yeast. Although the mechanism of poisoning is not known in detail, overexpression of j3-tubulin initially leads to the loss of all microtubules in the cell, followed by the appearance of dots of j!l-tubulin (but not utubulin) at or near the nucleus-a pattern consistent with the accumulation of g-tubulin at the spindle pole bodies. Later, as j3-tubulin accumulates, a novel, nonmicrotubule polymer forms. Eventually, the cells die, but as Weinstein and Solomon [22*] have recently shown, this is probably due to the loss of cellular microtubules rather than to the presence of abnormal polymers. Overexpression of j3-tubulin is lethal, even at relatively low levels (50% of cells lose all of their microtubules by the time the protein has accumulated to a Lifold excess over wild-type levels) [ 22.1. In contrast, overexpression of a-tubulin is only slightly deleterious and only then at much higher concentrations. Signilicantly, the lethality associated with moderate overexpression of j3-tubulin is completely suppressed by co-overexpression of a-tubulin, even when the total accumulation of tubulin dimer reaches 5-10 times the normal level. There is a limit, however, to how much tubulin a yeast cell can tolerate, and at 30-60 times the normal level, cell viability decreases signilicantly. On the basis of these observations, Burke and Solomon et al have proposed that the rate of formation of tubulin dimer is dependent on the rate of j3-tubulin synthesis. In this model, a-tubulin is synthesized in some excess of the @ubulin and is rapidly degraded when in the free subunit form. As a result, the cell never accumulates free j3-tubulin monomers that are otherwise poisonous and the rate of j3-tubulin synthesis determines the rate at which tubulin dimer can accumulate. Whether the rate of tubulin dimer synthesis and stoichiometry of a- and j3subunits is regulated in this way in other eukaryotic cells, is not yet known. Although the amounts of j3-tubulin synthesized in previous mammalian cell-expression experiments are probably sufficient to induce poisoning, the a-tubulin:j3-tubulin ratio is usually shown to be close to one, and cellular functions are usually not perturbed, suggesting that poisoning by overexpression of g-tubulin has so far not been a problem. The trick then may be to obtain expression without upsetting the u-$Isubunit ratio while maintaining the normal concentration of the intracellular tubulin pool. Recent experiments on
gene replacement in mopbila this might be done.
Drosophila resulmo functionally
of tubulin
isofomw
Murphy
provide insights on how
yields new and definitive tubulin isotypes that are not equivalent
In Drosophila, Raff and colleagues have shown that mutations in the testis-specilic j32-tubulin, the predominant j3-tubulin isotype in this tissue, disrupts several distinct processes in sperm differentiation [3,23.=]. These studies provided a clear demonstration of the tissue-specilic expression by certain isotypes, and showed that a single isotype could participate in a number of different intracellular functions, including meiosis, manchette formation, and assembly of the axoneme. Nevertheless, the work did not answer the question of the unique functional capabilities of isotypes. In a recent paper, Hoyle and Raff [23*=] have shown that two j!l-tubulin isotypes are not functionally equivalent by stably incorporating a developmentally regulated j3-tubulin isotype (83) into the male germ line of Drosqf&ikz A hybrid gene (8283) encoding the j33 protein was constructed by joining the 5’ promoter-containing sequences of the j32 gene to protein-coding and 3’ non-coding sequences of the 83 gene. When expressed in flies that completely lack the normal testis isoform, j33 only supported assembly of microtubules involved in the elongation of mitochondrial derivatives, but meiosis, nuclear shaping, and axoneme formation were deficient or did not occur (Fig. 2). Furthermore, when p3 protein expression exceeded 20% of the total tubulin pool in llies containing normal amounts of j32-tubulin, axoneme function was disrupted and ilies were sterile. Thus, the functional capacities of j32- and j33-isotypes are not equivalent. As the total amount of tubulin dimer was reported to be normal in the altered llies, this conclusion seems justified. Nevertheless, the presence of reduced amounts of tubulin in some of the fly constructs raises the question of to what extent underexpression or overexpression of tubulin influenced the phenotypes that were observed. One of the most interesting discoveries in this study is that the disruption of microtubule assembly and function depends, in a critical way, on the amount of j33tubulin that is expressed, exhibiting what might be called a ‘threshold effect’. In the case of co-expression of j33tubulin with wild-type j32tubulin, llies only became sterile when the relative proportion of j33-tubulin was greater than 20%; dies containing less than this amount were normal and fertile. It is indeed sulking that a gradual impairment was not observed in response to increasing amounts of j33-tubulin. Apparently, when the proportion of j33-tubulin is below 20%, the structure and function of microtubules are either una&cted or are close enough to normal that the flies are fertile. A similar effect was observed in earlier microinjection experiments where Prescott et al [24*] introduced tubulin from the myxamebae of Pbysurum into PtK2 cells, and reported a threshold effect in the response of injected cells during
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Fig. 2. The replacement of the gene for a testis-specific tubulin isotype in Drosophila shows that tubulin isotypes do not share the same functional capacities. (a) Elongated spermatids in a sterile male homozygous for a chimeric tubulin gene that directs expression of p3-tubulin and for a null mutation of the testis-specific tubulin. In the absence of P2-tubulin, the nuclei farrowed) remain round and are not aligned within the bundle of spermatids: The presence of nuclei of different size indicates abnormal chromosome segregation during meiosis. (b) Elongated spermatids in a fertile male that has two dimeric p3 genes and two normal 82 genes. Nuclei appear thesame as in a wild-type male. Published with permission 1239.
tests of microtubule stability. Thus, these studies emphasize the potential importance of a threshold effect, where an introduced isotype must accumulate to a critical percentage or threshold before effects on microtubule function are observed. The study of Hoyle and Raff [23*-l is also notable because the p3-isotype was shown to accumulate to relatively high levels: (more than 20% of the endogenous tubulin in co-expression experiments), a level of tubulin expression never before obtained in a higher eukaryote. An obvious difference in the experimental design is the use of the endogenous promoter-containing sequences of the 5’ non-coding region of the p2 gene to drive /33tubulin expression. This may indicate the importance of using the non-coding portions of the endogenous host tubulin genes to drive the expression of an introduced isotype. In particular, this approach might be useful in obtaining high levels of expression in transfected mammalian tissue culture cells.
for nucleation?
In order to find microtubule-interacting proteins in AS pergilhs, Oakley and Oakley and colleagues [ 25*,26*] undertook a search for exttagenic suppressors of a P-tubulin mutation and discovered a gene (m.@4), the product of which corresponds to an altogether new class of tubulin subunits. The sequence of this tubulin isotype only shows approximately 30% homology of amino acid sequence to either a- or P-tubulin, considerably lower than the 3642% identity that all other a- and P-tubulins reported to date are known to share with each other. Because the m@A product does not resemble either a- or ptubulin very closely, it has been designated as a new tubulin class called y-tubulin. Disruption of the m@4 gene is lethal as it strongly inhibits nuclear division and thus is essential for cell function in Rspergillus. Antibodies generated to a unique peptide sequence of y-tubulin label the spindle pole bodies and give faint staining of the central spindle and certain cytoplasmic spots (Fig. 3). Because of the unique location of y-tubulin in the spindle pole bodies, the authors conclude that it may play a role in microtubule nucleation. In addition, because it was identified as a (3-tubulin-interacting protein, the authors speculate that it is possible that the P-ends of microtubules are associated with the spindle pole body. Thus, y-tubutin may not only nucleate microtubules but also establish the structural polarity of microtubules in the cell. Work is presently underway to identify y-tubulin genes in vertebrate cells and thereby determine if the patterns of expression, distribution and mode of action of y-tubulin are conserved in other organisms.
Studies on purified tubulin isotypes: indication of the mechanisms by which isotypes confer specificity of function
A very successful strategy for evaluating the functional significance of tub&n isotypes has been to directly purify the proteins and examine their properties in vitro. Even more importantly, this approach may allow one to compare the mechanisms of assembly of different .isotypes [nucleation, growth, dynamics, and interaction with microtubule-associated proteins (MAPS)], to quantify these differences, and to predict the consequences of their expression in vivo. Among the best-studied examples are the tubulins from chicken erythrocytes and Antarctic fish, and preparations of isotype-depleted tubulin from mammalian brain. Together with other collaborators, Rothwell and myself demonstrated that chicken erythrocytes contain a unique @ubulin isotype that is associated with the marginal band in these cells [ 27.1. This divergent vertebrate isotype is expressed only in blood cells and differs from brain tubulin in many important respects. Erythrocyte tubulin is composed predominantly of class VI P-tubulin, an isotype not found in brain, whereas brain tubulin
Functions
of tubulin
isoforms
Murphy
that the dilferences in functional capacities of isotypes are reflected in their ability to bind MAPS with different affinities. By Scatchard analysis of equilibrium binding, we found that MAP-2 binds to microtubules with a K,-J of 0.1 @, with the binding to brain microtubules being twofold higher than to microtubules composed of erythrocyte tubulin. Thus, a potential for different binding afhmities for hL4F’s exists and may eventually provide insights into the functional significance of tubulin isotypes.
Fig. 3. Localization of y-tubulin in Aspergillus by immunofluorescence microscopy. Cermlings of Aspergillus are shown in phase contrast (a) and after double immunofluorescence staining with antibodies to reveal a-tubulin (b) and y-tubulin (c)*The y-tubulincontaining spots at the spindle poles are visible. Published with permission [25-l.
consists of a complex mixture of isotypes, including isotype classes I, II, III and IV. In comparison with brain microtubules, erythrocyte microtubules are more stable and less dynamic, longer and more flexible, and minimaLly af fected by large changes in ionic strength and pH. Knowing the assembly rate constants for brain and etythrocyte tubulin, we successfully predicted the composition of rncrotubules assembled from mixtures of these two tubulin isotypes [ 28.1. The two isotypes copolymerized eficiently, but when they polymerized under conditions that were close to steady state, microtubule copolymers also exhibited gradients in the ratio of tubulin isotypes along their lengths that differed at their ends by as much as 2530% (Fig. 4). The observations agree with previous reports on the homogeneous distribution of tubulin isoforms in cells and suggest that tubulin subunits by themselves have a limited capacity to segregate into distinct subsets of microtubules. Recently, we used brain and erythrocyte microtubules to compare the binding affinities of chicken brain MAP-2. Such a comparison allows one to test the hypothesis
Another remarkable isotype is the divergent u-subunit found in the brain tissue of Antarctic fish. As the microtubules in these organisms exist at or close to 0°C an extremely efficient mechanism of stabilization is necessary to counteract the instability of microtubules at low temperature. This is apparently accomplished through the expression of a highly divergent a-subunit that maintains a critical concentration for assembly comparable to that found in other organisms, but which polymerizes efficiently even at 5°C [29*]. The divergent a-tubulin comprises 55-65% of the total u-subunits and, although it is not known if the low-temperature isotype is the product of a unique gene or the product of extensive post-translational modifications, the range of temperatures supporting efficient tubulin assembly is considerably reduced. A comparison of the thermodynamic parameters of fish and mammalian tubulins by van t’Hoff analysis revealed sizeable increases in the entropy and enthalpy terms that determine the microtubule equilibrium for fish tubulin, indicating that it is the increased level of hydrophobic interactions between subunits that allows these microtubules to exist at low temperatures [ 30.1. Of all of the tubufin variants that have been isolated and studied so far, Antarctic fish tubulin is biochemically and biophysicalfy one of the most divergent, and future work should provide valuable insights on the role of a-tubulin in microtubule assembly. Even within the complex mixture of subunits that comprise mammalian ‘brain tub&n’, individual isotypes differ markedly in their in vitro assembly properties. In a recent paper, Banejee et al. [31*] used affinity chromatography and a specific monoclonal antibody to remove a class III, neuron-specific P-tub&n isotype from preparations of purified, bovine brain tubulin. Surprisingly, the isotype-depleted tubulin polymerized twice as rapidly and to a greater extent than the whole unfractionated sample when examined in a MAP-containing assembly buffer lacking stabilizers such as glycerol or taxol. The authors concluded that even moderate amounts of certain isotypes (class III P-tubulin comprises approximately 25% of the total cycled protein) are capable of influencing the properties of microtubule assembly. This finding also strongly suggests that the presence of the class III isotype may be important for the normal functioning of microtubules in neurons. Two such possibilities are that the presence of class LU tubulin (which retards self-assembly) may reduce the potential for spontaneous nucleation to control microtubule number, or that the isotype is segregated within neurons to determine the regional distribution of MAPS, a phenomenon that has already been well documented.
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Reversible tyrosination of tubulin: a post-translational modification that may regulate the dynamic activity of microtubules
several post-translational modifications of tubulin have now been identified, including tyrosination, acetylation, phosphorylation and, most recently, polyglutamylation. With the exception of phosphorylation, these modilications are notable for their unusual nature and for the fact they affect diverse but specific sites on a- and P-tubulin subunits. Whereas little is known yet about the functional sigriihcance of these modifications, including the newly discovered polyglutamylation [32*], there is compelling evidence that both tyrosination and acetylation of a-tubulin are associated with an increased degree of mi crotubule stability in uivo. In particular, the cause and effect relationship between tyrosination and microtubule stability has been analyzed in detail and has yielded some surprising results. Tubulin tyrosination refers to the reversible addition of tyrosine to the tyrosine-free carboxy-terminus of a-tubulin. The modilication (both addition and removal) occurs on the polymer itself and is controlled by tubulin-tyrosine ligase and a tubulin-carboxypeptidase, which respectively add and remove terminal tyrosine residues from a-tubulin. Detyrosination of tyrosine-containing tubulin (Qr-tubulin) produces tubulin ending in a glutamic acid residue (Glu-tub&n), and it is this detyrosinating activity that is associated with enhanced polymer stability. This modification occurs in a wide variety of organisms and cell types and correlates with changes observed in microtubule dynamic activities associated with mitosis and cell dilferentiation; it is thus believed to be ubiquitous and fundamental to understanding the control of microtubule function. The relationship of tyrosination with decreased stability is particularly evident in the growth cones of neurons [ 33*], where cellular domains with increased motility exhibit microtubules with greater amounts of Tyrtubulin. This correlation was made even more compelling in a recent study of muscle cell differentiation by Gundersen et al. [34-l, who showed that detyrosination to Glu-microtubules (MTs) closely paralleled a decrease in the dynamic activity of microtubules in syncytia resulting from myoblast fusion. Any doubts about this correlation were removed by Baas and Black [35*] who demonstrated approximately a 50fold difference in the nocodzole sensitivity of Glu- versus TyrMTs in the axons of cultured sympathetic neurons.
Fig. 4. Immuno-electron microscopy of a microtubule co-polymer that has elongated from the end of an erythrocyte microtubule seed in a preparation containing equal amounts of chicken brain and erythrocyte tubulin subunits. Labeling was obtained using an antibody, specific for the class VI p subunit from chicken erythrocytes. Although these divergent isotypes copolymerize efficiently, their capacity to assemble is not equivalent, as shown by the changing ratio of tubulin isotypes along the length of the elongating copolymer. Published with permission I28*1.
Functions
Immunofluorescence analyses clearly showed that labile microtubules were enriched in Tyr-tubulin, whereas stable microtubules contained only detyrosinated and acetylated a-tubulin, and that the relative proportions of the biochemical species corresponded precisely to the observed ratio of labile and stable microtubules in the axons. Baas and Black also showed that drug-resistant GluMTs appeared to nucleate the regrowth of labile Tyr-MT polymers, leading the authors to suggest that Glu-MTs might regulate microtubule dynamics in neurons. Detyrosination, however, does not increase microtubule stability directly.
Detyrosination: tubulin-stabilizing
indirect involvement mechanisms
in other
In a previous study by Khawaja et al [36*], it was noted that cultured kidney cells contain distinct subsets of Gluand Tyr-MTs that diifer in their sensitivity to microtubuledepolymerizing drugs. When permeabilized cell models, however, were treated with carboxypeptidase A to generate Glu-MTs from endogenous ‘Qr-MT’s, no change in the resistance of microtubules to dilution was observed, suggesting that detyrosination alone was insticient to confer enhanced stability. Webster et al [37*] have now examined the effect of detyrosination on microtubules in living fibroblasts by injecting an antibody to tubulin-tyrosine ligase, a treatment that blocks enzyme activity and results in the virtual elimination of ‘Iyr-MTs and the accumulation of Glu-MTs in the cell. Upon measuring microtubule dynamics in these cells, it was found that the turnover time was approximately 3 min in both treated and untreated cells, strongly indicating that detyrosination itself does not stabilize microtubules. The authors suggest that microtubule-capping proteins or MAPS could be the components that directly confer stability. Alternatively, the function of detyrosination may be to differentiate further, stable microtubules for specilic activities or to allow interaction with Glu-specific MAPS to maintain the stability. The observations of Baas and Black [35*] on the stability of Glu-MTs in axons support these ideas. It is possible that the factors that stabilize Glu-MTs (neuronal MARS) are absent or present in reduced amounts in cultured fibroblasts, but are more concentrated in axons. An interesting experiment, therefore, would be to microinject the antibody to tubulin-tyrosine ligase into neuronal cells. With the rapid pace of experiments in this field, we should soon have an answer.
Conclusions The papers reviewed here provide important new insights into the diversity and function of tubulin isofonns in cells. The molecular basis of isoform diversity and the
of tubulh
isoforms
Murphy
patterns of expression of tubulin isoforms are now understood in considerable detail. More importantly, the ‘functional significance of isotypes’ has come to mean the adaptation of the physical and biochemical properties of an isotype (or group of isotypes) to particular cellular environments, while the previous concept of one isotype-one function no longer seems applicable. A testis-specific P-tubulin isotype in DrnuqMa has emerged as a pArime example: the same isotype performs multiple functions in developing spennatocytes but cannot be replaced by another dissimilar isotype. Nevertheless, the extent to which isotypes have evolved to function in specialized environments remains unclear, and the physical
References
and recommended
reading
Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstanding interest 1.
FUL.TON C, SIMPSON PA: Selective Synthesis and Utiliaation of Flagellar Tubulin The Multi-Tubulin Hypothesis. In Cdl MotiUtyedited by Goldman R, Pollard T, Rosenbaum J [book]. New York: Cold Spring Harbor laboratories, 1976, pp 987-1005.
2.
STEPHENS
3.
SUU~VANKF: Struchue and Utuization of Tubulin Annu Rev Gel1 Bioll988, 4687-716.
4. .
RE: Structural Chemistry of the Axoneme: Evidence for Chemically and Functionally Unique Tubulin Dimem ln Outer Fibers. In MoIecules and Gd Movement edited by Inoue S, Stephens REI [book]. New York: Raven Press, 1975, pp 181-206.
Isotypes.
JOSHIHC, CIXVE~ DW: Diversity Among Tubulin Subunits: Toward What Functional End? Cell Motil Cytaske&ton 1990, 16:15+163. A useful, up-to-date review that focuses on tedundancyversus specilicity of function for tub&n isotypes and that includes references on the heterogeneity and disttibution of tubulin isofomw.
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0~ MT, ARAI T, HAMAGIJCHIY: Heterogeneity of Microtubules in Dividing Sea Urchin Fggs Revealed by Immunotluorescence Microscopy: Spindle Composed of Tubulin Isotypes Different tral Microtubules. Cell Moth Cyrarkelern
Microtubules from Those
are of As-
1990, 16:239-250. Striking examples of distinct tubukn isotype segregation within the mitcitic spindle by imrnunoguorescence microscopy. D~NOU~ET P, FIUJA~~EAUG, DE NECHAUD B, GROS F, DI GIA~RERARDINOL DiiferentiaI Axonai Transport of Isotubulins in the Motor Axons of the Rat Sciatic Nerve. / Cell Bid 1989, 108:%5-971. Several tubuIin isoforms are demonstrated to show diiTerent distdbutions in soluble and cytoskeletai fractions associated with the slow components of axonal transport 6.
.
7.
BURGO~~E RD, CAMBRAY-DEMN MA, LEWIS SA, SARKARS, COWAN NJz DitTerentiaI Distribution of g-TubuIin Isotypes in Cerebellum. EMSO] 1988, 72311-2319.
8.
LEwrs SA, COWAN NJ Complex Regulation and Functional Versatility of Mammalian (1- and g-tubuRn Isotypes During the
Ditlkrentiation
of Testis.
and
Muscle
CelJs.
J Cell Biol
1988, 106:20232033.
Tubulin Upstream Sequence Causes a Reduction fJ-Tubulin Level but Has No Effect on Microtubule Cell Motil C)&xkeleton 1990, 16:214-220.
in ber~4
Function.
A tub&n isotype normaiiy associated with spore formation accumulates to 50% but has no effect on microtubuie function when placed under the control of the constimtive g-tub&n gene. This paper includes carefui quantitation of protein synthesis. 18.
MAY GS: The Highly Divergent b-TubuRns of Aspergfllus nfdulans are Functionally Interchangeable. J Cdl Bioll989, 109:2267-2274. The minor g-tubuiin isotype normaUy associated with spore formation was manipulated to become the only Functional gtubuiin in hpeqillus No elkt on cell function was observed. .
SISODIASS. GAY DA, CLEVEIANDDW: In ofvo Discrimination Among g-Tubuiin Isotypes: Selective Degradation of a lope IV g-TubuIin Isotype Following Overexpression in Cultured Animal Cells. New Biologfit 1990, 266-76. A careful and interesting paper that analyzes the problem of tubulin isotype expression in transfected cells. A class IV g-tubuiin is synthesized but rapidiy degraded during cell expression, suggesting the presence of an additional mechanism for regulating expression in an isotype-speciiic manner.
19. .
9.
IDPATA MA Cuzv~lruv~ DW: In ofvo Microtubules are Copolymers of AvaiIabIe g-Tubuiin Isotypes: Localization of Each of Six Vertebrate g-TubuIin Isotypes Using PolycIonaI Antibodies F&cited by Synthetic Peptide Antigens. J Cell Biol 1987, 105:1707-1720.
BURKED, GA~DA~KAP, HARTWELLL Dominant Effects of Tubuiin Overexpression in Saccharomyces Cerevfsiae. Mol CeN Biol 1989, 9:104+1059. When a- and g-tubuiin are overexpressed in yeast, excess a-tubuiin is not detrimental but overexpression of 8tubuiin, or of both a- and 8tubuiins together, is lethal.
10.
AX DJ, REMOLONANM: Tubuhn Isotype Usage In Vfva A Unique Spatial Distribution of tbe Minor Neuronal-Specific g-TubuRn Isotype in Pbeochromocytoma CeiIs. Dev Biol
21. .
.
1989,
13Z398-409.
Shows that diiferences exist in the function of tubuiin isotypes in differentiated cells: ciass RI g-tubuiin is not incorporated into microtubules in diiIerentiating PC12 ceiis. 11. .
JOSHIHC, CLEVELANDDW: Differential Utilization of B-Tubulin Isotypes in DiIferentiating Neurites. J Cdl Biol 1989, 109z663673. Fii 8-tubukn isotypes exhibit diIferences in synthesis rates, assembly, and intraceUuIar localization in diIIerentiating neudtes of ~~12 ceiis.
12.
Sutuv~~ RF, CLEVELANDDW: IdentiIIcation of Conserved Isotype-DeIining Variable Region Sequences for Four Vertebrate p-Tubulin Pokypeptide Classes. Proc Natl Acud Sci USI 1986, 83:4327-4331.
13.
WANG D, V~UXANIX 4 LEWIS Sq COWAN NJ: The Mammalian g-Tub&n Repertoire: Hematopoietic Expression of a Novel, Heterologous g-TubuRn Isotype. J Cell Biol 1986, 103:19031910.
14.
RAPPEC: Genetics of Microtubule 99:1-10.
Systems. J Cell Bill 1984,
15. .
SAVAGEC, HAMEuN M, Cu~orn JG, COUL~~NA, AIBERT~ONDG, CHALFIEM: met- 7 is a g-TubuIin Gene Required for tbe Production of 15Protofdament Microtubuies in Caenorhabditfs elegans Genes Dev 1989, 3870-881. A dramatic example of the specificity of g-tubuiin function in Cueno-itis ekgans Mutation in the g-tubuiin gene, mer-7, results in touch-insensitive animals that lack microtubules in their six touchreceptor neurons. 16. .
SAWADAT, CAB~U.LF: Expression and Function of g-TubuIin Isotypes in Chinese Hamster Ovary Cells. J Biol Cbem 1989, 264:30133020. The authors show by 2-D gel electrophoresis and immunoiluorescence microscopy that three distinct g-tub&n isotypes in CHO cells are used interchangeabiy.
20. .
KATZW, WEINSTEINB, SOLOMONF: Regulation of Tubulin LeveIs and MIcrotubule Assembly in Saccharomyces cenwfsfae: Consequences of Altered TubuRn Gene Copy Number. Mol Cell Bid 1990, 10:5286-5294. An interesting paper showing that cells function normaiiy on 50% of the normal tubuiin level and that strains containing extra copies of tubuiin genes tubuiin downreguiate their tubuiin levels. The data support a model for mbuiin dimer formation that depends on the rate of g-tubuiin synthesis. 22. .
WEINSTEIN B, SOLOMON F: Phenotypic Consequences of TubuIin Overproduction in Saccharomyces Cerwfsfae. Differences between Alpha- and Beta-TubuRn. Mol Cell Biol 1990, 10:5296-5304. Overexpression of P-tub&n (as opposed to overexpression of a-tubuiin or to co.overexpression of both a- and 8tubuiin) is uniquely toxic and leads to rapid disassembly of endogenous microtubules and decreased viability. HOYLE HD, RWF EC: Two Drosophila Beta TubuIin Isoforms are not Functionally Equivalent. J Cell Biol 19$X1, 111:1009-1026. An impressive and comprehensive study employing genetics, microscopy, and biochemistry to show that two 8.Nbuiin isotypes in Drosophila are not functiona& equivalent. 23.
..
24. .
PREXO’IT AR, FOSTERKE, WARNRM, Guu K: Incorporation of Tubuiin from an EvolutionariIy Diverse Source, Pbysarum Polycephalum, into tbe Microtubules of a Mammalian CeU. J Cell Sci 1989, 92:59%05. One of the first demonstrations is that a threshold level of Nbuiin required is to observe alteration of microtubule function in microinjected cells. OAKLEYBR, OAKIXY CE, YWN Y, JUNGMK: Gamma-TubuIin is a Component of tbe Spindle Pole Body that is Essential for Microtubule Function in Aspeqiflus Nfdufans Cell 1990, 61:1289-1301. A new chss of tubuiin called y-tubuiin is localized in Azpeqillus to the spindle pole body. suggesting a role in nucleation and mitosis, and possibly the determination of polarity of nucleated microtubuies. 25. .
26.
17. .
MAY GS, WARINGRB, Moiuus NR: Increasing h&Z 8-TubuIin Synthesis by placing it Under the Control of a benA 8-
.
OAKLEY CE, OAJUEY BR Identification of Gamma-Tubuiin, a New Member of the TubuIin Superfamily Encoded by mfpA Gene of Aspeqfllus nfdulans Nature 1989, 338:662&$X
Functions Dramatic disclosure of the sequence for a new class of tubulins called y-tubulin. ROT~VEU SW, GRASSERWA, MURPHYDB: Tubulin Variants Exhibit DifTerent Assembly Properties. Ann NY&ad Sci 1986, 466:103110. Comparison of the biochemical properties and assembly characteristics, includ.ing the assembly rate constants, of brain and erythrocyte tubulin. 27.
.
BAKERHN, RO’IHWXUSW, GRASSER WA, Wus ICI’, MURPHYDB: . Copolymerization of Two Distinct Tubulin Isotypes During Microtuble Assembly in vitro. / Cell Biol 1990, 110:97-104. Mixtures of divergent tubulin isotypes co-assemble in vitro but the polymers exhibit gradients in the ratio of rubulin isotypes along their lengths. It is estimated that isotype segregation in uivo would require specialhed intracellular conditions. 28.
of tubulin
isoforms
Murphy
ROB~ONSJ, BURGOYNERD: Dierencial Localization of Tyrosinated, Detyrosinated, and Acetylated u-Tubulins in Neurites and Growth Cones of Dorsal Root Ganglion Neurons. Cell Motil Cytmkeleton 1989, 12:273282. The tyrosinated form of a-tubulin is shown to be enriched in labile microtubules found in the growth cones of cultured neurons. 33.
.
GUNDER~ENGG, KHAWAJAS, BULINSKIJC: Generation of a Stable, Posttranslationally Modified hlicrotubule Array is an Early Event in Myogenic Dilferentiation. / cell Bfof 1989, 109:22732288. This study correlates an increase in the detyrosination of microtubules with a decrease in microtubule dynamics in difTerentiating myoblasts. 34.
.
.
BAAS PW, BLACK MM: Individual Microtubules in the Axon Consist of Domains that Differ in both Composition and Stability. J Ceff Biol 1990, 111:495-509. Provides the most convincing demonstration so far for the correlation between detyrosinated microtubules and polymer stability, and estimates the difference to be approximately 50.fold.
DEIIUCH HW, JOHN~N m MARCHESE-RAGONA SP: Polymerization of Antarctic Fish Tubulins at Low Temperatures: Energetic Aspects. Biodemfshy 1989, 28:10085-10093. An interesting thermodynamic approach to describing (and explaining) the differences in the assembly of Antarctic tish tubulin.
KHAWAJAS, GUNDER~ENGG, BULINSKIJC: Enhanced Stability of Microtubules Enriched in Detyrosinated Tubulin is not a Direct Function of Detyrosination Level J Cd Bid 1988, 106:141-149. The authors treat cell models with carboxypeptidase to generate detyrosinated microtubules but find that there is no increased resistance of the modified polymers to dilution.
l-hm RH, DETIUCH HW: Dynamics of Antarctic Fiih Microtubules at Low Temperatures. Biodemfshy 1989, 28:508+5095. Pulse-chase experiments are used to show that Antarctic tish tubulin is dynamic under near-physiological conditions at 5°C. 29.
30.
.
31.
.
BANERJEEA, ROACH MC, TRCKA P, LUDUENARF: increased Microtubule Assembly in Bovine Brain Tub&n Lacking the Qpe Ill isotype of &Tubulin. J Biol Cbem 1990, 265:1794-1799.
Removal of a class II1 p-tubulin isotype from preparations of bovine brain tub&n results in preparations that polymerize faster and to a greater extent. The work shows that individual isotypes can affect microtubule assembly, even in complex mixtures of tubulin isotypes in
35.
.
36.
.
WEBS~FRDR, WEH~ANDJ, WEBERK, Bo~ls~ GG: Detyrosination of Alpha Tubulin does not Stabiie Microtubules in ufua J cell Bid 1990, 111:113122. The authors inject antibody to tubulin-tyrosine ligase into fibroblasts to generate Glu-MTs and find that both Glu-MTs and Tyr+lTs show the same level of resistance to anti-MT drugs. 37.
.
vih-a
EDDE B, ROSSIERJ, LE CAER J-P, DESBRWERE~E, GROS F, DENOUIET P: Posttranslational Glutamylation of a-Tubulin. Science 1990, 247:83+5. A surprising, new post-translational modification of tubulin found in neurons. 32.
.
DB Murphy, Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 N Wolfe Street, Baltimore, Maryland 21205, USA
51
of tubulin
Douglas The Johns Hopkins
University
School
isoforms
B. Murphy of Medicine,
The biological significance of tubulin isotypes lies in different chemical and physical environments. the origin and distribution of several new tubulin ways for studying their assembly and function
Current
Opinion
in Cell
Biology
Baltimore,
Maryland,
USA
in their ability to function Recent papers document isotypes and suggest new in specialized cells.
1991,
3:43-51
p-tubulins that are only used for mitosis in the plasmodium of Pbysarum, asexual sporulation in Aspergillus, etythropoiesis in vertebrates, and spermatocyte differentiation in Drosophila have already been identified [ 3,401.
Introduction Microtubules perform diverse and essential functions in all eukaryotic cells, and during recent years it has become clear that the u- and P-tubulin subunits of microtubules, together with certain microtubule-associated proteins (MAPS), are as diverse and complex as the functions of microtubules themselves.
In articles that appeared during the past year, Oka et al. [5*] convincingly demonstrated the existence of several immunologically distinct tubulin isoforms in the asters and central spindles of dividing sea urchin eggs (Fig. 11, and Denoulet et al. [6*] showed that distinct tubulin isoforms are preferentially associated with either the soluble or cytoskeletal fractions comprising the slow components of axonal transport in fat sciatic nerve.
Depending on the context of their expression, microtubules may be either dynamic and labile or stable and inert, and also serve as substrates for a wide assortment of MAPS, cross-linking proteins and cytoplasmic microtubule motors. Although the diversity in the physical properties of microtubules can in principle be explained by changes in the intracellular environment and microtubule-interacting proteins, early observations led to the hypothesis that the tubulin subunits themselves might occur in different biochemical isoforms, each associated with unique properties for performing certain functions in the cell [ 1,2]. To cite just a few examples,
It is now well established that both a- and P-tubulin represent families of structurally and biochemically distinguishable isofon-ns that have their origin in multiple diverse tubulin genes and in several speciIic forms of posttranslational modifications. For P-tubulin, the number of genes ranges from one in yeast and two in Aspergil1u.s and Chhmydomonas to four in Drcxophih and as many
Fig. 1. lmmunofluorescence staining of dividing echinoderm eggs reveals segregation of tubulin isoform in the mitotic, spindle. Eggs of the sand dollar, C. japonicas, labeled with the following isotype-specific antibodies: (a) YLl/2, an antibody that labels microtubules, the asubunits of which contain tyrosine at their carboxy-terminal ends, labels astral microtubules near the spindle poles; (b) Class II antibody, D2D6, which recognizes specific a-tubulin isoforms, labels primarily the central spindle; (c) Class Ill antibody, DMIB, specific for the carboxyl terminus of a vertebrate P-tubulin isotype, labels peripheral microtubules of the spindle asters. Published with permission IS*]. Abbreviations MAP-microtubule-associated @
Current
Biology
protein; Ltd
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MT-microtubule. 0955-0674
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. as six in vertebrates [ 31. The a-tubulins exhibit a similar degree of genetic complexity but have n& been studied as extensively. A striking feature of the genetic isotypes of tubulin is that many are expressed in specific tissues and cell types [ 7,8,9]. Even within single cells, tubulin isotypes may be non-uniformly distributed; for example, a class III p-tubulin &type has been found to be partially excluded from the microtubule cytoskeleton in the neurites of differentiating PC12 cells [,lO*,llm]. One of the present challenges is to distinguish whether isotypes perform spec&z functions, as Cleveland and Cowan first suggested [12,13], or whether they are redundant in their functions, as Ratf first proposed [14], their evolution resulting from selection that controls the time and place of tubulin expression during development. As this review of the literature covering the last two years will point out, the evidence is complex but appears to support both of these hypotheses.
Molecular and genetic approaches: testing for the requirements and properties of isotypes ‘Wo molecular and genetic approaches have been employed to examine the requirement for tubulin isotypes in viva: analysis of organisms with mutated or replaced tubulin genes, and introduction of exotic tubulins into tissue culture cells by transfection with tubulin DNA sequences. The deletion or mutation of tubulin genes in a genetically amenable organism puts forward the following question: because cells contain multiple isotypes, does ablation of one isotype disrupt a specific cell process? In Drosopbiila and Gzenorbabditis, mutation of tubulin genes has been shown to have striking and specific effects. As discussed later in more detail, mutations in a single B-tubulin isotype in LIros@ka arrest spermatocyte development and result in male sterility [ 141. In a recent analysis of mutations in Gzenorbabdie Savage et ul [ W] discovered another tissue-specific p-tubulin gene designated m-7. The microtubules in this organism contain 11 protolilaments, the single known exception being the microtubules in six touch-receptor neurons that contain 15 protofilaments. Mutations in m-7 resulted in touch-insensitive animals that lack the 15 protofilament microtubules in their touch-receptor neurons. This is one of the most dramatic examples relating tubulin heterogeneity to the formation of a set of microtubules that is both st.ructuraUy distinct and is located in a specific cell type. III contrast to this extreme example, however, Sawada and Cabral [16] recently showed that P-tubulin isotypes are used interchangeably in vertebrate tissue culture cells-a result that has been observed previously in at least two other organisms. In lower organisms such as yeasts and Aspeqilluswhich contain, respectively, two a- and two g-tubulin genes, the
replacement of one tubulin gene with another has no observable effect on cell function [ 31. Recently, Morns and May and their collaborators [ 17’,18*] have focused their attention on Aspergiks, where one of two P-tubulin isotypes is normally expressed only in structures associated with spore formation. As in the case of a-tub&n gene substitutions in yeast, the replacement of the general P-tubulin (benA) with the conidiation-specific one (tubC) and vice wrsu (the experiment has now been carried out in both directions in A.speygik.x) yielded no observable phenotype, and the tubulin pool size and a-tubulin$tubulin ratio were normal in the modified cells. Given that the two P-tubulins show a 17% divergence in their ammo acid sequences, and the fact that Rspergi1Iu.s contams several distinct cell types and exhibits a number of microtubule-dependent processes, the lack of an observable phenotype is puzzling. As May suggests, perhaps differences in cell function would have been observed under different environmental conditions, or perhaps the rationale for the existence of two j3-tubulin genes is not in having different sequences available, but in order to line-tune the time and place of tubulin expression. These ideas have their merits, but could the results mean that it is the ap-tubulin dimer or even the a-subunit itself that is the critical parameter that needs to be examined? It follows that it would be interesting to determine whether aand fi-tubulin form isotype-specific dimer pairs. If such a relationship is confirmed, the key experiment would be to replace both the g- and corresponding a-subunits. Other recent reports also point to this possibility, as described later. An alternative strategy has been to introduce exotic tubulin DNA sequences into cells by transfection. The rationale here is that an introduced isotype with distinct (and possibly inappropriate) biochemical properties would disrupt some cell function or possibly produce a distinct intracellular distribution of microtubules. Although a number of different isotypes and cell types have been employed, the results thus far are not definitive [3]. The introduced isotypes co-mingle with the endogenous isotypes and become incorporated in all of the microtubules, and cellular functions are not disrupted. A common problem encountered with this method has been that the introduced tubulin is expressed only at the level of a few per cent of the endogenous tub&n. Acting on the belief that low amounts of protein might bias the interpretation of the results (and indeed there is evidence that this is the case), investigators are using more efficient cell expression systems. In a recent study, Sisodia et al. [19*] used a method of gene amplification to overexpress chicken class IV @ubulin in Chinese hamster ovary (CHO) cells. Although transcription of class N mFWA was increased three-fourfold relative to that of the endogenous tubulin isolypes, the class N isotype never accumulated to greater than 10% of the total tubulin protein. The RNA and tubulin protein were synthesized at a rapid rate, but the newly synthesized P-tubulin never accumulated, suggesting that it was continuously and rapidly degraded.
Functions
Part of the di5culty in obtaining large amounts of expressed protein may lie in the autoregulatory mechanism controlling tubulin expression, but this does not totally account for the problem since, during the time the chicken class N sequences were being overexpressed, there was a compensating reduction in the transcription of the endogenous class N g-tubulin message. Sisodia et al concluded that tubulin synthesis might be a&ted by other isotype-specilic regulatory mechanisms that have not yet been identilied. If overexpression of P-tubulin did occur, there is the possibility that in the absence of compensating amounts of endogenous cc-tubulin, excess subunits would poison the cell as Burke et aC [ 20*], Katz et al. [ 21.1, and Weinstein and Solomon [22*] have shown during tubulin overexpression in yeast. Although the mechanism of poisoning is not known in detail, overexpression of j3-tubulin initially leads to the loss of all microtubules in the cell, followed by the appearance of dots of j!l-tubulin (but not utubulin) at or near the nucleus-a pattern consistent with the accumulation of g-tubulin at the spindle pole bodies. Later, as j3-tubulin accumulates, a novel, nonmicrotubule polymer forms. Eventually, the cells die, but as Weinstein and Solomon [22*] have recently shown, this is probably due to the loss of cellular microtubules rather than to the presence of abnormal polymers. Overexpression of j3-tubulin is lethal, even at relatively low levels (50% of cells lose all of their microtubules by the time the protein has accumulated to a Lifold excess over wild-type levels) [ 22.1. In contrast, overexpression of a-tubulin is only slightly deleterious and only then at much higher concentrations. Signilicantly, the lethality associated with moderate overexpression of j3-tubulin is completely suppressed by co-overexpression of a-tubulin, even when the total accumulation of tubulin dimer reaches 5-10 times the normal level. There is a limit, however, to how much tubulin a yeast cell can tolerate, and at 30-60 times the normal level, cell viability decreases signilicantly. On the basis of these observations, Burke and Solomon et al have proposed that the rate of formation of tubulin dimer is dependent on the rate of j3-tubulin synthesis. In this model, a-tubulin is synthesized in some excess of the @ubulin and is rapidly degraded when in the free subunit form. As a result, the cell never accumulates free j3-tubulin monomers that are otherwise poisonous and the rate of j3-tubulin synthesis determines the rate at which tubulin dimer can accumulate. Whether the rate of tubulin dimer synthesis and stoichiometry of a- and j3subunits is regulated in this way in other eukaryotic cells, is not yet known. Although the amounts of j3-tubulin synthesized in previous mammalian cell-expression experiments are probably sufficient to induce poisoning, the a-tubulin:j3-tubulin ratio is usually shown to be close to one, and cellular functions are usually not perturbed, suggesting that poisoning by overexpression of g-tubulin has so far not been a problem. The trick then may be to obtain expression without upsetting the u-$Isubunit ratio while maintaining the normal concentration of the intracellular tubulin pool. Recent experiments on
gene replacement in mopbila this might be done.
Drosophila resulmo functionally
of tubulin
isofomw
Murphy
provide insights on how
yields new and definitive tubulin isotypes that are not equivalent
In Drosophila, Raff and colleagues have shown that mutations in the testis-specilic j32-tubulin, the predominant j3-tubulin isotype in this tissue, disrupts several distinct processes in sperm differentiation [3,23.=]. These studies provided a clear demonstration of the tissue-specilic expression by certain isotypes, and showed that a single isotype could participate in a number of different intracellular functions, including meiosis, manchette formation, and assembly of the axoneme. Nevertheless, the work did not answer the question of the unique functional capabilities of isotypes. In a recent paper, Hoyle and Raff [23*=] have shown that two j!l-tubulin isotypes are not functionally equivalent by stably incorporating a developmentally regulated j3-tubulin isotype (83) into the male germ line of Drosqf&ikz A hybrid gene (8283) encoding the j33 protein was constructed by joining the 5’ promoter-containing sequences of the j32 gene to protein-coding and 3’ non-coding sequences of the 83 gene. When expressed in flies that completely lack the normal testis isoform, j33 only supported assembly of microtubules involved in the elongation of mitochondrial derivatives, but meiosis, nuclear shaping, and axoneme formation were deficient or did not occur (Fig. 2). Furthermore, when p3 protein expression exceeded 20% of the total tubulin pool in llies containing normal amounts of j32-tubulin, axoneme function was disrupted and ilies were sterile. Thus, the functional capacities of j32- and j33-isotypes are not equivalent. As the total amount of tubulin dimer was reported to be normal in the altered llies, this conclusion seems justified. Nevertheless, the presence of reduced amounts of tubulin in some of the fly constructs raises the question of to what extent underexpression or overexpression of tubulin influenced the phenotypes that were observed. One of the most interesting discoveries in this study is that the disruption of microtubule assembly and function depends, in a critical way, on the amount of j33tubulin that is expressed, exhibiting what might be called a ‘threshold effect’. In the case of co-expression of j33tubulin with wild-type j32tubulin, llies only became sterile when the relative proportion of j33-tubulin was greater than 20%; dies containing less than this amount were normal and fertile. It is indeed sulking that a gradual impairment was not observed in response to increasing amounts of j33-tubulin. Apparently, when the proportion of j33-tubulin is below 20%, the structure and function of microtubules are either una&cted or are close enough to normal that the flies are fertile. A similar effect was observed in earlier microinjection experiments where Prescott et al [24*] introduced tubulin from the myxamebae of Pbysurum into PtK2 cells, and reported a threshold effect in the response of injected cells during
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Fig. 2. The replacement of the gene for a testis-specific tubulin isotype in Drosophila shows that tubulin isotypes do not share the same functional capacities. (a) Elongated spermatids in a sterile male homozygous for a chimeric tubulin gene that directs expression of p3-tubulin and for a null mutation of the testis-specific tubulin. In the absence of P2-tubulin, the nuclei farrowed) remain round and are not aligned within the bundle of spermatids: The presence of nuclei of different size indicates abnormal chromosome segregation during meiosis. (b) Elongated spermatids in a fertile male that has two dimeric p3 genes and two normal 82 genes. Nuclei appear thesame as in a wild-type male. Published with permission 1239.
tests of microtubule stability. Thus, these studies emphasize the potential importance of a threshold effect, where an introduced isotype must accumulate to a critical percentage or threshold before effects on microtubule function are observed. The study of Hoyle and Raff [23*-l is also notable because the p3-isotype was shown to accumulate to relatively high levels: (more than 20% of the endogenous tubulin in co-expression experiments), a level of tubulin expression never before obtained in a higher eukaryote. An obvious difference in the experimental design is the use of the endogenous promoter-containing sequences of the 5’ non-coding region of the p2 gene to drive /33tubulin expression. This may indicate the importance of using the non-coding portions of the endogenous host tubulin genes to drive the expression of an introduced isotype. In particular, this approach might be useful in obtaining high levels of expression in transfected mammalian tissue culture cells.
for nucleation?
In order to find microtubule-interacting proteins in AS pergilhs, Oakley and Oakley and colleagues [ 25*,26*] undertook a search for exttagenic suppressors of a P-tubulin mutation and discovered a gene (m.@4), the product of which corresponds to an altogether new class of tubulin subunits. The sequence of this tubulin isotype only shows approximately 30% homology of amino acid sequence to either a- or P-tubulin, considerably lower than the 3642% identity that all other a- and P-tubulins reported to date are known to share with each other. Because the m@A product does not resemble either a- or ptubulin very closely, it has been designated as a new tubulin class called y-tubulin. Disruption of the m@4 gene is lethal as it strongly inhibits nuclear division and thus is essential for cell function in Rspergillus. Antibodies generated to a unique peptide sequence of y-tubulin label the spindle pole bodies and give faint staining of the central spindle and certain cytoplasmic spots (Fig. 3). Because of the unique location of y-tubulin in the spindle pole bodies, the authors conclude that it may play a role in microtubule nucleation. In addition, because it was identified as a (3-tubulin-interacting protein, the authors speculate that it is possible that the P-ends of microtubules are associated with the spindle pole body. Thus, y-tubutin may not only nucleate microtubules but also establish the structural polarity of microtubules in the cell. Work is presently underway to identify y-tubulin genes in vertebrate cells and thereby determine if the patterns of expression, distribution and mode of action of y-tubulin are conserved in other organisms.
Studies on purified tubulin isotypes: indication of the mechanisms by which isotypes confer specificity of function
A very successful strategy for evaluating the functional significance of tub&n isotypes has been to directly purify the proteins and examine their properties in vitro. Even more importantly, this approach may allow one to compare the mechanisms of assembly of different .isotypes [nucleation, growth, dynamics, and interaction with microtubule-associated proteins (MAPS)], to quantify these differences, and to predict the consequences of their expression in vivo. Among the best-studied examples are the tubulins from chicken erythrocytes and Antarctic fish, and preparations of isotype-depleted tubulin from mammalian brain. Together with other collaborators, Rothwell and myself demonstrated that chicken erythrocytes contain a unique @ubulin isotype that is associated with the marginal band in these cells [ 27.1. This divergent vertebrate isotype is expressed only in blood cells and differs from brain tubulin in many important respects. Erythrocyte tubulin is composed predominantly of class VI P-tubulin, an isotype not found in brain, whereas brain tubulin
Functions
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Murphy
that the dilferences in functional capacities of isotypes are reflected in their ability to bind MAPS with different affinities. By Scatchard analysis of equilibrium binding, we found that MAP-2 binds to microtubules with a K,-J of 0.1 @, with the binding to brain microtubules being twofold higher than to microtubules composed of erythrocyte tubulin. Thus, a potential for different binding afhmities for hL4F’s exists and may eventually provide insights into the functional significance of tubulin isotypes.
Fig. 3. Localization of y-tubulin in Aspergillus by immunofluorescence microscopy. Cermlings of Aspergillus are shown in phase contrast (a) and after double immunofluorescence staining with antibodies to reveal a-tubulin (b) and y-tubulin (c)*The y-tubulincontaining spots at the spindle poles are visible. Published with permission [25-l.
consists of a complex mixture of isotypes, including isotype classes I, II, III and IV. In comparison with brain microtubules, erythrocyte microtubules are more stable and less dynamic, longer and more flexible, and minimaLly af fected by large changes in ionic strength and pH. Knowing the assembly rate constants for brain and etythrocyte tubulin, we successfully predicted the composition of rncrotubules assembled from mixtures of these two tubulin isotypes [ 28.1. The two isotypes copolymerized eficiently, but when they polymerized under conditions that were close to steady state, microtubule copolymers also exhibited gradients in the ratio of tubulin isotypes along their lengths that differed at their ends by as much as 2530% (Fig. 4). The observations agree with previous reports on the homogeneous distribution of tubulin isoforms in cells and suggest that tubulin subunits by themselves have a limited capacity to segregate into distinct subsets of microtubules. Recently, we used brain and erythrocyte microtubules to compare the binding affinities of chicken brain MAP-2. Such a comparison allows one to test the hypothesis
Another remarkable isotype is the divergent u-subunit found in the brain tissue of Antarctic fish. As the microtubules in these organisms exist at or close to 0°C an extremely efficient mechanism of stabilization is necessary to counteract the instability of microtubules at low temperature. This is apparently accomplished through the expression of a highly divergent a-subunit that maintains a critical concentration for assembly comparable to that found in other organisms, but which polymerizes efficiently even at 5°C [29*]. The divergent a-tubulin comprises 55-65% of the total u-subunits and, although it is not known if the low-temperature isotype is the product of a unique gene or the product of extensive post-translational modifications, the range of temperatures supporting efficient tubulin assembly is considerably reduced. A comparison of the thermodynamic parameters of fish and mammalian tubulins by van t’Hoff analysis revealed sizeable increases in the entropy and enthalpy terms that determine the microtubule equilibrium for fish tubulin, indicating that it is the increased level of hydrophobic interactions between subunits that allows these microtubules to exist at low temperatures [ 30.1. Of all of the tubufin variants that have been isolated and studied so far, Antarctic fish tubulin is biochemically and biophysicalfy one of the most divergent, and future work should provide valuable insights on the role of a-tubulin in microtubule assembly. Even within the complex mixture of subunits that comprise mammalian ‘brain tub&n’, individual isotypes differ markedly in their in vitro assembly properties. In a recent paper, Banejee et al. [31*] used affinity chromatography and a specific monoclonal antibody to remove a class III, neuron-specific P-tub&n isotype from preparations of purified, bovine brain tubulin. Surprisingly, the isotype-depleted tubulin polymerized twice as rapidly and to a greater extent than the whole unfractionated sample when examined in a MAP-containing assembly buffer lacking stabilizers such as glycerol or taxol. The authors concluded that even moderate amounts of certain isotypes (class III P-tubulin comprises approximately 25% of the total cycled protein) are capable of influencing the properties of microtubule assembly. This finding also strongly suggests that the presence of the class III isotype may be important for the normal functioning of microtubules in neurons. Two such possibilities are that the presence of class LU tubulin (which retards self-assembly) may reduce the potential for spontaneous nucleation to control microtubule number, or that the isotype is segregated within neurons to determine the regional distribution of MAPS, a phenomenon that has already been well documented.
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*
Reversible tyrosination of tubulin: a post-translational modification that may regulate the dynamic activity of microtubules
several post-translational modifications of tubulin have now been identified, including tyrosination, acetylation, phosphorylation and, most recently, polyglutamylation. With the exception of phosphorylation, these modilications are notable for their unusual nature and for the fact they affect diverse but specific sites on a- and P-tubulin subunits. Whereas little is known yet about the functional sigriihcance of these modifications, including the newly discovered polyglutamylation [32*], there is compelling evidence that both tyrosination and acetylation of a-tubulin are associated with an increased degree of mi crotubule stability in uivo. In particular, the cause and effect relationship between tyrosination and microtubule stability has been analyzed in detail and has yielded some surprising results. Tubulin tyrosination refers to the reversible addition of tyrosine to the tyrosine-free carboxy-terminus of a-tubulin. The modilication (both addition and removal) occurs on the polymer itself and is controlled by tubulin-tyrosine ligase and a tubulin-carboxypeptidase, which respectively add and remove terminal tyrosine residues from a-tubulin. Detyrosination of tyrosine-containing tubulin (Qr-tubulin) produces tubulin ending in a glutamic acid residue (Glu-tub&n), and it is this detyrosinating activity that is associated with enhanced polymer stability. This modification occurs in a wide variety of organisms and cell types and correlates with changes observed in microtubule dynamic activities associated with mitosis and cell dilferentiation; it is thus believed to be ubiquitous and fundamental to understanding the control of microtubule function. The relationship of tyrosination with decreased stability is particularly evident in the growth cones of neurons [ 33*], where cellular domains with increased motility exhibit microtubules with greater amounts of Tyrtubulin. This correlation was made even more compelling in a recent study of muscle cell differentiation by Gundersen et al. [34-l, who showed that detyrosination to Glu-microtubules (MTs) closely paralleled a decrease in the dynamic activity of microtubules in syncytia resulting from myoblast fusion. Any doubts about this correlation were removed by Baas and Black [35*] who demonstrated approximately a 50fold difference in the nocodzole sensitivity of Glu- versus TyrMTs in the axons of cultured sympathetic neurons.
Fig. 4. Immuno-electron microscopy of a microtubule co-polymer that has elongated from the end of an erythrocyte microtubule seed in a preparation containing equal amounts of chicken brain and erythrocyte tubulin subunits. Labeling was obtained using an antibody, specific for the class VI p subunit from chicken erythrocytes. Although these divergent isotypes copolymerize efficiently, their capacity to assemble is not equivalent, as shown by the changing ratio of tubulin isotypes along the length of the elongating copolymer. Published with permission I28*1.
Functions
Immunofluorescence analyses clearly showed that labile microtubules were enriched in Tyr-tubulin, whereas stable microtubules contained only detyrosinated and acetylated a-tubulin, and that the relative proportions of the biochemical species corresponded precisely to the observed ratio of labile and stable microtubules in the axons. Baas and Black also showed that drug-resistant GluMTs appeared to nucleate the regrowth of labile Tyr-MT polymers, leading the authors to suggest that Glu-MTs might regulate microtubule dynamics in neurons. Detyrosination, however, does not increase microtubule stability directly.
Detyrosination: tubulin-stabilizing
indirect involvement mechanisms
in other
In a previous study by Khawaja et al [36*], it was noted that cultured kidney cells contain distinct subsets of Gluand Tyr-MTs that diifer in their sensitivity to microtubuledepolymerizing drugs. When permeabilized cell models, however, were treated with carboxypeptidase A to generate Glu-MTs from endogenous ‘Qr-MT’s, no change in the resistance of microtubules to dilution was observed, suggesting that detyrosination alone was insticient to confer enhanced stability. Webster et al [37*] have now examined the effect of detyrosination on microtubules in living fibroblasts by injecting an antibody to tubulin-tyrosine ligase, a treatment that blocks enzyme activity and results in the virtual elimination of ‘Iyr-MTs and the accumulation of Glu-MTs in the cell. Upon measuring microtubule dynamics in these cells, it was found that the turnover time was approximately 3 min in both treated and untreated cells, strongly indicating that detyrosination itself does not stabilize microtubules. The authors suggest that microtubule-capping proteins or MAPS could be the components that directly confer stability. Alternatively, the function of detyrosination may be to differentiate further, stable microtubules for specilic activities or to allow interaction with Glu-specific MAPS to maintain the stability. The observations of Baas and Black [35*] on the stability of Glu-MTs in axons support these ideas. It is possible that the factors that stabilize Glu-MTs (neuronal MARS) are absent or present in reduced amounts in cultured fibroblasts, but are more concentrated in axons. An interesting experiment, therefore, would be to microinject the antibody to tubulin-tyrosine ligase into neuronal cells. With the rapid pace of experiments in this field, we should soon have an answer.
Conclusions The papers reviewed here provide important new insights into the diversity and function of tubulin isofonns in cells. The molecular basis of isoform diversity and the
of tubulh
isoforms
Murphy
patterns of expression of tubulin isoforms are now understood in considerable detail. More importantly, the ‘functional significance of isotypes’ has come to mean the adaptation of the physical and biochemical properties of an isotype (or group of isotypes) to particular cellular environments, while the previous concept of one isotype-one function no longer seems applicable. A testis-specific P-tubulin isotype in DrnuqMa has emerged as a pArime example: the same isotype performs multiple functions in developing spennatocytes but cannot be replaced by another dissimilar isotype. Nevertheless, the extent to which isotypes have evolved to function in specialized environments remains unclear, and the physical
References
and recommended
reading
Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstanding interest 1.
FUL.TON C, SIMPSON PA: Selective Synthesis and Utiliaation of Flagellar Tubulin The Multi-Tubulin Hypothesis. In Cdl MotiUtyedited by Goldman R, Pollard T, Rosenbaum J [book]. New York: Cold Spring Harbor laboratories, 1976, pp 987-1005.
2.
STEPHENS
3.
SUU~VANKF: Struchue and Utuization of Tubulin Annu Rev Gel1 Bioll988, 4687-716.
4. .
RE: Structural Chemistry of the Axoneme: Evidence for Chemically and Functionally Unique Tubulin Dimem ln Outer Fibers. In MoIecules and Gd Movement edited by Inoue S, Stephens REI [book]. New York: Raven Press, 1975, pp 181-206.
Isotypes.
JOSHIHC, CIXVE~ DW: Diversity Among Tubulin Subunits: Toward What Functional End? Cell Motil Cytaske&ton 1990, 16:15+163. A useful, up-to-date review that focuses on tedundancyversus specilicity of function for tub&n isotypes and that includes references on the heterogeneity and disttibution of tubulin isofomw.
49
50
Cytoplasm 5.
.
and cell motility
0~ MT, ARAI T, HAMAGIJCHIY: Heterogeneity of Microtubules in Dividing Sea Urchin Fggs Revealed by Immunotluorescence Microscopy: Spindle Composed of Tubulin Isotypes Different tral Microtubules. Cell Moth Cyrarkelern
Microtubules from Those
are of As-
1990, 16:239-250. Striking examples of distinct tubukn isotype segregation within the mitcitic spindle by imrnunoguorescence microscopy. D~NOU~ET P, FIUJA~~EAUG, DE NECHAUD B, GROS F, DI GIA~RERARDINOL DiiferentiaI Axonai Transport of Isotubulins in the Motor Axons of the Rat Sciatic Nerve. / Cell Bid 1989, 108:%5-971. Several tubuIin isoforms are demonstrated to show diiTerent distdbutions in soluble and cytoskeletai fractions associated with the slow components of axonal transport 6.
.
7.
BURGO~~E RD, CAMBRAY-DEMN MA, LEWIS SA, SARKARS, COWAN NJz DitTerentiaI Distribution of g-TubuIin Isotypes in Cerebellum. EMSO] 1988, 72311-2319.
8.
LEwrs SA, COWAN NJ Complex Regulation and Functional Versatility of Mammalian (1- and g-tubuRn Isotypes During the
Ditlkrentiation
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and
Muscle
CelJs.
J Cell Biol
1988, 106:20232033.
Tubulin Upstream Sequence Causes a Reduction fJ-Tubulin Level but Has No Effect on Microtubule Cell Motil C)&xkeleton 1990, 16:214-220.
in ber~4
Function.
A tub&n isotype normaiiy associated with spore formation accumulates to 50% but has no effect on microtubuie function when placed under the control of the constimtive g-tub&n gene. This paper includes carefui quantitation of protein synthesis. 18.
MAY GS: The Highly Divergent b-TubuRns of Aspergfllus nfdulans are Functionally Interchangeable. J Cdl Bioll989, 109:2267-2274. The minor g-tubuiin isotype normaUy associated with spore formation was manipulated to become the only Functional gtubuiin in hpeqillus No elkt on cell function was observed. .
SISODIASS. GAY DA, CLEVEIANDDW: In ofvo Discrimination Among g-Tubuiin Isotypes: Selective Degradation of a lope IV g-TubuIin Isotype Following Overexpression in Cultured Animal Cells. New Biologfit 1990, 266-76. A careful and interesting paper that analyzes the problem of tubulin isotype expression in transfected cells. A class IV g-tubuiin is synthesized but rapidiy degraded during cell expression, suggesting the presence of an additional mechanism for regulating expression in an isotype-speciiic manner.
19. .
9.
IDPATA MA Cuzv~lruv~ DW: In ofvo Microtubules are Copolymers of AvaiIabIe g-Tubuiin Isotypes: Localization of Each of Six Vertebrate g-TubuIin Isotypes Using PolycIonaI Antibodies F&cited by Synthetic Peptide Antigens. J Cell Biol 1987, 105:1707-1720.
BURKED, GA~DA~KAP, HARTWELLL Dominant Effects of Tubuiin Overexpression in Saccharomyces Cerevfsiae. Mol CeN Biol 1989, 9:104+1059. When a- and g-tubuiin are overexpressed in yeast, excess a-tubuiin is not detrimental but overexpression of 8tubuiin, or of both a- and 8tubuiins together, is lethal.
10.
AX DJ, REMOLONANM: Tubuhn Isotype Usage In Vfva A Unique Spatial Distribution of tbe Minor Neuronal-Specific g-TubuRn Isotype in Pbeochromocytoma CeiIs. Dev Biol
21. .
.
1989,
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Shows that diiferences exist in the function of tubuiin isotypes in differentiated cells: ciass RI g-tubuiin is not incorporated into microtubules in diiIerentiating PC12 ceiis. 11. .
JOSHIHC, CLEVELANDDW: Differential Utilization of B-Tubulin Isotypes in DiIferentiating Neurites. J Cdl Biol 1989, 109z663673. Fii 8-tubukn isotypes exhibit diIferences in synthesis rates, assembly, and intraceUuIar localization in diIIerentiating neudtes of ~~12 ceiis.
12.
Sutuv~~ RF, CLEVELANDDW: IdentiIIcation of Conserved Isotype-DeIining Variable Region Sequences for Four Vertebrate p-Tubulin Pokypeptide Classes. Proc Natl Acud Sci USI 1986, 83:4327-4331.
13.
WANG D, V~UXANIX 4 LEWIS Sq COWAN NJ: The Mammalian g-Tub&n Repertoire: Hematopoietic Expression of a Novel, Heterologous g-TubuRn Isotype. J Cell Biol 1986, 103:19031910.
14.
RAPPEC: Genetics of Microtubule 99:1-10.
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15. .
SAVAGEC, HAMEuN M, Cu~orn JG, COUL~~NA, AIBERT~ONDG, CHALFIEM: met- 7 is a g-TubuIin Gene Required for tbe Production of 15Protofdament Microtubuies in Caenorhabditfs elegans Genes Dev 1989, 3870-881. A dramatic example of the specificity of g-tubuiin function in Cueno-itis ekgans Mutation in the g-tubuiin gene, mer-7, results in touch-insensitive animals that lack microtubules in their six touchreceptor neurons. 16. .
SAWADAT, CAB~U.LF: Expression and Function of g-TubuIin Isotypes in Chinese Hamster Ovary Cells. J Biol Cbem 1989, 264:30133020. The authors show by 2-D gel electrophoresis and immunoiluorescence microscopy that three distinct g-tub&n isotypes in CHO cells are used interchangeabiy.
20. .
KATZW, WEINSTEINB, SOLOMONF: Regulation of Tubulin LeveIs and MIcrotubule Assembly in Saccharomyces cenwfsfae: Consequences of Altered TubuRn Gene Copy Number. Mol Cell Bid 1990, 10:5286-5294. An interesting paper showing that cells function normaiiy on 50% of the normal tubuiin level and that strains containing extra copies of tubuiin genes tubuiin downreguiate their tubuiin levels. The data support a model for mbuiin dimer formation that depends on the rate of g-tubuiin synthesis. 22. .
WEINSTEIN B, SOLOMON F: Phenotypic Consequences of TubuIin Overproduction in Saccharomyces Cerwfsfae. Differences between Alpha- and Beta-TubuRn. Mol Cell Biol 1990, 10:5296-5304. Overexpression of P-tub&n (as opposed to overexpression of a-tubuiin or to co.overexpression of both a- and 8tubuiin) is uniquely toxic and leads to rapid disassembly of endogenous microtubules and decreased viability. HOYLE HD, RWF EC: Two Drosophila Beta TubuIin Isoforms are not Functionally Equivalent. J Cell Biol 19$X1, 111:1009-1026. An impressive and comprehensive study employing genetics, microscopy, and biochemistry to show that two 8.Nbuiin isotypes in Drosophila are not functiona& equivalent. 23.
..
24. .
PREXO’IT AR, FOSTERKE, WARNRM, Guu K: Incorporation of Tubuiin from an EvolutionariIy Diverse Source, Pbysarum Polycephalum, into tbe Microtubules of a Mammalian CeU. J Cell Sci 1989, 92:59%05. One of the first demonstrations is that a threshold level of Nbuiin required is to observe alteration of microtubule function in microinjected cells. OAKLEYBR, OAKIXY CE, YWN Y, JUNGMK: Gamma-TubuIin is a Component of tbe Spindle Pole Body that is Essential for Microtubule Function in Aspeqiflus Nfdufans Cell 1990, 61:1289-1301. A new chss of tubuiin called y-tubuiin is localized in Azpeqillus to the spindle pole body. suggesting a role in nucleation and mitosis, and possibly the determination of polarity of nucleated microtubuies. 25. .
26.
17. .
MAY GS, WARINGRB, Moiuus NR: Increasing h&Z 8-TubuIin Synthesis by placing it Under the Control of a benA 8-
.
OAKLEY CE, OAJUEY BR Identification of Gamma-Tubuiin, a New Member of the TubuIin Superfamily Encoded by mfpA Gene of Aspeqfllus nfdulans Nature 1989, 338:662&$X
Functions Dramatic disclosure of the sequence for a new class of tubulins called y-tubulin. ROT~VEU SW, GRASSERWA, MURPHYDB: Tubulin Variants Exhibit DifTerent Assembly Properties. Ann NY&ad Sci 1986, 466:103110. Comparison of the biochemical properties and assembly characteristics, includ.ing the assembly rate constants, of brain and erythrocyte tubulin. 27.
.
BAKERHN, RO’IHWXUSW, GRASSER WA, Wus ICI’, MURPHYDB: . Copolymerization of Two Distinct Tubulin Isotypes During Microtuble Assembly in vitro. / Cell Biol 1990, 110:97-104. Mixtures of divergent tubulin isotypes co-assemble in vitro but the polymers exhibit gradients in the ratio of rubulin isotypes along their lengths. It is estimated that isotype segregation in uivo would require specialhed intracellular conditions. 28.
of tubulin
isoforms
Murphy
ROB~ONSJ, BURGOYNERD: Dierencial Localization of Tyrosinated, Detyrosinated, and Acetylated u-Tubulins in Neurites and Growth Cones of Dorsal Root Ganglion Neurons. Cell Motil Cytmkeleton 1989, 12:273282. The tyrosinated form of a-tubulin is shown to be enriched in labile microtubules found in the growth cones of cultured neurons. 33.
.
GUNDER~ENGG, KHAWAJAS, BULINSKIJC: Generation of a Stable, Posttranslationally Modified hlicrotubule Array is an Early Event in Myogenic Dilferentiation. / cell Bfof 1989, 109:22732288. This study correlates an increase in the detyrosination of microtubules with a decrease in microtubule dynamics in difTerentiating myoblasts. 34.
.
.
BAAS PW, BLACK MM: Individual Microtubules in the Axon Consist of Domains that Differ in both Composition and Stability. J Ceff Biol 1990, 111:495-509. Provides the most convincing demonstration so far for the correlation between detyrosinated microtubules and polymer stability, and estimates the difference to be approximately 50.fold.
DEIIUCH HW, JOHN~N m MARCHESE-RAGONA SP: Polymerization of Antarctic Fish Tubulins at Low Temperatures: Energetic Aspects. Biodemfshy 1989, 28:10085-10093. An interesting thermodynamic approach to describing (and explaining) the differences in the assembly of Antarctic tish tubulin.
KHAWAJAS, GUNDER~ENGG, BULINSKIJC: Enhanced Stability of Microtubules Enriched in Detyrosinated Tubulin is not a Direct Function of Detyrosination Level J Cd Bid 1988, 106:141-149. The authors treat cell models with carboxypeptidase to generate detyrosinated microtubules but find that there is no increased resistance of the modified polymers to dilution.
l-hm RH, DETIUCH HW: Dynamics of Antarctic Fiih Microtubules at Low Temperatures. Biodemfshy 1989, 28:508+5095. Pulse-chase experiments are used to show that Antarctic tish tubulin is dynamic under near-physiological conditions at 5°C. 29.
30.
.
31.
.
BANERJEEA, ROACH MC, TRCKA P, LUDUENARF: increased Microtubule Assembly in Bovine Brain Tub&n Lacking the Qpe Ill isotype of &Tubulin. J Biol Cbem 1990, 265:1794-1799.
Removal of a class II1 p-tubulin isotype from preparations of bovine brain tub&n results in preparations that polymerize faster and to a greater extent. The work shows that individual isotypes can affect microtubule assembly, even in complex mixtures of tubulin isotypes in
35.
.
36.
.
WEBS~FRDR, WEH~ANDJ, WEBERK, Bo~ls~ GG: Detyrosination of Alpha Tubulin does not Stabiie Microtubules in ufua J cell Bid 1990, 111:113122. The authors inject antibody to tubulin-tyrosine ligase into fibroblasts to generate Glu-MTs and find that both Glu-MTs and Tyr+lTs show the same level of resistance to anti-MT drugs. 37.
.
vih-a
EDDE B, ROSSIERJ, LE CAER J-P, DESBRWERE~E, GROS F, DENOUIET P: Posttranslational Glutamylation of a-Tubulin. Science 1990, 247:83+5. A surprising, new post-translational modification of tubulin found in neurons. 32.
.
DB Murphy, Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 N Wolfe Street, Baltimore, Maryland 21205, USA
51