To determine the role of α4-tubulin in microtubule formation and how E82K-α4-tubulin exerts its toxic effect, two sets of in vitro microtubule polymerization assays were carried out. Extracts were prepared from 0-hour- to 1-hour-old eggs of +/- females (control) and Kavar18c/- virgin females, and polymerization of tubulin was induced in the egg extracts by addition of GTP. The forming microtubules were stabilized with taxol. The microtubules were pelleted and the types of proteins in the pellets were separated by SDS-PAGE. Western blot analysis was used to detect α1-tubulin and α3-tubulin with DM1A antibody, and α4-tubulin with its specific anti-α4-tubulin antibody. To distinguish the different α-tubulin isoforms fluorescently labeled secondary antibodies were used. Results of the in vitro tubulin-polymerization show that egg cytoplasm of the Kavar18c/- and the +/- females contain roughly equal amounts of total α-tubulin. Western blot analysis did not detect α-tubulin in the supernatant, indicating that both the α4-tubulin and E82K-α4-tubulin become incorporated into the forming microtubules. Apparently, , E82K-α4-tubulin does not exert its toxic effect by blocking microtubule formation: it does become incorporated into the microtubules but, however, keeps them short (Venkei, 2006).
To confirm this possibility, extracts were prepared from eggs of +/- females (control), Kavar18c/- and kavar0/- females, and tubulin polymerization was induced in the presence as well as in the absence of taxol. Whereas in the absence of taxol only nucleation occurs and no microtubules form in extract of the Kavar18c/- females, microtubules do form in these extracts when taxol is present - although they are shorter than those in the control females (4.6 µm versus 9.9 µ), showing that taxol, a microtubule stabilizing agent, partially compensates the microtubule-destabilizing action of E82K-α4-tubulin (Venkei, 2006).
Unexpectedly, the microtubules grew to an almost equal size in egg extract of control (+/-) and kavar0/- females. It appears that equally long microtubules form if the time available for tubulin polymerization is sufficiently long, irrespectively of the presence or absence of α4-tubulin (Venkei, 2006).
To test the effect of α4-tubulin on microtubule polymerization, the kinetics of tubulin polymerization in egg extracts of virgin +/- (control) and kavar0/- females was studied, and the length-distribution of the forming microtubules plotted against the time allowed for tubulin polymerization was analyzed. There is a remarkable difference in kinetics of microtubule formation in the two egg extracts. By 2 minutes after the initiation of tubulin polymerization, nucleation seeds appear in both preparations and their numbers do not differ significantly over a field of view. However, whereas microtubules with measurable length appear after 5 minutes in the control, at this time still only nucleation seeds are present in the kavar0/- egg extract. During the early phase of elongation even the longest microtubules forming in absence α4-tubulin are shorter than most of the microtubules forming in the presence of α4-tubulin. Analysis of polymerization kinetics curves revealed very similar average microtubule growth rates. However, whereas in the control preparations the maximum rate is reached by 15 minutes, it took 25 minutes in absence of α4-tubulin, indicating that α4-tubulin accelerates the formation of microtubules. The difference in the distribution of microtubules of different length in the two types of preparations gradually decreases and disappears by about 30 minutes. Results of the in vitro tubulin-polymerization kinetics indicate that α4-tubulin is required for rapid tubulin polymerization (Venkei, 2006).
Microtubules (MTs) are essential for cell division, shape, intracellular transport, and polarity. MT stability is regulated by many factors, including MT-associated proteins and proteins controlling the amount of free tubulin heterodimers available for polymerization. Tubulin-binding cofactors are potential key regulators of free tubulin concentration, since they are required for alpha-beta-tubulin dimerization in vitro. This paper shows that mutation of the Drosophila tubulin-binding cofactor B (TBCB) affects the levels of both alpha- and beta-tubulins and dramatically destabilizes the MT network in different fly tissues. However, this study found that dTBCB is dispensable for the early MT-dependent steps of oogenesis, including cell division, and that dTBCB is not required for mitosis in several tissues. In striking contrast, the absence of dTBCB during later stages of oogenesis causes major defects in cell polarity. dTBCB is required for the polarized localization of the axis-determining mRNAs within the oocyte and for the apico-basal polarity of the surrounding follicle cells. These results establish a developmental function for the dTBCB gene that is essential for viability and MT-dependent cell polarity, but not cell division (Baffet, 2012).
The TBCB protein is part of an evolutionarily conserved tubulin-folding pathway crucial for the formation of the tubulin heterodimers. Indeed, TBCs are thought to regulate MT dynamics by modulating the concentration of dimers available for polymerization (Lopez-Fanarraga, 2001). Using Drosophila as a model system, this study looked at the requirement for TBCB during development. dTBCB wsa shown to be required for MT integrity and is essential for viability, MT-associated transport, and cell polarity (Baffet, 2012).
TBCs are mostly cytoplasmic proteins, in accordance with their tubulin-chaperoning function. However, TBCE also accumulates at the Golgi apparatus of motor neurons, where it is essential for axonal tubulin routing (Schaefer, 2007). TBCD is concentrated at the centrosome, midbody, and cell junctions, where it participates in centriologenesis, spindle organization, cell abscission, and epithelial cell structure (Cunningham, 2008; Shultz, 2008; Fanarraga, 2010). The current results indicate that dTBCB is largely cytoplasmic but also partly overlaps with MTs, corroborating some previous studies in different species. This partial colocalization is particularly clear in developing embryos and in S2 cells. Such distribution is not observed with dTBCE, the α-TBC interacting with TBCB. This possibly indicates that dTBCB fulfills additional functions independent of the other TBCs. Further experiments willbe required to address these potential functions (Baffet, 2012).
dTBCB is required for the integrity of the MT network in different tissues. This is consistent with data obtained in yeast knockout, but not in mammalian knockdown cells. TBCB has been shown, together with TBCE, to stimulate tubulin heterodimer dissociation in vitro. In accordance with this, it was found that strong dTBCB overexpression in S2 cells triggers MT depletion. The increased MT density observed in mammalian microglia, after TBCB depletion by RNAi, might therefore be due to a reduced tubulin heterodimer dissociation. The different phenotypes reported in previous studies might be due to species specificity or to the efficiency of the technical approaches, with the mutational approach probably being more efficient than RNAi. It is proposed that severely impaired tubulin dimerization is the cause of the strong MT defects in yeast and Drosophila. In mammalian knockdown cells, however, a sufficient pool of tubulin heterodimers is produced, and only the dissociation of the tubulin heterodimer seems affected. Altogether these results suggest that TBCB is required for tubulin dimerization and for tubulin heterodimer dissociation in vivo. The latter function might be more sensitive to variations in TBCB concentration and could be used to fine-tune MT polymerization in a spatiotemporal manner (Baffet, 2012).
The α-TBC TBCE, a direct partner of TBCB, is required for viability and MT formation in Drosophila but does not affect α-tubulin levels (Jin, 2009). In dTBCB1 mutant egg chambers and larvae, however, a strong reduction was observed of α-tubulin levels. This suggests that the monomeric α-tubulins, which failed to bind dTBCB and to dimerize, are unstable. This is consistent with the fact that monomeric tubulins have been observed to be very unstable molecules in vitro. It was also observed that the β-tubulin levels were reduced in dTBCB1, although TBCB is known to associate specifically with α-tubulin. This suggests that the monomeric form of β-tubulin in Drosophila is unstable, even in the presence of its putative dedicated chaperone, dTBCA. It is interesting to note that knocking down mammalian TBCA also induces a decrease in both α- and β-tubulin levels. In dTBCB1 mutant tissues, the observed tubulin levels decrease most likely causes the MT destabilization. However, dTBCB does not seem absolutely required to form tubulin heterodimers in vivo, since wa significant pool of dimers is still present in mutant extracts. Overall the current results suggest that dTBCB enhances tubulin dimer formation in vivo to promote MT assembly (Baffet, 2012).
In plants and yeasts, TBCB is essential for cell division. In flies, dTBCB is expressed throughout cell cycle, and during mitosis it colocalizes to some extend with the MT spindle.Surprisingly, dTBCB knockout in diverse tissues does not prevent cell division, suggesting that dTBCB is not essential for mitosis. It was observed that mitotic spindles do form in mutant neuroblasts and allow mitosis, even if the overall cell cycle is slower than normal. Therefore the remaining levels of α- and β-tubulin are sufficient to fulfill cell division. Similarly, depletion of Drosophila TBCE decreases MT levels without precluding cell proliferation proceeding in embryos (Jin, 2009). Likewise, even though a null allele of α-tubulin 84B is cell-lethal, a hypomorphic mutation can affect larval viability without preventing cell proliferation). This suggests that mitosis in flies is a robust process, resistant to a significant decrease in α- and β-tubulin amounts. It is possible that, at least in Drosophila, TBCs are not essentials to sustain the MT polymerization level necessary for mitosis (Baffet, 2012).
This study has found that dTBCB is required for cell polarity in the ovary. In the oocyte and in the overlying epithelial follicle cells, the MT-based transport is particularly important for the asymmetric distribution of determinants. The drop in MT level observed in dTBCB mutant cells correlates with defects in localization of apico-lateral polarity proteins in epithelial cells, defects of mRNAs, and nucleus-polarized transport in oocyte at midoogenesis. However, low MT density does not trigger obvious polarity defects in early oogenesis polarization steps, since Orb accumulation occurs normally in mutant oocytes. It therefore seems that there is an interesting differential requirement for dTBCB between early and late stages of oogenesis that is not due to the persistence of the protein. This may point to a higher robustness of the MT-dependent processes in early stages of oogenesis: a partial depolymerization of the MT network may affect late but not early events. Early polarity maintenance could be due to the smaller size of the early cysts that probably need a lower MT-nucleation activity to produce a functional MT network (Baffet, 2012).
The simultaneous decreases of the expression levels of dTBCB and the ubiquitously expressed α-tubulin 84B prevent egg hatching and cause cell polarity defects in ovarian epithelial cells, suggesting that dTBCB regulates polarity most likely by controlling tubulin/MT integrity. Interestingly, this heteroallelic combination does not impair the formation/division of the follicle cell monolayer but affects its apico-basal polarity. These results confirm that mitotic cells are less sensitive to MT destabilization than interphasic cells, probably because of the much higher MT nucleation activity during mitosis. In accordance with this, it is interesting to note that nocodazol treatment depolymerizes inter phasic MT networks much more efficiently than mitotic spindle (Baffet, 2012).
In humans, the presence of a mutated α-tubulin defective for TBCB binding has been correlated with the brain malformation lissencephaly, while high dTBCB levels have been reported in breast tumors . The results of this study may be of significant importance in understanding the molecular basis of the development of these pathologies (Baffet, 2012).
To visualize α4-tubulin throughout the cleavage cycles, a polyclonal anti-α4-tubulin antibody was generated by making use of the unique 14 amino acid long C-terminal segment of α4-tubulin. This antibody is specific for α4-tubulin and does not recognize α1-tubulin and α3-tubulin, the evolutionally highly conserved and constitutively expressed isoforms. The monoclonal anti-α-tubulin antibody DM1A, however, recognizes α1-tubulin and α3-tubulin but not α4-tubulin and, thus, the two antibodies allow side-by-side detection of the different α-tubulin types (Venkei, 2006).
All three α-tubulin isoforms are components of all types of microtubules throughout the cleavage divisions. However, there is a significant enrichment of α4-tubulin in the so-called interpolar microtubules that embrace the nuclear envelope during interphase and early prophase, and push the daughter centrosomes along the nuclear perimeter to opposite poles (see also Scholey, 2003; Cytrynbaum, 2003). The α4-tubulin molecules are incorporated into the first embryonic spindle apparatus where they appear evenly distributed over the microtubules. Notably, E82K-α4-tubulin also becomes incorporated into the microtubule tassels in eggs of the Kavar18c/- females, however, it does not seem to support the formation of long microtubules (Venkei, 2006).
Two major alpha-tubulin isotypes are present during Drosophila embryogenesis: an evolutionarily divergent maternal isotype that is synthesized only in the ovary and deposited in the oocyte and a highly conserved constitutive isotype that is both maternally supplied and zygotically synthesized. A maternal isotype-specific antibody and a monoclonal antibody that recognizes both the maternal and constitutive isotypes were characterized and used to determine the distribution and abundance of alpha-tubulins during embryogenesis. Both isotypes are abundant and assemble into all classes of microtubules from the syncytial blastoderm stage until completion of germ band retraction. During subsequent development, however, the maternal isotype is retained only in the developing CNS, and later in a subset of connective fibers within the CNS. In contrast, total alpha-tubulin levels remain high in essentially all tissues throughout embryogenesis, indicating that most tissues selectively accumulate the constitutive isotype. To determine if selective accumulation of the constitutive isotype requires zygotic synthesis of this protein, mutant embryos that do not contain functional constitutive alpha-tubulin genes were examined. In these embryos, as in wild type, the maternal isotype decreases to background levels in tissues that retain high levels of the constitutive isotype. The constitutive isotype therefore appears to be more stable than the maternal isotype in most tissues. Differences in isotype stability may play an important role in determining the developmental pattern of isotype accumulation in Drosophila embryos (Theurkauf, 1992).
Transcripts from the four different Drosophila alpha-tubulin genes were detected by filter hybridization experiments that used subcloned fragments from each gene as hybridization probes. These hybridization experiments demonstrated that each gene is transcribed. All of the transcripts are found on polysomes and are long enough to encode an alpha-tubulin protein. The hybridization studies were extended to examine the developmental pattern of RNA concentrations. The concentration of RNAs from the alpha 2 and alpha 4 genes vary independently and dramatically, while those from alpha 1 and alpha 3 have parallel variations. It is concluded that at the RNA level of expression, two alpha-tubulin genes are regulated in parallel and two genes are not. It is hypothesized that the different concentration patterns reflect different functions for the protein products of each gene (Kalfayan, 1982).
Three alpha-tubulin proteins contribute to microtubules during oogenesis and early embryogenesis in Drosophila melanogaster: alpha TUB84B, alpha TUB84D, and alpha TUB67C. alpha TUB67C is unique in two respects. It is a structurally divergent alpha-tubulin, sharing only 67% amino acid identity with the generic isotypes alpha TUB84B and alpha TUB84D, and its expression is exclusively maternal. The genetic analysis of the alpha Tub67C gene described in this study demonstrates that alpha TUB67C is required for nuclear division in the oocyte and early embryo. Both meiosis and cleavage-stage mitoses are severely affected by mutations that result in a substantial decrease in the ratio of alpha TUB67C/alpha TUB84B+alpha TUB84D. A large increase in this ratio, achieved by increasing the gene dosage of alpha Tub67C, has little or no effect on meiosis, but severely disrupts mitotic spindle function. Thus, both classes of alpha-tubulin isotype present in the mature oocyte, alpha TUB67C and alpha TUB84B/84D, are essential for normal spindle function in early Drosophila development. These alpha-tubulins provide the first example of tubulin isotypes known to be coexpressed in wild-type animals whose encoded variation is required for the normal function of a microtubule array (Matthews, 1993; full text of article).
Drosophila melanogaster oocytes heterozygous for mutations in the alpha-tubulin 67C gene (alphatub67C) display defects in centromere positioning during prometaphase of meiosis I. The centromeres do not migrate to the poleward edges of the chromatin mass, and the chromatin fails to stretch during spindle lengthening. These results suggest that the poleward forces acting at the kinetochore are compromised in the alphatub67C mutants. Genetic studies demonstrate that these mutations also strongly and specifically decrease the fidelity of achiasmate chromosome segregation. Proper centromere orientation, chromatin elongation, and faithful segregation can all be restored by a decrease in the amount of the Nod chromokinesin. These results suggest that the accurate segregation of achiasmate chromosomes requires the proper balancing of forces acting on the chromosomes during prometaphase (Matthies, 1999; full test of article).
The dominant-negative female-sterile KavarD mutations and their revertant kavarr alleles identify the alphaTubulin67C gene of Drosophila melanogaster, which codes for the maternally provided alpha4-tubulin isoform. The mutations result in the formation of monopolar, collapsed spindles (each with two nearby centrosomes, a tassel of microtubules and overcondensed chromosomes), thus revealing a novel function for alpha4-tubulin in spindle maintenance and elongation. Molecular features of the two KavarD alleles and a kavarnull allele are described and models for their actions are discussed (Venkei, 2005).
Eggs of the Kavar18c/+ females are fertilized as in wild type, the centrosome divides and daughter centrosomes form. In wild-type embryos the daughter centrosomes move apart to opposite poles over the perimeter of the nuclear envelope and form a spindle. In eggs of the Kavar18c/+ females separation of the daughter centrosomes comes to a standstill when they are 3-4 µm apart and, although the centrosomes nucleate microtubules, they remain short (at 5-8 µm), straight and form a tassel that appears as a monopolar spindle (Venkei, 2005). Embryogenesis is terminated at this point inside eggs of the Kavar18c/+ females irrespective of the genotype of the embryo (Venkei, 2006).
Very similar, if not identical, defects emerge inside eggs of the kavar0/- females, which lack functional αTub67C genes (Venkei, 2005). The mutant phenotype, i.e. the formation of a single monopolar spindle in every egg of the Kavar18c/+, the kavar0/- and the Kavar18c/- females may originate through failure of the daughter centrosomes to separate or the collapse of the first cleavage spindle. This implies involvement of α4-tubulin in separation of the daughter centrosomes or in maintenance and/or elongation of at least the first cleavage spindle. The similar defects seen in eggs of the Kavar18c/+ and the kavar0/- females show that α4-tubulin and E82K-α4-tubulin molecules participate in the same process, and since E82K-α4-tubulin hinders function of α4-tubulin, Kavar18c is a dominant-negative mutation (Venkei, 2006).
To reveal the role of α4-tubulin, small cytoplasm samples from eggs of wild-type (control) and Kavar18c/- hemizygous females were injected into live embryos whose microtubules were highlighted by GFP-tagged α-tubulin. Two types of cytoplasm injections were done. In the first set of injections, the detailed effect of the injected cytoplasm was not followed over time. The injected embryos were engaged in the seventh to ninth cleavage cycle. Toxicity of the Kavar18c/- derived egg cytoplasm was apparent: while larvae hatched from almost all of the 185 embryos injected with wild-type egg cytoplasm, not a single larva hatched from the 138 embryos that had been injected with E82K-α4-tubulin-containing cytoplasm. In the second set of experiments, the cytoplasm samples were injected into embryos in the tenth to eleventh cleavage cycle that had their nuclei already in the egg cortex and the effects of the injected cytoplasm was analyzed in time-lapse optical sections. In this experiment 20 embryos were injected with-wild type egg cytoplasm, and 30 embryos with cytoplasm from Kavar18c/- females. The Kavar18c/- egg cytoplasm had no effect on the already established mitotic spindles and did not disturb events of metaphase, anaphase or telophase. The observation excludes the possibility that the monopolar spindles originate through spindle collapse (Venkei, 2006).
Injection of cytoplasm containing E82K-α4-tubulin invariably slowed down the separation of daughter centrosomes. Depending on - most probably - the local concentration of E82K-α4-tubulin, changes in the microtubule formation pattern were abnormal: in about 50% of the injected embryos the partially separated centrosomes nucleated microtubules that remained short and straight, and spindles never formed over the affected nuclei. The defect is very similar to that seen inside eggs of the Kavar18c/+ females. In less severe cases, the slowed down centrosome failed to space the nuclei properly and as a consequence tripolar spindles formed by recruiting a centrosome of the adjoining nucleus (Venkei, 2006).
Surprisingly, the injected egg cytoplasm derived from Kavar18c/- females did not harm the 16 embryos that had accomplished the twelfth cleavage division or were engaged in the thirteenth (last) cleavage division. To clarify the unexpected effect of E82K-α4-tubulin, UAS-Kavar18c and (as control) UAS-αTub67C transgenes, which allow ectopic expression of E82K-α4-tubulin or normal α4-tubulin, were constructed, and expressed in different cell types with different tissue specific Gal4 drivers. Notice that, α4-tubulin is synthesized during oogenesis and is used in the course of early embryogenesis, and the αTub67C gene is not expressed throughout the larval and pupal life (Theurkauf, 1992). It is presumed that ectopic expression of E82K-α4-tubulin would cause an arrest in cell proliferation and that for example the elav-Gal4; UAS-Kavar18c combination (in which E82K-α4-tubulin appears, e.g. in the neuroblast cells) would not survive to adulthood. Similarly, the ey-Gal4; UAS-Kavar18c flies (in which E82K-α4-tubulin is produced in the eye discs) were expected not to have eyes. A set of Gal4 drivers was selected that ensured the production of E82K-α4-tubulin in several different cell types. In all crosses, the Gal4; UAS-Kavar18c zygotes developed to adult at the expected proportion without any delay and there was no indication of cell death or developmental abnormality. The αTub84B-Gal4 driver - in which Gal4 is produced under the promoter of the constitutively expressed α1-tubulin-encoding αTub84B gene - ensures production of E82K-α4-tubulin in all the cells at all developmental stages and yet, the αTub84B-Gal4; UAS-Kavar18c zygotes are fully viable. The lack of zygotic death or abnormal development is certainly not the consequence of nonfunctional UAS-Kavar18c transgene because when driven by the αTub84B-Gal4 or the female germ-line-specific nanos-Gal4 driver, females are sterile and their embryos showed defects described for the hypomorph αTub67C- mutant alleles (Matthews, 1993). In conclusion, although E82K-α4-tubulin was present in eye disc cells of late third instar ey-Gal4; UAS-Kavar18c larvae eyes of the developing adults were normal. The above result clearly shows that ectopic expression of E82K-α4-tubulin does not reduce cell viability (Venkei, 2006).
A possible explanation for nontoxicity of the ectopically expressed E82K-α4-tubulin is that it is not incorporated into the microtubules. To decide whether the ectopically expressed E82K-α4-tubulin (and also the wild-type α4-tubulin) molecules are incorporated into microtubules of neuroblasts and whether they affect the length of microtubules, elav-Gal4; UAS-Kavar18c larvae and, as a control, elav-Gal4; UAS-αTub67C, were generated; the ventral ganglia were dissected and the neuroblast cells were analyzed. E82K-α4-tubulin (like wild-type α4-tubulin) becomes incorporated into the spindle microtubules and does not alter spindle shape or size. The longest microtubules are 6-8 µm and appear fully functional (Venkei, 2006).
Reference names in red indicate recommended papers.
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Venkei, Z. and Szabad, J. (2005). The KavarD dominant female-sterile mutations of Drosophila reveal a role for the maternally provided alpha-tubulin4 isoform in cleavage spindle maintenance and elongation. Mol. Genet. Genomics 273(4): 283-9. 15864652
Venkei, Z., Gaspar, I., Toth, G. and Szabad, J. (2006). alpha4-Tubulin is involved in rapid formation of long microtubules to push apart the daughter centrosomes during earlyx Drosophila embryogenesis. J. Cell Sci. 119(Pt 15): 3238-48. 16847053
date revised: 25 May 2013
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