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Vol. 19, Issue 1, 368-377, January 2008
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-Tubulins in DrosophilaDepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706
Submitted August 19, 2007;
Revised October 25, 2007;
Accepted November 2, 2007
Monitoring Editor: Sandra Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Tubulin is an indispensable component of the animal centrosome and is required for proper microtubule organization. Within the cell, -tubulin exists in a multiprotein complex containing between two (some yeasts) and six or more (metazoa) additional highly conserved proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). -Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named the -tubulin ring complex (TuRC). Curiously, many organisms (including humans) have two distinct -tubulin genes. In Drosophila, where the two -tubulin isotypes have been studied most extensively, the -tubulin genes are developmentally regulated: the "maternal" -tubulin isotype (named Tub37CD according to its location on the genetic map) is expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the meiotic and early embryonic cleavages. The second -tubulin isotype (Tub23C) is ubiquitously expressed and persists in most of the cells of the adult fly. In those rare cases where both -tubulins coexist in the same cell, they show distinct subcellular distributions and cell-cycle-dependent changes: Tub37CD mainly localizes to the centrosome, where its levels vary only slightly with the cell cycle. In contrast, the level of Tub23C at the centrosome increases at the beginning of mitosis, and Tub23C also associates with spindle pole microtubules. Here, we show that Tub23C forms discrete complexes that closely resemble the complexes formed by Tub37CD. Surprisingly, however, Tub23C associates with a distinct, longer splice variant of Dgrip84. This may reflect a role for Dgrip84 in regulating the activity and/or the location of the -tubulin complexes formed with Tub37CD and Tub23C. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Although efforts to generate an inventory of centrosomal components have made great progress in recent years (for example, Andersen et al., 2003), the molecular organization of the centrosome itself is not well understood, and mechanistic descriptions of how centrosomal proteins contribute to microtubule formation and organization are yet to be obtained.
-Tubulin (Oakley and Oakley, 1989) is an indispensable component of the animal centrosome required for microtubule nucleation and organization in all eukaryotes (reviewed in Wiese and Zheng, 2006). Human -tubulin can restore viability to Schizosaccharomyces pombe lacking endogenous -tubulin (Horio and Oakley, 1994), suggesting that -tubulins are functionally conserved in phylogenetically distant organisms. Within the cell, -tubulin exists in a multiprotein complex containing between two (in the case of Saccharomyces cerevisiae) and five or more (S. pombe, Xenopus laevis, Drosophila melanogaster, and humans) additional proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). -Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named -tubulin ring complex (TuRC; Zheng et al., 1995). The non--tubulin TuRC components are themselves highly conserved (Murphy et al., 1998, 2001; Anders et al., 2006; Tassin et al., 1998; Martin et al., 1998; Haren et al., 2006; Lüders et al., 2006). One of the major subcomplexes of the TuRC, which has been named the -tubulin small complex (TuSC; Oegema et al., 1999), appears to be a tetramer composed of two molecules of -tubulin and one molecule each of two other Grips known as Spc97p and Spc98p in S. cerevisiae, Dgrip84 and Dgrip91 in Drosophila melanogaster, or GCP2 and GCP3 in mammals (reviewed in Wiese and Zheng, 2006). The tetramer was first described in S. cerevisiae (Knop and Schiebel, 1997), where it may be the only soluble -tubulin complex. The metazoan TuRC is thought to be composed of six or seven tetramers held together and capped by several additional proteins that copurify with -tubulin from Xenopus egg extracts or Drosophila embryos (Zheng et al., 1995; Oegema et al., 1999; Gunawardane et al., 2003), but subcomplexes between -tubulin and non-TuSC Grips may also contribute to TuRC structure (Gunawardane et al., 2000).
Consistent with a role for the TuRC in microtubule nucleation, -tubulin and its binding partners are localized to the PCM at the minus ends of the microtubules (Moritz et al., 1995a,b; reviewed in Wiese and Zheng, 2006; Moritz and Agard, 2001). However, the molecular mechanism(s) for the function of the TuRC in microtubule nucleation are still unclear. In addition to facilitating nucleation of microtubules, -tubulin also seems to play a role in interphase microtubule organization (Jung et al., 2001; Horio and Oakley, 2003), spindle assembly (Paluh et al., 2000; Prigozhina et al., 2001; Vogel and Snyder, 2000a,b; Sampaio et al., 2001; Barbosa et al., 2003; Lüders et al., 2006), and spindle assembly checkpoint regulation (Vardy and Toda, 2000; Hendrickson et al., 2001; Vardy et al., 2002; Müller et al., 2006; reviewed by Blagden and Glover, 2003). Little is known to date about how -tubulin or the Grips participate in each of these functions.
Curiously, the genomes of several organisms, including humans and flies, encode two distinct -tubulin genes (Zheng et al., 1991; Wilson et al., 1997; Tavosanis et al., 1997; Wise et al., 2000). In Drosophila, where the two -tubulin isotypes have been studied most extensively, the -tubulin genes are developmentally regulated: the "maternal" -tubulin isotype (named Tub37CD according to its location on the genetic map) is highly expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the early embryonic cleavages (Wilson et al., 1997; Llamazares et al., 1999). Consistent with this idea, mutation of Tub37CD results in female sterility (Tavosanis et al., 1997). In contrast, Tub23C is essentially ubiquitous and is required for viability, microtubule organization during somatic mitotic divisions, and male meiosis (Sunkel et al., 1995; Sampaio et al., 2001). Although both isotypes are present in early embryos, only Tub37CD is detected at centrosomes in syncytial embryos, whereas punctate structures containing Tub23C distribute throughout the cytoplasm (Wilson et al., 1997). Later during development, both Tub23C and Tub37CD localize to centrosomes in cellularized embryos (Tavosanis et al., 1997; Wilson et al., 1997; Raynaud-Messina et al., 2001). The extent to which each -tubulin isotype contributes to the assembly of female meiotic spindles remains to be resolved (Tavosanis et al., 1997; Wilson and Borisy, 1998; Llamazares et al., 1999).
Differences in the timing of their association with the centrosome have also been reported for the two -tubulin isotypes in Drosophila tissue culture cells (Raynaud-Messina et al., 2001). In Kc23 cells, the centrosomal levels of Tub37CD have been reported to vary only slightly throughout the cell cycle, whereas centrosomal Tub23C levels increased sharply during late G2. Furthermore, Tub23C was also recruited to the microtubules of the mitotic spindle, whereas Tub37CD was never found on spindle microtubules. This suggests the existence of mechanisms that allow the cell to distinguish between the two -tubulin isotypes. Here, we set out to gain a better understanding of the biochemistry of Tub23C. We report that Drosophila Tub23C isolated from tissue culture cells forms bona fide ring complexes that include most of the same Grips as the TuRCs formed by Tub37CD. However, we were surprised to find that Tub23C and Tub37CD associate with distinct splice variants of Dgrip84/GCP2 that differ by 74 amino acids in their amino termini. We speculate that Dgrip84 may be involved in regulating the subcellular localization of -tubulin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibody Production
Dgrip84 antibodies were a kind gift from Y. Zheng (Carnegie Institution of Washington, Baltimore, MD) and are described in Oegema et al. (1999). Synthetic peptides (made by the Protein Synthesis facility of the University of Wisconsin, Madison Biotech Center) corresponding to the COOH-terminal 17 amino acids of Tub23C (Cys-PVDSKSEDSRSVTSAGS; GenBank Accession no. P23257) or Tub37CD (Cys-QIDYPQWSPAVEASKAG; GenBank Accession no. P42271) were used to raise and affinity purify rabbit polyclonal antibodies as described elsewhere (Field et al., 1998). Peptides contained an additional N-terminal cysteine residue to facilitate coupling to KLH for antibody production, or to SulfoLink coupling gel (Pierce Biotechnology, Rockford, IL) for affinity purification. Specific antibodies were purified as described (Kellogg and Alberts, 1992), except that before affinity purification of the antisera with the immunogenic peptide, each serum was passed over a column corresponding to the nonimmunogenic -tubulin isotype. This procedure depleted potential cross-reacting antibodies and thus ascertained the specificity of the affinity-purified antibodies.
Drosophila S2 Cell Tissue Culture
Schneider S2 cells (Schneider, 1972) were cultured in Schneider's Drosophila medium as described by the supplier (Invitrogen, Carlsbad, CA). Cells to be used for extract preparation were grown for 4 d at 27°C in 
70 ml Drosophila medium in T-150 flasks. Cells were collected by centrifugation and washed twice with PBS, the volume of the cell pellet was estimated, and the cells were stored at –80°C until use.
PEG Precipitation
PEG (polyethylene glycol P-2139; average mol wt 5–8000; Sigma Chemical Co., St. Louis, MO) was added to a final concentration of 1–5% (from a 30% stock in H100) to clarified Drosophila embryo or S2 cell extract. The mixture was incubated on ice for 20 min, spun at 17,000 rpm for 10 min in a JA20 rotor (Beckman, Fullerton, CA), and the supernatant was removed. The pellets were resuspended in a volume corresponding the original extract volume of H200 plus 0.05% NP-40 and 100 µM GTP and were clarified at 17,000 rpm for 10 min in a JA20 rotor. Equivalent portions of supernatant and pellet fractions were then analyzed by sucrose gradient fractionation, SDS-PAGE, and Western blotting.
Immunoisolation of -Tubulin–containing Complexes from Drosophila Embryo Extract
Drosophila embryo extract was prepared by homogenizing Drosophila embryos in homogenization buffer as described (Moritz et al., 1998; Oegema et al., 1999). Embryo extract was frozen in aliquots in liquid nitrogen, stored at –80°C, and used within 2 wk of preparation. Clarified extract was prepared by centrifugation of thawed crude extract for 1 h at 50,000 rpm in a rotor (SW55; Beckman) at 4°C. -Tubulin complexes were immunoprecipitated from the supernatant essentially as described (Oegema et al., 1999), except that the PEG precipitation step was omitted. Briefly, anti--tubulin antibody (23C or 37CD; 8 µg/ml) was incubated with the supernatant at 4°C for 1 h with gentle rotation. Per 12 ml of clarified extract, 35 µl of protein A affiprep beads (Bio-Rad Laboratories, Hercules, CA) were equilibrated in wash buffer (H200 plus 0.05% NP-40, 100 µM GTP, and 1 mM PMSF) and added to the antibody-extract mix. After a further incubation for 1 h with gentle rotation, the beads were collected and washed six times with wash buffer and were incubated for 16–18 h at 4°C with an equal volume of EB containing the respective peptide. -Tubulin complexes were collected by loading an additional 40 µl of EB onto the beads and collecting the supernatant.
Immunoisolation of -Tubulin–containing Complexes from Drosophila Tissue Culture Cells
Drosophila tissue culture cell extract was prepared by resuspending thawed S2 cell pellets in 3 vol of cell lysis buffer, brief vortexing, and incubation on ice for 10 min. Cell debris was removed by a 10-min spin (at 4°C) at 2400 rpm in a Beckman table-top centrifuge. The supernatant was then incubated with antibody and processed as described above for the immunoisolation of -tubulin complexes from embryo extract.
Sucrose Gradient Sedimentation
Sucrose gradients (5–40%) were poured and run as described by Oegema et al. (1999). Briefly, gradients were poured by hand as step gradients (five steps of equal volume) in H100 plus 1 mM GTP and allowed to diffuse into continuous gradients. After centrifugation at 50,000 rpm in a Beckman TLS-55 rotor for 4.5 h at 4°C, gradients were fractionated by hand (16 fractions plus pellet). Fractions were separated on a 10% SDS-PAGE gel and analyzed by Western blot or Coomassie stain, as indicated. For some experiments, each fraction was precipitated by addition of 10% trichloroacetic acid (TCA) and centrifugation (30 min) in an Eppendorf centrifuge after a 30-min incubation on ice. Pellets were resuspended in SDS-sample buffer and analyzed by SDS-PAGE and Western blot.
Electron Microscopy
Negative staining electron microscopy of peptide-eluted complexes was described elsewhere (Zheng et al., 1995; Wiese and Zheng, 2000). Samples were viewed at 80 kV in a Philips Tecnai 12 microscope (Mahwah, NJ) equipped with a cooled CCD camera.
RT-PCR to Detect Dgrip84/GCP2 Isoforms
Total RNA was extracted from 100 µl of frozen 0–4 h Drosophila embryos or 107 S2 cells by homogenizing the cells in Trizol reagent (Invitrogen) and extracting the RNA according to the manufacturer's recommendations.
The following primers were used to reverse transcribe Dgrip84 mRNA (all isoforms) and amplify specific regions of the gene to distinguish the isoforms: the first strand cDNA template was synthesized from the extracted RNA using the ImPromII-Reverse Transcription System (Promega, Madison, WI) according to the manufacturer's recommendations and using the Dgrip84/GCP2 gene-specific primer, 5'-CTCTACACACATCTTGTC-3'. Isoform-specific PCR primers used to differentiate the Dgrip84 isoforms were as follows: To distinguish the 5' end of the "A"-isoform from B/C using three PCR primers: forward primer 5'-AAAGAGCTCAATTGCC-3' (specific for the 5' untranslated region of isoform A) combined with reverse primer 5'-TCGAGCAGCATACTC-3' (amplifies all isoforms) gives an 885-base pair band if isoform A is present, no product for isoform B or C; forward primer 5'-CAGTCGATCTAGCCGTTG-3'(specific for isoforms B and C, because this primer targets a region that is spliced out in isoform A) with reverse primer 5'-TCGAGCAGCATACTC-3'(amplifies all isoforms) gives an 855-base pair band if isoform B or C is present. To test for the presence of the 3' insertion that distinguishes isoform C from isoforms A and B, we used forward primer 5'-GCTAGTCTCTATCGAG-3' and reverse primer 5'-CTGTTGATCTGCTTGAGG-3'. This primer pair amplifies a 500-base pair band if the insert is present, or a 400-base pair band if the insert is absent. PCR products were cloned and sequenced to confirm their identity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tub23C and its interacting proteins we began by raising antibodies specific for Tub23C or Tub37CD (Figure 1). Sequence alignments and molecular modeling (Figure 1, A and B) revealed that the differences between Tub23C and Tub37CD (a total of 81 amino acids) did not cluster to defined regions within the -tubulin molecule but were distributed across most of the surfaces (Figures 1B and Supplementary Figure S1). The biggest difference between Tub23C and Tub37CD was a 14-amino acid insertion near the C-terminus of Tub23C. We took advantage of this difference to generate -tubulin isotype-specific antibodies by immunizing rabbits with synthetic peptides corresponding to the C-terminal 17 amino acids of each -tubulin isotype (see Materials and Methods). Dot-blot analysis confirmed that the affinity-purified antibodies were specific for their respective immunogenic peptides and did not cross-react with the nonimmunogenic peptide (Figure 1D). As expected, the antibodies recognized bands of 50 kDa (Tub37CD antibodies) and 52 kDa (Tub23C antibodies), respectively, on Western blots of embryo extracts (Figure 1E) or of purified -tubulin complexes (Figure 1F). In addition, the Tub23C antibodies recognized a band of 50 kDa in S2 (Drosophila tissue culture) cell extracts, whereas no band corresponding to -tubulin was recognized by the Tub37CD antibodies in S2 cell extracts (Figure 1E). This suggests that our S2 cell line does not express Tub37CD, or only undetectably low levels.
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Tub23C formed TuRC-like complexes. For this, we first asked whether the Dgrips that interact with Tub37CD in early Drosophila embryos also exist in cells that only express Tub23C. Extracts of Drosophila tissue culture cells (Schneider S2 or Kc) or embryos were Western blotted and probed for individual TuRC proteins (Figure 2A). This analysis revealed that Dgrip163/GCP6, Dgrip128/GCP5, Dgrip98/GCP3, and Tub23C migrated similarly in extracts made from embryos or tissue culture cells. In contrast, Dgrip84/GCP2 behaved differently in embryos and tissue culture cells: no band corresponding to the embryonic Dgrip84 was found in the tissue culture cell lines. Instead, two novel bands appeared that migrated at 72 and 100 kDa (Figure 2Af). To determine which, if either, of the novel bands might be a subunit of the TuRC, we analyzed extracts of S2 (Figures 2B and 3B) or Kc (Figure 3C) cells on 5–40% sucrose gradients and probed Western blots of gradient fractions for various TuRC subunits. This analysis showed that the 72-kDa band migrated near the top of the sucrose gradient and thus appeared to be unrelated to the TuRC or the TuSC. Whether or how the 72-kDa band is related to Dgrip84 is not yet clear. In contrast, the 100-kDa band migrated similar to Tub23C in tissue culture cells (Figure 2B) and like Dgrip84 in embryos (Figure 3D). Thus, our results strongly suggested that the 100-kDa band may be the Dgrip84 of somatic cells.
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Western blots of staged embryos probed with anti-Dgrip84 antibodies showed that Dgrip84 (marked "D" in Figure 4D) migrated more slowly in older embryos compared with younger ones. The slower-migrating form of Dgrip84 was detectable within 8–12 h of development, and its relative levels increased as the levels of the shorter isoform (marked "C" in Figure 4D) slowly declined.
To determine whether the appearance of the slower-migrating Dgrip84 protein could be explained by a switch to a longer Dgrip84 isoform, we examined the isoforms expressed in staged embryos by Northern blot analysis. Northern blots showed that the shorter isoform of Dgrip84 expressed in early embryos (0–4 h) was replaced by a longer isoform later in development that was also present in S2 cells (Figure 4E). To identify the specific isoforms present in early embryos and S2 cells, we first designed a strategy to identify the isoform by RT-PCR using specific primers (diagramed in Supplementary Figure S2). Our results were then confirmed by direct sequencing of the RT-PCR product. Consistent with results from previous work (Oegema et al., 1999; Gunawardane et al., 2000), we found that the isoform present in early embryos lacked both the N-terminal extension and the C-terminal insertion. This suggested that the embryos expressed mainly the "C"-isoform of Dgrip84. S2 cells, on the other hand, expressed mRNAs that contained the N-terminal extension. However, we were surprised to find no evidence for the C-terminal insertion. This suggested that the major isoform of Dgrip84 expressed in S2 cells is the previously unknown, novel "D" splice variant of Dgrip84 (Figure 4A). Together, these results suggested that the expression of Dgrip84 splice variants is developmentally regulated.
To confirm that the longer Dgrip84 splice variant is indeed a component of the -tubulin complex, we used our -tubulin isoform-specific or Dgrip84 antibodies to immunoprecipitate protein complexes from embryos or S2 cells and compared these by Coomassie-stained SDS-PAGE gels and Western blot (Figure 5). With the exception of the band that corresponds to Dgrip84, similar sets of proteins were precipitated from S2 tissue culture cells and from 0- to 3-h embryos using antibodies directed against Dgrip84 (Figure 5Aa), and their protein profile closely resembled the Tub37CD and Tub23C complexes isolated from embryos and S2 cells, respectively (Figure 5Ab; Oegema et al., 1999). This suggested that both Dgrip84 slice variants are components of -tubulin complexes. One important difference between S2 and embryonic -tubulin complexes was the absence of the band corresponding to the embryonic Dgrip84 and a concomitant appearance of a 100-kDa band in the S2 -tubulin complexes. Thus, consistent with the findings described above, the -tubulin complexes isolated from embryos or S2 cells differed by the associated Dgrip84 splice variant.
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-tubulin complexes in greater detail. Using the Tub23C antibody, we adapted the strategy for isolating Tub37CD complexes from early embryo extracts (Oegema et al., 1999) to isolate -tubulin complexes from S2 cell extracts (Figure 5Ab). In this strategy, -tubulin complexes are immunoprecipitated using the affinity-purified peptide antibodies, and the complexes are then eluted from the affinity beads with the specific synthetic peptide against which the antibodies were raised. This allowed us to examine the complexes by negative staining electron microscopy (Figure 5B), which showed that the purified Tub23C complexes appeared as 25-nm rings that closely resembled the embryonic TuRC37CD (Zheng et al., 1995; Oegema et al., 1999). We therefore concluded that Tub23C formed a "canonical" TuRC in S2 cells.
The purified complexes were further analyzed by Western blotting with antibodies directed against embryonic Dgrips (Figure 5C). The Dgrips we tested (163/GCP6, 128/GCP5, and 98/GCP3) all copurified with Tub23C complexes isolated from late embryos (8–16 h) or S2 cells and migrated similarly on a 10% SDS-PAGE gel as their early embryonic counterparts (Figure 5C). Consistent with the results described above, the -tubulin complexes contained distinct but immunologically related Dgrip84 isoform(s), depending on which -tubulin isotype was present: Tub37CD correlated with a faster migrating Dgrip84, whereas the presence of Tub23C correlated with a slower migrating isoform. Interestingly, a small amount of Tub23C copurified with Tub37CD and vice versa (Figure 5C; see also Figures 1F and 6C), suggesting that some TuRCs may contain both -tubulin isotypes.
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Tub23C present in early embryos and Tub23C present in late embryos or S2 cells (Figures 5 and 6). Although the Tub23C antibodies immunoprecipitated a TuRC-like complex from late embryos (Figure 5C) or S2 cells (Figure 5, Ab and C), they failed to precipitate Tub23C, or a TuRC-like complex, from early embryos (Figure 6A). To understand whether Tub23C exists in a TuRC-like complex in early embryos, we examined embryo extracts (0–8 h) by sucrose gradient analysis and Western blotting (Figure 6B). Under the conditions used (100 mM salt, 5–40% sucrose; 16 fractions), Tub37CD distributed mainly in two peaks that corresponded to the previously characterized TuRC (fractions 13–15) and TuSC (fractions 6–8; Oegema et al., 1999). Tub23C showed a distinct (albeit overlapping) distribution: only a small portion of Tub23C was found in the same fractions as the TuRC37CD. Instead, Tub23C peaked in fractions 11 and 12 and was distributed broadly in fractions 4–9 without forming a distinct peak (Figure 6B). Surprisingly, the "large" Tub23C complex (fractions 11 and 12) was noticeably smaller than the TuRC37CD. Similarly, the broad distribution of Tub23C in the lower fractions (4–9) suggested that Tub23C might form a smaller complex, perhaps resembling the TuSC, but this complex may be distinct from and/or more heterogeneous than the TuSC37CD. We concluded that Tub23C migrated as a large complex in early embryos, but that this complex is mostly distinct from the TuRC (Figure 6, A and B). These conclusions were further supported by experiments using differential precipitation of extract proteins with increasing concentrations of polyethylene glycol (PEG). Tub37CD and its associated proteins precipitated nearly quantitatively in the presence of 2.5% PEG, whereas a significant portion of Tub23C remained in the supernatant under these conditions. Sucrose gradient analysis of the supernatant and pellet fractions from the 2.5% PEG precipitation showed that the Tub37CD and Tub23C complexes could be separated from one another under these conditions (Figure 6C). Thus, we concluded that although both -tubulin isotypes are present in complexes in early embryos, Tub23C complexes are mostly distinct from the TuRC37CD.
In contrast, Tub23C comigrated with the Dgrips in tissue culture cells (Figure 3; see also Figure 2B), consistent with the observation that it forms a TuRC in these cells (see above). Although it is not yet clear at which developmental stage Tub23C switches from the "embryonic" complex to become part of the TuRC, or how this process is regulated, these findings support the idea that the formation of TuRCs containing Tub23C is developmentally controlled.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tub23C and Tub37CD in tissue culture cells and syncytial embryos (Sunkel et al., 1995; Wilson et al., 1997; Raynaud-Messina et al., 2001, 2004) prompted us to examine the molecular compositions of the complexes formed by each of the two Drosophila -tubulin isotypes. We reasoned that differences in their localization might be regulated by intrinsic differences between the -tubulin isotypes or their binding partners, or both.
We analyzed -tubulin complexes in two different systems: in Drosophila embryos, where Tub37CD is the major -tubulin isotype, and in Schneider S2 tissue culture cells, where Tub23C is the major -tubulin isotype and Tub37C plays only a minor role, if any (Raynaud-Messina et al., 2004). Based on the observation that -tubulin and its interacting proteins are highly conserved among distantly related species, it was reasonable to suspect that Tub23C forms complexes in Drosophila cells that closely resemble the TuRC37CD. Here, we provide the first experimental evidence that this is indeed the case. We show that previously identified -tubulin–interacting proteins (Dgrips) copurify with Tub23C isolated from tissue culture cells, and together these proteins form a ring-shaped complex that closely resembles the TuRC37CD when viewed by negative staining electron microscopy. Our studies revealed two unexpected results: 1) Tub23C forms a large complex in early embryos that is distinct from the TuRC, and 2) expression of different Dgrip84 isoforms is developmentally regulated and correlates with expression of different -tubulin isoforms. These findings support the notion that the two Drosophila -tubulins interact with different binding partners and perhaps interact differently with similar binding partners.
What Is the Role of the Embryonic Tub23C Complex?
In embryos, Tub23C distributes in punctate patterns not associated with centrosomes, whereas Tub37CD localizes to centrosomes and spindle poles during early embryonic divisions (Wilson et al., 1997). It has been postulated that only Tub37CD is utilized at centrosomes during embryonic cleavages (Wilson et al., 1997). Consistent with this, female meiosis and nuclear divisions during early embryogenesis specifically require Tub37CD, and Tub37CD mutants are female sterile despite the presence of Tub23C in the embryos (Tavosanis et al., 1997; Tavosanis and Gonzales, 2003). This suggests that the -tubulin isotypes are not functionally redundant during embryogenesis. Our results provide a potential biochemical explanation for these observations. We propose that the embryonic Tub23C complex may represent a type of ‘storage form’ of Tub23C. This is suggested by the observation that Tub23C could not be immunoprecipitated from early embryos, indicating that the epitope recognized by the antibody was not accessible. We speculate that the storage particle might include molecular chaperones of the TriC protein family, which have been shown to interact with and aid in the folding of -tubulin (Melki et al., 1993). We found that the large Tub23C complexes persisted for at least the first 8 h of development, suggesting that the availability of Tub23C is very tightly regulated during early fly development. Full analysis of the embryonic Tub23C complex awaits methods to purify the complex that do not rely on antibody affinity chromatography. How the association of Tub23C with the putative storage particle is regulated and how Tub37CD escapes recruitment to the particle remains a mystery.
What Is the Importance of the Different Isoforms of Dgrip84/GCP2 in Drosophila?
To date, Drosophila Dgrip84 is the only member of the GCP2 protein family that has been reported to exist as more than one splice variant. Database analysis reveals that at least two Drosophila species possess the 74-amino acid N-terminal extension, D. melanogaster and D. pseudoobscura. It is possible that unique structural features of one or the other Dgrip84 isoform facilitate the particularly rapid centrosome assembly of the early Drosophila embryo, as has previously been postulated for Tub37CD (Wilson et al., 1997). Consistent with this hypothesis, Dgrip84 has recently been implicated in centrosome assembly and separation (Colombié et al., 2006). GCP2 (and GCP3) family members have also been implicated in TuRC recruitment to the spindle pole body in S. cerevisiae and to the centrosome in metazoa (Knop and Schiebel, 1997, 1998; Nguyen et al., 1998; Barbosa et al., 2000; Vinh et al., 2002; Takahashi et al., 2002; Zimmerman et al., 2004; Kawaguchi and Zheng, 2004; Vérollet et al., 2006). The precise role of GCP2 family members in TuRC function and recruitment remains to be elucidated.
Our analysis of Dgrip84 isoforms showed that the isoform expressed in embryos corresponds to the shortest splice variant, whereas the isoform expressed in older embryos and S2 cells corresponds to a previously unreported variant of Dgrip84 that has an N-terminal extension but lacks the reported C-terminal insertion. To date, we have been unable to find experimental evidence for the existence of Dgrip84 isoforms that possess the C-terminal insertion (isoforms A and B). It is possible that these isoforms are expressed only at very low levels or that they are expressed only in certain tissues. The roles of the extension or the insertion in Dgrip84 function are not yet clear, but we noted that the developmental switch to the longer isoform of Dgrip84 correlated with the appearance of TuRCs composed of Tub23C. Although thus far this is merely a correlation, it is tempting to speculate that the N-terminal Dgrip84 extension aids in folding or assembly of the somatic TuRC, or is required for incorporation of Tub23C into the TuRC. Experiments to address these questions are currently underway.
Chimeric TuRCs to Fine-Tune Microtubule Organization?
The metazoan TuRC contains 12–15 -tubulin molecules (Zheng et al., 1995; reviewed in Jeng and Stearns, 1999; Wiese and Zheng, 2006), most of which are thought to be arranged in tetrameric subcomplexes (TuSCs) that contain two copies of -tubulin. Having two -tubulin isotypes in the same cell prompts a number of important questions: can both -tubulin isotypes coexist in the same TuSC? Can both isotypes coexist in the same TuRC? How does the cell distinguish between the -tubulin isotypes? Do the -tubulin isotypes perform different functions? How does the cell transition from using primarily one isotype to using the other, and how does the activity of a chimeric TuRC (for which we found evidence by both sucrose gradient analysis and analysis of purified complexes) or TuSC differ from each of the homogenous versions? The results presented here are beginning to provide answers to some of these questions, but gaining a true understanding of how -tubulin complexes that differ only slightly in their composition are perhaps used to fine-tune microtubule organization will be the subject of future studies.
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ACKNOWLEDGMENTS |
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Tub37CD antibodies, S. Chitteni-Pattu for the negative staining EM, and A. Steinberg and L. Vanderploeg for help with figure preparation. C.W. is a Scholar of the Sidney Kimmel Foundation for Cancer Research. Work in the Wiese lab was further supported by funds from UW-Madison WISELI, the UW-Madison Graduate School, and the Department of Biochemistry. |
Footnotes |
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Address correspondence to: Christiane Wiese (wiese{at}biochem.wisc.edu)
Abbreviations used: tub, -tubulin; TuRC, -tubulin ring complex; TuSC, -tubulin small complex; MT, microtubule.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and β tubulin within the microtubule lattice, as illustrated in (C). The snapshots illustrate two important points: first, the differences between the two 
