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Institut National de la Santé et de la Recherche Médicale UMR384, Laboratoire de Biochimie, UFR Médecine, 28, place Henri Dunant, 63001 Clermont-Fd, France
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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All these different populations of microtubules present specific dynamics during mitosis, indicating a differential control. How these subpopulations of microtubules are dynamically regulated through mitosis is one of the fundamental questions in understanding cell division. Non-motor microtubule-associated proteins (MAPs) are major effectors of this control (for review see Merdes & Cleveland 1997; Andersen 2000). They may influence different aspects of microtubule behaviour such as nucleation, stability or organization. These MAPs are the targets of different mitotic regulators including phosphorylation by kinases (do Carmo Avides et al. 2001; Terada et al. 2003), the action of the small GTPase Ran (Gruss et al. 2001; Wiese et al. 2001) or the control of their stability in time and space (Juang et al. 1997).
The Drosophila toucan (toc) gene was first described for its developmental involvement in oogenesis (Grammont et al. 1997). During this process the Toc protein localization is related to the microtubule network (Grammont et al. 2000). More recent work has shown that Toc is also present on spindles during embryonic syncytial mitoses and toc gene mutations lead to a very early blockage of these divisions (Debec et al. 2001). Spindles present important defects and are blocked in prometaphase or metaphase. The precocity and the strength of this phenotype did not allow investigation of the Toc protein function through mitosis. Finally, how Toc contributes to mitosis at the molecular level has not been investigated.
In this study we show that Toc is a MAP, interacting directly with microtubules by its central basic domain, and that the over-expression of Toc in mammalian cells induces a stabilization of their microtubule network. We also performed a detailed study of the Toc function using inducible in vivo RNAi technique in syncytial embryos. Toc protein depletion disrupts several mitotic processes, including formation of the metaphase spindle, chromosome segregation and spindle elongation during late mitosis. The observed defects are specific to spindle microtubules and do not concern astral microtubules. Taken together, these data show that Toc is a MAP required for spindle microtubule stability.
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Results |
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With the aim of characterizing the function of the Toucan protein, we decided to study its behaviour in a heterologous system using transient transfections in mammalian cells. In interphase Cos 7 cells, the Toucan protein localizes to the microtubules (Fig. 1A). The same observation was also made in HeLa and Kb cells. This localization is uniform throughout the microtubule network, indicating that Toc protein has no particular affinity for one particular extremity of the microtubules. The presence of Toc protein on microtubules was confirmed by direct observation of living cells expressing a GFP-Toucan fusion protein (data not shown). Unfortunately, we were not able to observe the localization of Toc during mitosis in these cells as its expression seemed to induce a blockage in G2 phase. The capacity of the over-expressed Toc protein to localize to the whole microtubule network in a heterologous system suggests a direct binding of the Toc protein to the microtubules.
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To test whether the CBD of Toc (amino acids 1000–1534) is able to interact directly with microtubules in vitro, it was fused to the GST protein, expressed in bacteria and then purified. This domain was submitted to overlay blotting with in vitro polymerized MAP-free microtubules (Fig. 1D). Microtubules were retained by purified GST-CBD whereas none were bound by proteins such as GST or BSA. As a control, we also tested another part of the Toc protein (GST-Toc610–948), which did not localize to microtubules in mammalian cells, and observed that it did not retain microtubules in this assay. These results indicate that not only does Toc co-localize with but also directly binds to microtubules, fulfilling the criteria of a bona fide MAP.
The Toucan protein induces stabilization of the microtubules in mammalian cells
A microtubule bundling appeared in cells with a high Toc protein level, based on signal intensity of the Toucan protein staining (about 35% of the cells) (Fig. 2A). Generally, these microtubule bundles formed concentric rings around the nucleus. This bundling, as it has been shown with other MAPs over-expressed in cultured cells, can reflect microtubule stabilization properties (Brandt & Lee 1993; Manabe et al. 2002).
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In vivo inducible RNAi against toucan leads to dose-sensitive depletion of Toc protein
The Toc protein has been shown to be required for syncytial mitosis (Debec et al. 2001). Embryos laid by mutant females for the toc gene stop their development after only a few divisions and present abnormal spindles blocked in pseudometaphase. This early blockage prevents the study of a possible involvement of the Toc protein during later phases of mitosis.
To define the precise in vivo function of the Toc protein, we constructed transgenic flies with an inducible RNAi transgene against the toc gene using the UAS/Gal4 expression system. An inverted repeat of about 1 kb corresponding to the coding region for the C-terminal part of the Toc protein was introduced in flies under the control of a UASp promoter. Expression of one copy of this transgene using one copy of an 
4tub-Gal4-VP16 driver in mothers led to a strong decrease of Toc protein level in the embryos (Fig. 3A). Toc protein became nearly undetectable when both transgenes were homozygous, indicating that the RNAi effect is dose dependent.
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Toucan protein is required for the formation of the central spindle during anaphase
Intermediate Toc-RNAi generally reach postmigration divisions where mitotic defects appear, probably when Toc protein becomes limiting (Table 1). In anaphase from wild-type embryos and most of the intermediate Toc-RNAi embryos, chromosomes reached the spindle poles and were close to the centrosomes, indicating that anaphase A had occurred normally. During anaphase B and telophase in wild-type embryos, interpolar microtubules were present between the chromosome sets and formed the central spindle (Fig. 4A). In intermediate Toc-RNAi mutants, no interpolar microtubules could be detected and chromosome sets presented a bilobed shape, indicating that their separation was not properly achieved (Fig. 4B).
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We also compared the size of the spindles from the metaphase to the end of the 9th syncytial mitosis by measuring the distance between centrosomes in wild-type and intermediate Toc-RNAi embryos presenting a central spindle formation defect. At metaphase, Toc-RNAi spindles appeared slightly shorter than wild-type spindles (23%) (Fig. 4D). During anaphase, these spindles almost did not elongate (+26%) whereas wild-type spindles increased their length strongly (+50%). These measurements correspond to those obtained by inhibition of motor proteins that act on the spindles for their elongation during late mitotic phases (Sharp et al. 1999, 2000). Therefore, it is in agreement with an absence of functional interpolar microtubules in Toc-RNAi mutants. These experiments led us to conclude that the Toc protein is required after metaphase to maintain interpolar microtubules and to form the central spindle.
The Toucan protein is required for chromosome segregation during anaphase A
In wild-type embryos, during anaphase A both sets of chromosomes separate and reach the vicinity of centrosomes (Fig. 4E). In strong and intermediate Toc-RNAi mutant embryos, we observed the formation of an elongated mass of chromosomes between the centrosomes (Fig. 4F). No microtubules were detected between the centrosomes in such spindles. The ovoid aspect of the chromosomes indicated that chromosome segregation had begun during anaphase A but was not completed, since the chromosome sets did not reach the vicinity of the centrosomes. The absence of spindle microtubules together with the incomplete chromosome segregation strongly suggests that the kinetochore microtubules were affected by Toc depletion. We never observed embryos showing a wild-type central spindle with a chromosome segregation defect. This could mean that complete migration of the chromosomes to the poles is required in order to maintain interpolar microtubules and enable spindle elongation. Nevertheless, it has recently been shown that abnormal chromosome segregation could occur without defect in spindle elongation (Rogers et al. 2004). Moreover, central spindle formation defects occurred without segregation defects in Toc-RNAi embryo (Fig. 4B). All together, these observations suggest that the chromosome segregation defect observed in Toc-RNAi embryos is always associated with the absence of formation of the central spindle, but that these two phenotypes are independent. In contrast to these spindle defects, astral microtubules were not affected by the depletion of the Toc protein. This observation indicates that Toc specifically acts on spindle microtubules during mitosis.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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In vivo inducible RNAi can inactivate Drosophila gene function which is expressed zygotically (Lam & Thummel 2000). In this article, we show that it is possible to deplete early embryos of a protein encoded by a maternal mRNA by inducing expression of an inverted repeat in the germ-line. This technique can be successfully used to generate loss of function phenotypes in syncytial embryos. Moreover, we also observed similar depletion of the Toc protein present in the germ-line of the Toc-RNAi females (V.M., unpublished observation). To our knowledge, this study is the first report of an mRNA that can be efficiently depleted in early embryos as well as in germ-line during oogenesis by the in vivo inducible RNAi technique.
This approach leads to dose-sensitive depletion of the target protein, which enables weak to strong loss of function conditions to be obtained for the gene studied. These characteristics are of particular interest for a gene involved in syncytial divisions. These divisions are one of the major models for the genetic analysis of mitotic processes, and many of the Drosophila genes involved in these processes have been identified by hypomorphic mutations leading to maternal-dependent embryonic lethality mutations (Sunkel & Glover 1988; Inoue et al. 2000). Hypomorphic conditions rarely induce systematic defects during the first phases of mitosis, and enable the role of a protein to be studied during the later phases. Depletion of a specific protein in syncytial embryos can also be obtained by antibody injections (Sharp et al. 1999; Gergely et al. 2000). The defects induced by injection appear to be dose-sensitive, depending on the distance from the injection site. This technique is of particular interest for time-lapse experiments because it allows the precortical divisions to be completed before protein depletion. However, this technique requires certainty of the antibody specificity and blocking activity. Here, we demonstrate that germ-line inducible RNAi provides an interesting approach for achieving conditional, hypomorphic and dose-sensitive protein depletion in syncytial embryos, and could be a good alternative method for studying mitotic genes for which no mutants are available.
The Toc protein is a new Drosophila mitotic microtubule-associated protein
This article characterizes the Drosophila Toc protein as a MAP. In vivo, Toc co-localizes with spindle microtubules during syncytial mitosis. Furthermore, during oogenesis, Toc follows the modifications of the microtubule network in the oocyte (Grammont et al. 2000). We show that Toc protein localizes to microtubules in mammalian cells via its central basic domain, and that this domain interacts directly with microtubules in vitro. As for Toc, most of the characterized microtubule-binding domains of MAPs are basic, interacting directly with the acidic carboxy-terminal domain of tubulin (Littauer et al. 1986). However, the Toc central basic domain shows no sequence homology with other known MAPs and therefore represents a new microtubule-binding domain.
Toc presents microtubule stabilizing properties
In this article we have carefully described the defects induced by partial Toc protein depletion on fixed embryos as well as on living embryos. All the observed mitotic defects can be explained by a destabilization of microtubules. First, the Toc protein is required for the formation of a normal bipolar metaphase spindle. When the Toc protein is depleted, spindles present broad poles and fail to properly align chromosomes to the metaphase plate. Centrosomes in these spindles do not stay anchored to the spindle poles, confirming the phenotypes observed in toc mutants (Debec et al. 2001). Although Toc is weakly present at centrosomes, most of the protein is concentrated on spindle microtubules, suggesting that this localization corresponds to its major site of action. The organization of spindle poles and anchoring of centrosomes depend on a protein matrix that undergoes dynamic interactions with microtubules. Toc could be a component of this matrix, acting on microtubule organization. Nevertheless, a lower depletion of Toc protein leads to functional and well-organized but significantly shorter metaphase spindles than in wild-type embryos. This result indicates that the Toc protein acts more on the length of microtubules, and thus on their stability, than on their organization. We propose that in the absence of Toc, the depolymerization of microtubule minus-ends is quicker and that the spindle pole matrix loses contact with them, leading to the broad poles and loss of centrosome anchoring observed in toc mutant embryos.
In this article we also present a role for Toc in spindle midzone and chromosome segregation. These phenotypes have not been observed in toc mutant embryos because spindles are blocked very early during mitosis. We observed mitotic figures with an incomplete segregation of the chromosome sets and no microtubules between centrosomes and chromosomes. This phenotype could be due to a quicker depolymerization of kinetochore microtubules at one extremity. We also characterized an absence of interpolar microtubules leading to abnormal spindle elongation during anaphase B in Toc-RNAi embryos. Time-lapse experiments clearly show that spindle microtubules depolymerize after the onset of anaphase and do not form a central spindle. Interestingly, very similar phenotypes concerning spindle microtubules and chromosome segregation have been observed after inhibition of dmEB1, a protein that influences microtubule dynamics (Rogers et al. 2002). These similarities support the hypothesis that Toc protein acts on microtubule stability.
Toc protein is strongly present in syncytial embryos but is not detectable in later stages of development, suggesting that Toc could act specifically during syncytial mitosis. Interestingly, syncytial mitoses are very fast, and analysis of their microtubule polar flux shows that microtubule dynamics are quicker than in the other mitotic models (Brust-Mascher & Scholey 2002; Maddox et al. 2002). This property could mean that specific factors such as Toc could be necessary at specific places and times in these embryos in order to ensure a normal mitotic process.
In contrast to spindle defects due to Toc depletion, the over-expression of Toc in mammalian cells leads to a strong stabilization of microtubules. This ability to stabilize microtubules in a heterologous system, whereas Toc has no known ortholog, suggests that this property is intrinsic to Toc. These results therefore reinforce the hypothesis that Toc is required for the stability of the spindle microtubules.
Toc acts differentially on mitotic microtubule subpopulations
Loss of function of toc disturbs both interpolar and kinetochore microtubule subpopulations. In contrast, the astral microtubules, which actively polymerize during anaphase in syncytial embryos, are not affected by Toc depletion. These defects are correlated with the localization of Toc, which is present on spindle but not on astral microtubule (Debec et al. 2001). Two hypotheses could explain this specificity of action. First, differential post-translation modifications between microtubule subpopulations could explain a specific interaction of Toc with one but not the other. Similar mechanisms have been observed for interphasic MAPs whose binding to microtubules is regulated by tubulin glutamylation (Boucher et al. 1994; Bonnet et al. 2001). Interestingly, glutamylation during mitosis in mammalian cells is limited to spindle microtubules and does not extend to astral microtubules (Bobinnec et al. 1998; Kann et al. 2003). Alternatively, MAPs can be activated and recruited to specific regions of the mitotic apparatus through their direct regulation by mitotic regulators (do Carmo Avides et al. 2001; Gruss et al. 2001; Wiese et al. 2001; Terada et al. 2003). Consequently, it would now be interesting to identify the molecular mechanisms that control the subcellular localization and the activity of the Toc protein throughout the cell cycle, and particularly during mitosis.
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Experimental procedures |
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For Toc-RNAi construct, an inverted repeat corresponding to the coding sequence of the last exon of the toc cDNA was realized (n 5601–6650 on gb|Y14157). Two fragments, with the same 5' position, of 1.05 and 1.1 kb in length, were PCR-generated with an XbaI site in 5' and an EcoRI site in 3'. Both fragments were cloned in one step into the XbaI site of the pUASp vector (Rorth 1998). UASp-Toc-RNAi construct was introduced into the Drosophila genome by P-element-mediated transformation (Rubin & Spradling 1982). Two insertions were obtained, named UASp-Toc-RNAiBC1 and UASp-Toc-RNAiBC2. The germ-line expression of both transgenes give similar phenotypes, but with a weaker effect with UASp-Toc-RNAiBC1. Results presented here were obtained with UASp-Toc-RNAiBC2.
The full-length and all deleted Toc constructs for expression in mammalian cells, except the construct 1001–1534, were obtained by cloning into pCI-neo (Promega). The construction 1001–1534 was introduced in-frame into the pSG5-FLAG vector.
Glutathione-S-transferase (GST) fusion proteins were generated by subcloning in frame DNA sequences coding amino acids 610–948 (Toc610–948) and 1000–1534 (CBD) of the Toc protein into the pGEX-4T-1 plasmid (Amersham).
Determination of embryo lethality
Embryo lethality tests were performed at 25 °C by crossing young virgin females of the indicated genotype with Canton S males. Eggs were collected over 12 h and the numbers of hatched and unhatched eggs were counted 30 h after, and this was repeated over three days. For each genotype, a minimum of 300 eggs were scored. For suppression tests with APC mutants, experiments were repeated three times from independent crosses.
Cell culture, transfection, drug treatment and immunostaining
COS-7, HeLa and Kb cells were maintained in DMEM supplemented with 10% foetal calf serum. Exponentially growing cells were plated on coverslips and transfected the next day with the indicated constructs using Lipofectamine (Invitrogen). Indirect immunofluorescence was performed 24 h later, as described elsewhere.
In microtubule depolymerization experiments, exponentially growing COS-7 were plated on coverslips and transfected the next day with the full-length Toc protein construct. After 20–24 h of transfection, cells were treated with 10 µM nocodazole or 10 µM colchicine or 5 µg/mL colcemid for indicated times before fixing and processing cells for immunofluorescence. Microtubule networks of at least 300 cells were analysed in each experiment for each drug and each time point.
Tubulin and acetylated tubulin were detected using DM1A (mouse, 1/5000) (Sigma) and 6-11B-1 (1/1000) (Sigma), respectively. Full-length Toc protein and all the constructs containing the 950 first amino acids were detected using anti-Toc80 antibody (rabbit, 1/1000) (Grammont et al. 2000). Toc protein constructs containing the C-terminal extremity were detected using a rabbit anti-Toc antibody raised against amino acids 1972–2176 (1/1000). Flag tagged construct 1001–1534 was detected using anti-Flag M5 antibody (mouse, 1/500) (Sigma). Secondary antibodies used were anti-mouse FITC (goat, 1/500) (Jackson) and anti-rabbit Cy3 (goat, 1/500) (Jackson). Cell observation was realized on an epifluorescence Zeiss Axiophot microscope.
Embryo immunostaining
Embryo immunofluorescence staining was performed using classical procedures as previously described in Debec et al. (2001). Image acquisition was realized on an Olympus FV300 confocal microscope.
Time-lapse confocal microscopy and quantitative image analysis
All images were acquired on an Olympus FV300 confocal microscope. Each image resulted from the accumulation of three z-series performed with 1 µM steps to counteract poor focus variation of spindles, and new images were acquired every 20 or 30 s. The distance between spindle poles was determined using Fluoview software (Olympus). In all cases the through-space distance between spindle poles was determined (the length of a straight line drawn between the middle of each spindle pole). The data shown are the average of the measurements of four independent spindles.
Microtubule overlay assay
Microtubule overlay assays were performed as described elsewhere (Saunders et al. 1997). 500 ng per lane of recombinant Toc-CBD, recombinant Toc610–948, BSA and GST were fractionated by 10% SDS-PAGE and blotted onto Optitran membrane (Schleicher & Schuell). The membranes were preincubated in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 5% low-fat powdered milk for 1 h, and then washed three times for 15 min in overlay buffer (0.1 M Pipes/NaOH, pH 6.6, 5 mM EGTA, 1 mM MgSO4, 0.9 M glycerol, 1 mM DTT, 1 mM PMSF). MAP-free bovine brain tubulin (Molecular Probes) was polymerized at a concentration of 2 µg/mL in overlay buffer by addition of GTP to a final concentration of 1 mM, and incubated at 37 °C for 30 min. The buffer containing polymerized microtubules was added to the membranes for incubation for 1 h at 37 °C with addition of taxol at a final concentration of 10 µM for the final 30 min. The blots were then washed three times with TBST and the bound tubulin was detected using anti--tubulin antibody (Sigma) and ECL Plus (Amersham).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: jl.couderc{at}inserm.u-clermont1.fr
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References |
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Received: 20 September 2004
Accepted: 15 October 2004
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