Blackwell Publishing, Ltd. Oxford, UK GTC Genes to Cells 1356-2443 © Blackwell Publishing Ltd ? 2004 9?Original Article Control of spindle dynamics by Toucan V Mirouse et al.
The
Drosophila
Toucan protein is a new mitotic
microtubule-associated protein required for spindle microtubule stability
Vincent Mirouse, Bernard Dastugue and Jean-Louis Couderc*
Institut National de la Santé et de la Recherche Médicale UMR384, Laboratoire de Biochimie, UFR Médecine, 28, place Henri Dunant, 63001 Clermont-Fd, France
Mitotic spindle dynamics are highly dependent on proteins that interact with microtubules to influence their organization or stability. Here, we show that the Drosophila Toucan protein interacts directly with microtubules. Its localization to the microtubule network when it is expressed in mammalian cells and its direct interaction with microtubules in vitro are dependent on its central basic domain. Moreover, Toc expression in mammalian cells strongly protects microtubules from depolymerization. By using in vivo inducible RNAi in syncytial embryos, we generated a dose-sensitive loss of function of toucan, demonstrating that this technique is an efficient method for inactivating a maternal transcript. This enabled us to accurately characterize several new mitotic defects from the early to the late phases of mitosis, depending on Toucan depletion level. Toucan is required for metaphase spindle formation and centrosome anchoring to the poles. Then, during anaphase, Toc depletion affects kinetochore microtubules and therefore chromosome segregation. Toc is also necessary for central spindle formation by the interpolar microtubules. In contrast, astral microtubules are not disturbed by Toc depletion. Taken together, our results show that Toucan is a microtubule-associated protein specifically required for the stability of spindle microtubules throughout mitosis.
Introduction
Equal partition of the genetic material from one nucleus to two daughter nuclei requires a self-organizing bipolar machine known as the mitotic spindle. This highly dynamic structure is composed of at least three different subpopu-lations of microtubules. The kinetochore microtubules interact with chromosome centromeres with their plus-ends whereas their minus-ends are concentrated at spindle poles, near centrosomes. During prometaphase, these microtubules allow the alignment of the chromosomes at the metaphase plate. Then, during anaphase A, they drive the segregation of sister chromatids to opposite spindle poles. The interpolar microtubules form a framework of two partially overlapping radial arrays, orientated with their minus-ends focused at spindle poles and their plus-ends at the central part of the spindle. During anaphase B these microtubules elongate and slide in an anti-parallel motion, pushing the spindle poles apart. The kinetochore and interpolar microtubules form the spindle microtubules by opposition with astral microtubules. These are formed from the centrosomes
and their plus-ends extend to the cytoplasm. The main purpose of the astral microtubules is to position the spindle in the cell, even if additional functions have been shown. 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 nuclea-tion, 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 localiza-tion 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
Communicated by: Claude Desplan
V Mirouse et al.
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, chromo-some 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.
Results
The Toucan protein is a microtubule-associated protein
With the aim of characterizing the function of the Toucan protein, we decided to study its behaviour in a heter-ologous system using transient transfections in mammalian cells. In interphase Cos 7 cells, the Toucan protein local-izes 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.
We determined which domain of the Toc protein is necessary for the microtubule localization by performing different deletions of the Toc protein. These truncated proteins were expressed in mammalian cells using transient transfections, and their localization was analysed by double immunofluorescence (Fig. 1C). Toc protein deleted for the C-terminal or N-terminal parts still localized to microtubules. These results are confirmed by the diffused
localization of the N-Terminal and C-terminal parts of Toc when they are expressed alone. Deletion of the central part of the Toc protein containing a basic domain also led to a diffused staining. Finally, when this central basic domain (CBD) was expressed alone, it was able to localize to microtubules (Fig. 1B). Thus, the central basic domain is necessary and sufficient for the localization of the Toc protein to microtubules in mammalian cells.
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 Figure 1 Toucan interacts directly with microtubules by its central basic domain. (A) Cos7 cell over-expressing Toc protein stained for Toc (red), tubulin (green) and DNA (blue). Toc presents a perfect co-localization with microtubules. (B) Cos7 cell over-expressing the central basic domain (CBD) of Toc (red). This domain is present in the nucleus and the cytoplasm where it co-localizes with microtubules. (C) Schematic representation of the Toc protein. The green box represents CBD (pI = 11.7) and the blue box represents a predicted coiled-coil domain. Red boxes represent regions conserved with more than 30% of identity in the
Anopheles gambiae (Ag) ortholog of Toc. Transfections of Toc
deletion constructs in Cos7 cells show that only the proteins containing the CBD localize to microtubules. (D) Microtubule overlay blotting assay. Equivalent quantities of BSA, GST, GST-CBD and GST-Toc610-948 were separated by SDS-PAGE, then subjected to Coomassie staining or overlay blotting with MAP-free purified and in vitro polymerized microtubules. Anti-tubulin antibody reveals that microtubules have been retained only by the CBD recombinant protein.
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).
To investigate this apparent stabilization effect of the Toc protein on microtubules in mammalian cells, we tested whether it can protect microtubules from the effect of microtubule-depolymerizing drugs. We used an anti-acetylated-tubulin as acetylated tubulin is an indicator of stable microtubules. In untreated cos-7 cells, acetylated microtubules were observed in all the cells, whether expressing the Toc protein or not (Fig. 2B). When the cells were fixed 12 h after nocodazole, colchicine or col-cemid treatment, acetylated tubulin was present only in cells expressing Toc (Fig. 2D). These results show that the Toc protein induces microtubule stabilization. Their level of Toc expression, their proportion, and the aspect of their microtubules suggested that these drug-resistant cells could correspond to those in which the Toc protein has induced microtubule bundling. It was therefore unclear whether the fact that the Toc protein prevents drug-induced depolymerization was related to the bundling. The microtubule stabilizing effect of Toc was quantified at various times after nocodazole treatment by comparing the number of transfected and untransfected cells with intact microtubules. The most striking effect of Toc on microtubule stability was seen 10 min after noc-odazole treatment, with 75% of Toc expressing cells with an intact network whereas only 20% of untransfected cells still have microtubules. The proportion of trans-fected cells with resistant microtubules and the aspect of these microtubules showed that the stabilizing effect of Toc is not due to the microtubule bundling effect (Fig. 2C,E) and that, more probably, the bundling is a consequence of a strong microtubule stabilization.
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 Figure 2 The Toucan protein induces microtubule stabilization in mammalian cells. (A, B, C) Toc is shown in red and DNA in blue. Green represents tubulin in (A) and acetylated tubulin in (B) and (C). (A) Toc over-expressing cell with microtubule bundles. (B and C) COS-7 cells were transfected with Toc and either untreated (B) or treated (C) with 10 µm nocodazole for 20 min. Acetylated tubulin is observed in all the untreated cells but only in Toc-expressing cells after nocodazole treatment. (D) Transfected cells were subjected to different microtubule depolymerizing treatments during 12 h before fixation, and the number of cells with intact microtubules was then scored. Untransfected cells never showed acetyl-tubulin staining whereas a significant percentage of cells with intact microtubules was observed with all the tested drugs. (E) COS-7 cells transfected with Toc and untransfected cells were scored for an intact microtubule network at the indicated times after nocodazole treatment. Transfected cells with intact microtubules were plotted against the time of nocodazole treatment.
V Mirouse et al.
transgene against the toc gene using the UAS/Gal4 expres-sion system. An inverted repeat of about 1 kb corre-sponding to the coding region for the C-terminal part of the Toc protein was introduced in flies under the con-trol of a UASp promoter. Expression of one copy of this transgene using one copy of an α4tub-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.
We also examined the viability of the embryos using all different combinations of drivers and RNAi transgenes ( Table 1). Embryos from females that are homozygous either for the driver or for the Toc-RNAi transgene presented a normal viability, whereas a sublethality appeared (36.3%) when the flies contained one copy of each transgene. We define this genotype as weak Toc-RNAi mutant. This lethality increased to approximately 90% with two copies of one transgene and one copy of the other, and we will refer to these flies as inter-mediate Toc-RNAi mutant. The maternal lethality became complete when both transgenes were homozygous, and we will call this genotype strong Toc-RNAi mutant. There was a correlation between the level of Toc protein and the percentage of embryos that reach the larval stage. Observa-tion of the embryos after DNA and microtubule staining indicated that strong Toc-RNAi mutant leads to an early lethality during premigration divisions. The more frequent phenotype observed in these embryos is an early blockage ( premigration divisions) of the syncytial mitoses in a pseu-dometaphase state. Compared to wild-type metaphase (Fig. 3B), the mitotic spindles were shorter, displayed broad poles and did not seem to present centrosomes in their vicinity (Fig. 3C). This is an exact phenocopy of the defects described for toc mutations (Debec et al. 2001), confirming that Toc is required for spindle formation and centrosome anchor-ing. We also tested whether Toc-RNAi interacted geneti-cally with toc alleles. One copy of these hypomorphic alleles strongly increased the lethality of weak Toc-RNAi mutant embryos (Table 1). This dominant genetic interaction confirmed that the embryo lethality observed in Toc-RNAi mutants is due to the depletion of the Toc protein.
Taken together, these results show that it is possible to deplete a protein encoded by a maternal mRNA by inducing expression of an inverted repeat in the germ-line and that this technique can be successfully used to generate loss of function phenotypes.
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
Figure 3 In vivo inducible RNAi against the toc mRNA strongly
decreases Toc protein level and leads to defects in metaphase spindle formation.Western blot with extracts from 0 to 1 h embryos laid by females of the following genotypes: line 1, one copy of Gal4 driver and UAS-Toc-RNAi; 2, two copies of each; 3, wild type. Note the dose-sensitive effect of Toc-RNAi on the Toc protein. (B) Wild-type metaphase spindles with DNA in red and tubulin in green. (C) Abnormal pseudometaphase spindles observed during syncytial divisions in strong Toc-RNAi mutants with overcondensed chromosomes and broad spindle poles. Bars: 10 µm.
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).
To understand the chronology of these defects, we performed time-lapse experiments (movies 1 and 2) using living embryos expressing a GFP-tubulin fusion protein (Grieder et al. 2000). In intermediate Toc-RNAi embryos, after several wild-type cell cycles, defects appeared abruptly during one of the cortical mitosis. At such a mitosis spindles appeared normal until metaphase (Fig. 4C). As anaphase started, all the microtubules began to depolymerize, and rapidly no microtubule could be detected between the centrosomes (Fig. 4C′ and 4C″). This differed dramatically with wild-type embryos, in which interpolar microtubules were maintained and elongated to form the central spindle. At the end of mitosis, the formation of elongated nuclei extending from one centrosome to the other was observed through the negative staining of tubulin (Fig. 4C″′).
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 con-clude 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 centro-somes (Fig. 4E). In strong and intermediate Toc-RNAi mutant embryos, we observed the formation of an elon-gated mass of chromosomes between the centrosomes (Fig. 4F). No microtubules were detected between the Table 1 Expression of Toc-RNAi transgene induces embryo lethality and mitotic defects. UASp-Toc-RNAi transgene is indicated as Toc-RNAi and α4tub-Gal4-VP16 driver as Gal4. For the determination of the hatching rate more than 300 eggs were counted for each phenotype. Stage corresponds to the first developmental stage in which defects appear. The most frequent defects were assessed by observation of more than 100 fixed embryos with microtubule and DNA staining. The expression of Toc-RNAi transgene in the germ-line leads to embryo lethality. However, the lethality level is dependent on the number of copies of the Gal4 driver and the UASp-Toc-RNAi transgenes. In consequence, we have defined the different genotypes as described at the right of the table. Toc-UASp-Toc-RNAi also presents dominant genetic interaction with toc mutations, indicating that the lethality is due to depletion of the toc gene product.
Genotype
% of
hatching eggs Stage Most frequent defect Phenotype
Copies of Gal4
Copies of
Toc-RNAi toc alleles
2 0 +/+ 97.5 Nd None
Wild type
0 2 +/+ 96.3 Nd None
1 1 +/+ 63.7 Syncytial blastoderm Anaphase (rare) Weak Toc-RNAi
2 1 +/+ 14.5 Syncytial blastoderm Anaphase (frequent)
IntermediateToc-RNAi
1 2 +/+ 11.0 Syncytial blastoderm Anaphase (frequent)
2 2 +/+ 0 Preblastoderm Metaphase/centrosome
anchoring
Strong Toc-RNAi
1 1 +/tocBA34
39.3 Syncytial blastoderm Anaphase
1 1 +/tocPR3A
26.7 Syncytial blastoderm Anaphase Nd, not determined.
V Mirouse et al.
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.
Figure 4 The Toucan protein is required for the formation of the central spindle during anaphase B and chromosome segregation. (A) Anaphase B syncytial mitoses from WT (A) and intermediate Toc-RNAi (B) embryos with DNA in red and tubulin in green. Absence of spindle tubulin staining in Toc-RNAi embryo indicates the absence of central spindle. The chromosome sets have finished their motion to the poles but are not well separated. (C) Images from a time-lapse experiment from metaphase to the end of mitosis in an intermediate Toc-RNAi embryo expressing a tubulin-GFP fusion protein. The spindle is normal until metaphase but spindle microtubules disappear after anaphase onset, leading to the formation of elongated nuclei from one centrosome to the other. (D) Spindle length in µm during the 9th syncytial mitosis from metaphase to the end of mitosis in WT embryos (squares) and in Toc-RNAi embryos in which the central spindle was absent (triangles). Time scale: 20 s. Arrow indicates anaphase onset. Error bars represent the standard deviation of the average lengths. At metaphase, spindles of Toc-RNAi mutant are quite shorter than in WT embryos. There is no significant elongation of Toc-RNAi spindles during the later mitotic phases. (E) Early WT anaphase B in which both sets of chromosomes have reached the vicinity of centrosomes. (B) End of mitoses in a Toc-RNAi embryo in which the sets of chromosomes form an ovoid mass indicating that chromosome segregation started but was not completed. No interpolar or kinetochore microtubules were observed. Arrows indicate the position of the centrosomes, and arrowheads indicate the extremity of chromosome sets.
Discussion
Silencing of a maternal transcript by inducible RNAi
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 mater-nal 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 pro-cesses, and many of the Drosophila genes involved in these processes have been identified by hypomorphic muta-tions 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 oogene-sis, 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 there-fore 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. Neverthe-less, 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 micro-tubules, 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 pheno-types have not been observed in toc mutant embryos because spindles are blocked very early during mitosis. We observed mitotic figures with an incomplete segre-gation of the chromosome sets and no microtubules between centrosomes and chromosomes. This pheno-type could be due to a quicker depolymerization of kinetochore microtubules at one extremity. We also
V Mirouse et al.
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 concern-ing 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, sug-gesting that Toc could act specifically during syncytial mitosis. Interestingly, syncytial mitoses are very fast, and analysis of their microtubule polar flux shows that micro-tubule 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 stabi-lize 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 localiza-tion 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 sub-populations 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 lim-ited 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.
Experimental procedures
Toucan constructs
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
Dro-sophila genome by P-element-mediated transformation (Rubin &
Spradling 1982). Two insertions were obtained, named UASp-Toc-RNAiBC1
and UASp-Toc-RNAiBC2
. The germ-line expres-sion 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 gener-ated 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 supple-mented 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
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), respect-ively. 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 polymer-ized 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
Acknowledgements
We are grateful to Antoine Guichet and the Bloogminton Stock Center for providing fly strains. We thank Alain Debec, Muriel Grammont and Olivier Bardot for critical reading of the manu-script. This research was supported by the Institut National de la Santé et de la Recherche Médicale. V. M. is supported by a fellow-ship from the MRES (Ministère de la Recherche et de l’Enseigne-ment Supérieur).
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Received: 20 September 2004 Accepted: 15 October 2004
