Functionally Unequal Centrosomes Drive Spindle Orientation in Asymmetrically Dividing Drosophila Neural Stem Cells

Functionally Unequal Centrosomes Drive Spindle Orientation in Asymmetrically Dividing Drosophila Neural Stem Cells Elena Rebollo1, Paula Sampaio2, Jens Januschke1, Salud Llamazares1, Hanne Varmark, 1,4 and Cayetano González1,3,* 1 Cell Division Group, Institute for Research in Biomedicine, Parc Cientíic Barcelona, C/ Josep Samitier 1-5, 08028 Barcelona, Spain 2 Advanced Light Microscopy Facility, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Campo Alegre 823, P4150-180 Porto, Portugal 3 Institució Catalana de Recerca i Estudis Avanc¸ ats, Passeig Lluis Companys 23, 08010 Barcelona, Spain 4 Present address: Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Biotech II, Worcester, MA 01605, USA. *Correspondence: [email protected] SUMMARY Stem cell asymmetric division requires tight control of spindle orientation. To study this key process, we have recorded Drosophila larval neural stem cells (NBs) engineered to express fluorescent reporters for microtubules, pericentriolar material (PCM), and centrioles. We have found that early in the cell cycle, the two centrosomes become unequal: one organizes an aster that stays near the apical cortex for most of the cell cycle, while the other loses PCM and microtubule-organizing activity, and moves extensively throughout the cell until shortly before mitosis when, located near the basal cortex, it recruits PCM and organizes the second mitotic aster. Upon division, the apical centrosome remains in the stem cell, while the other goes into the differentiating daughter. Apical aster maintenance requires the function of Pins. These results reveal that spindle orientation in Drosophila larval NBs is determined very early in the cell cycle, and is mediated by asymmetric centrosome function. INTRODUCTION Like the stem cells in other higher eukaryotes, Drosophila neural stem cells (NBs) undergo asymmetric division whereby one of the two daughters retains stem cell identity, while the other enters a program of differentiation. The molecular mechanisms that drive asymmetric division in these cells have been the subject of intensive studies in the last decade. Briefly, the localization at the apical cortex of the Par complex, which includes Bazooka (Baz) (Kuchinke et al., 1998; Schober et al., 1999; Wodarz et al., 1999), Par-6 (Petronczki and Knoblich, 2001), and atypical protein kinase C (aPKC) (Rolls et al., 2003; Wodarz et al., 2000), drives the basal localization of the adaptor proteins Miranda (Ikeshima-Kataoka et al., 1997; Shen et al., 1997) and Partner of Numb (Lu et al., 1998; Roegiers et al., 2001). These, in turn, mediate the accumulation of cell fate determinants such as Numb (Ikeshima-Kataoka et al., 1997; Rhyu et al., 1994; Shen et al., 1997), Prospero (Hirata et al., 1995; Knoblich et al., 1995; Spana and Doe, 1995), and Brat (Betschinger et al., 2006; Lee et al., 2006) at the basal cortex of the NB. Consequently, upon cell division, the determinants end up mostly within the small ganglion mother cell (GMC) that buds off the basal side of the NB, resulting in the unequal developmental fate of the two daughters. A key step in the asymmetric segregation of the determinants is spindle orientation that must be in line with the polarity axis of the cell (Betschinger and Knoblich, 2004; Kaltschmidt and Brand, 2002). This process is governed by Inscuteable (Kaltschmidt et al., 2000; Schaefer et al., 2000), another protein of the apical complex that binds to Baz and aPKC (Schober et al., 1999; Wodarz et al., 1999) and mediates the organization of a complex that includes Pins (Schaefer et al., 2000; Yu et al., 2000), G (Schaefer et al., 2001), and Mushroom body defective (Mud), the protein thought to ultimately mediate spindle orientation through direct interaction with one of the two mitotic asters that is thereby singled out as the apical aster (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006). It is unclear, however, how the system discriminates between the two asters of the NB, so that only one engages in this interaction (Kaltschmidt and Brand, 2002). Unlike embryonic NBs where, after assembly, the spindle rotates 90 degrees to align along the polarity axis (Kaltschmidt et al., 2000), larval NBs assemble the spindle already aligned and no rotation occurs (Savoian and Rieder, 2002; Siller et al., 2006). The role of aPKC in spindle orientation seems also to be different between larval and embryonic NBs (Rolls et al., 2003; Wodarz et al., 2000). Directed assembly without rotation has been observed as well in grasshopper embryonic neural stem cells and in Drosophila germline stem cells (Kawamura, 1960; Yamashita and Fuller, 2005; Yamashita et al., 2003). To get a mechanistic insight into how such directionality is achieved, we have followed the centrosome cycle in Drosophila larval NBs. RESULTS The Microtubule-Organizing Center that Enters the Differentiating Daughter Cell Is Assembled Late in the Cell Cycle, near the Basal Cortex, and Away from the Apical Aster We first recorded Drosophila larval NBs expressing fluorescent microtubule and pericentriolar material (PCM) markers. Typically, in cells that divide symmetrically, the single interphase microtubule-organizing center (MTOC) splits into two that segregate from one another around the nucleus (Blagden and Glover, 2003). The recordings obtained from asymmetrically dividing NBs expressing GFP-α-tubulin (Rebollo et al., 2004) revealed a different process (Figure 1A; see Movie S1 in Supplemental Data available with this article online). For most of the cell cycle, Drosophila larval NBs contain a single major aster, which is permanently located on the apical side, opposite to the GMCs that remain near the basal side of the NB (Figure 1A, -610 to -290, arrowhead). It is only with the onset of prophase, shortly before nuclear envelope breakdown (NEB), that a second mitotic aster can be observed (Figure 1A, -40, arrow). Notably, assembly of this second aster seems to occur independently of the first, as it appears suddenly at a basal position. After NEB (Figure 1A, 00), the spindle assembles (Figure 1A, 80), and when the cell divides the basal aster is inherited by the GMC (Figure 1A, 160, arrow and asterisk), while the apical aster remains in the NB. Similar observations were obtained from NBs expressing a fusion protein between green fluorescent protein (GFP) and the microtubule-binding protein EB1 (Rogers et al., 2002) (Figure 1B; Movie S2). A single apical MTOC (Figure 1B, -160, arrowhead) can be observed until a second aster is suddenly organized, shortly before mitosis, near the basal cortex, opposite to the first with which it seems to bear no relation (Figure 1B, -70, -40). NEB then follows (Figure 1B, 00), the spindle is assembled (Figure 1B, 30), and, after mitosis, the last appearing aster is incorporated into the small daughter cell (Figure 1A, 100). Therefore, in Drosophila neural stem cells, the apical aster is in place throughout the cycle and the second is only assembled late, before mitosis, at the basal side, away from the first. We then followed the PCM in NBs expressing GFP-Cnn (Centrosomin) (Megraw et al., 2002; Movie S3). After cytokinesis, a single GFP-Cnn dot can be observed in the NB, located near the apical side of the nucleus. Later, this single dot of PCM splits into two (Figure 1C, -710, double arrowhead), suggesting that centrosome duplication may have taken place. One of these two GFP-Cnn dots disappears later (Figure 1C, -630), so that for most of the cell cycle, a single dot of GFP-Cnn can be observed, always at the apical side. A second focus of PCM material is only observed shortly before NEB, always located near the basal cortex of the NB (Figure 1C, -140, arrow; Figures 1D and 1E), thus coinciding in time and place with the formation of the second aster as described above. This second focus of PCM matures quickly, recruiting significant GFP-Cnn amounts before NEB takes place (Figure 1C, 00). It is this late-appearing centrosome that is the one that enters the resulting GMC as the stem cell divides (Figure 1C, 200, asterisk and arrow). The process is then repeated all over again in the next cell cycle of the NB (Figure 1C, 200 to 320). Similar results were obtained from NBs expressing a second PCM marker, Minispindles, fused to GFP (Barros et al., 2005; Movie S4). Two alternative hypotheses could account for the observed behavior of centrosomes and asters in larval NBs. One possibility could be that, rather than resulting from the classic duplication pathway, the centrosome destined to the GMC would assemble de novo, near the basal cortex, where it would organize the late-appearing aster. De novo centrosome assembly is a well-documented process, both under natural (Magnuson and Epstein, 1984; Palazzo et al., 1992; Riparbelli and Callaini, 2003; Szollosi et al., 1972) and experimental (La Terra et al., 2005; Marshall et al., 2001) conditions. A second hypothesis that may account for our observations is that after duplication, the MTOC activity of one of the resulting centrosomes could be transiently downregulated until shortly before NEB, when located near the basal cortex. Transient downregulation of MTOC activity has been observed in the paternal centrosome of Spisula oocytes (Wu and Palazzo, 1999). Soon after Duplication, One of the Centrosomes Becomes a Downregulated MTOC that Moves Extensively through the NB for Most of the Cell Cycle To discriminate between these two hypotheses, we recorded NBs expressing the centriole marker YFP-Asl (see Figure S1). Soon after cytokinesis, the single YFP-Asl signal associated with the spindle pole that remains in the stem cell splits into two (Figure 2A, -590 to -560, arrowhead; Movie S5) that migrate together toward the apical cortex (Figure 2A, -510, blue and green tracings). They stay there until one of them starts a series of seemingly unrestricted movements across the cell that last for a significant fraction of the cell cycle (Figure 2A, -290 to -110, blue tracing). Shortly before NEB (Figure 2A, 00), the migrating YFP-Asl dot becomes stabilized at the basal side of the nucleus, where it remains as the spindle is assembled (Figure 2A, 120), and cleavage takes it into the resulting GMC (Figure 2A, 170, asterisk and blue line). The same process takes place again in the following cell cycle (Figure 2A, 300 to 650). Only short-range movements can be observed in the centrosome that remains apical through the cell cycle and stays within the NB after cytokinesis (Figure 2A, -290 to 170, green line). These results rule out the “de novo assembly” hypothesis and substantiate the view that, following centrosome duplication, the MTOC activity of one of the resulting centrosomes is downregulated for most of the cell cycle and upregulated again only when the centrosome is located basally, soon before spindle assembly. These observations are somewhat reminiscent of the situation reported in HeLa cells, where the daughter centriole is not associated with an aster and moves during interphase (Piel et al., 2000). Significant differences apply, however, regarding the timing of events, the extent of migration, the position of centrosomes within the cell, the presence of PCM in the moving centriole of HeLa cells, and, most importantly, the fact that unequal centrosome behavior in NBs is tightly correlated with the polarity axis of these cells. To better resolve centrosome movement in Drosophila larval NBs, we acquired additional movies at 60 s intervals (Figure 2B; Movie S6). Like in the previous recording, following duplication, the two YFP-Asl foci migrate together, at an average maximum speed of 0.02±0.01μm/s (n=11) (Figure 2C), to the apical cortex, where they stay until one of them starts to move. From then on, these time-resolved series reveal a tendency in the pattern of movement of the YFP-Asl signal. At first, migration takes place in the apical hemisphere and is mostly accounted for by back and forth movements to the proximity of the apical centrosome. This results in aster-like tracings (Figure 2B, -1050 to -500) that may suggest some involvement of the apical aster in guiding centrosome migration at this stage (Piel et al., 2000). Later on, movement is largely restricted to the basal half of the cell and seems more irregular (Figure 2B, -180 to 00), perhaps reflecting the involvement of the more randomly oriented actin filaments (Piel et al., 2000). Interestingly, the two centrosomes remain functionally different even when migration of the downregulated centrosome takes it very close to the active centrosome, strongly suggesting that once acquired, differential centrosome identity is not primarily governed by spatial cues. Centriole speed can reach peaks of up to 0.92μm/s (Figure 2C). Pins Is Required to Maintain Apical Centrosome Identity in Larval NBs Because NB spindle alignment depends on Pins (Schaefer et al., 2000), we decided to follow centrosome movement and MTOC activity in pins mutant NBs. The recordings of two clusters of pinsP89/pinsP62 mutant cells (hereafter referred to as pins) expressing either YFP-Asl (Movies S7 and S8) or GFP-α-tubulin (Movie S9) are summarized in Figure 3. Following NB division (Figure 3A, -2690, asterisks), YFP-Asl reveals that the first stages of centrosome movement in pins NBs occur like in the wild-type: a highly correlated behavior, as they move in parallel toward the cortex (green and blue traces in Figure 3A, -2600), where they remain until one of them starts moving around the cell (Figure 3A, -2140). Consistently, GFP-αtubulin recordings in pins NBs show that soon after mitosis (Figure 3B, -1300), a prominent aster (arrowhead) moves to and becomes stabilized at the cortex of the NB, opposite the newly formed GMC (Figure 3B, -980). However, a clear difference with wild-type NBs can be observed in pins cells at a later time point, when the cortical YFP-Asl signal loses its stable position and starts moving. Migration of this centrosome is at first restricted to one side of the cell (Figure 3A, -1250), but later takes place throughout the entire cell, to the extent that the trajectories from both centrosomes become fully intermingled (Figure 3A, -120). Accordingly, at this stage, the cortical aster is disassembled (Figure 3B, -890 to -850). Before NEB (Figure 3A, -120), both centrosomes settle down, coinciding with the assembly of the two asters (Figure 3B, -50, arrows). After spindle assembly (Figure 3A, 230; Figure 3B, 120), cell division takes place (Figure 3A, 380; Figure 3B, 350), cleaving the NB into two cells of similar size. These results reveal that the stability of the apical aster, but not its initial assembly, requires Pins and suggest that Pins mediates spindle orientation by maintaining apical centrosome identity. Consistent with such functional connection between unequal centrosomes and the asymmetric division machinery in NBs, we have found that the two centrosomes of GMCs— the daughter cells, which divide symmetrically—are functionally equal. Interestingly, their behavior is reminiscent of the behavior of the late-maturating, highly motile basal centrosome of the NB, from which they derive (Figure S2; Movie S10). DISCUSSION A schematic summary of our results is shown in Figure 4. Immediately after cytokinesis, the single dot revealed by both PCM (red) and centriole (yellow) reporters splits in two, strongly suggesting that centrosome duplication has taken place. The YFP-Asl marker, like other centriolar markers in Drosophila (Basto et al., 2006; Martinez-Campos et al., 2004), does not allow for resolution of individual centrioles within a centrosome in larval NBs. Therefore, timing of centriole duplication in these cells remains uncertain. The two resulting centrosomes migrate together within the single major aster of the cell to the apical cortex. Later on, one of the centrosomes loses PCM and starts to migrate. At this early time point in the cell cycle, unequal centrosome fate is already established: one, apical, will remain in the stem cell; the other will go into the differentiating daughter. Migration of the downregulated centrosome (revealed by the centriolar marker), initially within the apical side of the cell and more basally later on, occupies most of the cell cycle and is the most variable stage, its duration being dependent on cell-cycle length (45.1±27.0 min; n=9). The apical centrosome organizes the only aster found in the NB for most of the cell cycle. As mitosis onset approaches (10.3±2.1 min before NEB; n=9), the moving downregulated centrosome becomes stabilized at the basal side and starts to accumulate PCM and organize the second aster. As a direct consequence, the spindle is assembled already in alignment with the polarity axis of the cell. In Drosophila male germline stem cells, one of the centrosomes is also consistently located adjacent to the hub from early interphase onward (Yamashita and Fuller, 2005). Only this centrosome maintains a robust aster through the cell cycle. The other, associated with only a few microtubules, moves away from the hub and is inherited by the gonialblast (Yamashita et al., 2007). In these cells, the oldest centriole is always in the centrosome that is proximal to the hub and is therefore retained by the stem cell (Yamashita et al., 2007). We have not yet been able to determine which of the two centrosomes contains the oldest centriole in larval neural stem cells. In pins NBs, unequal centrosome fate and function are established, but, eventually, the stable, aster-forming apical centrosome is downregulated and starts to behave like the other, migrating across the cell. Like the other too, it organizes an aster only shortly before NEB. The place of assembly of the two asters in pins mutant NBs is not fixed and consequently spindle orientation is randomized, and so is the size difference between the two daughter cells (Izumi et al., 2004, 2006; Siller et al., 2006). It is still unclear how NB polarity is maintained from one cycle to the next (Yu et al., 2006), as a distinct Baz apical crescent is only assembled at prophase (Siller et al., 2006). The permanent positioning of the NB centrosome in the apical side of the cell, through the cell cycle, suggests that it could be contributing to specifying the apical cortex after mitosis. In summary, four main conclusions can be derived from these observations: 1. The two centrosomes of asymmetrically dividing Drosophila larval NBs become unequal early in the cell cycle in terms of mobility, MTOC activity, and fate. 2. Such elaborated unequal centrosome regulation provides a means to position the asters, thus ensuring spindle alignment along the polarity axis of the cell. 3. Pins contributes to spindle orientation in NBs by preventing the downregulation of the MTOC capability of the apical centrosome, thus maintaining the apical aster in place. 4. Spindle orientation is predetermined and can be accurately predicted as soon as the aster reaches the apical cortex during the initial stages of the cell cycle. Altogether, our observations reveal that asymmetry in Drosophila neural stem cells goes beyond the polarized localization of a number of protein complexes during mitosis and may affect entire organelles such as the centrosome, which exerts a major effect on cell architecture and function throughout the cell cycle. EXPERIMENTAL PROCEDURES Fly Stocks and DNA Constructs All fly strains used in this work are described in FlyBase. GFP-EB1 was obtained by polymerase chain reaction on Eb1 cDNA (RE41362 obtained from the Drosophila Genomics Resource Center), sequenced, and subsequently EcoR1/Xba1 cloned into pUASpmGFP6. NB expression of the UAS constructs was driven by the gal4 enhancer-trap strain Mz1061 (Ito et al., 1995). To obtain the YFP-Asl fusion, the CG2919 gene was amplified from an embryonic cDNA library (Brown and Kafatos, 1988) using primers 5 -ATTTGCGGCCGCTATG′ AACACGCCAGGTATAAGCCTCTTTC-3 and 5 -ATTTGCGGCCGCT′ ′ TAGCTGTGACCATTGCCTTTGGG-3 that span the entire open reading frame, and NotI cloned′ into the EYFP-C1 vector (Clontech), modified by introducing additional NotI and NheI sites into the BglII and EcoR1 sites. The resulting fusion was then cut with NheI, cloned into the SpeI site of the Drosophila polyubiquitin transformation vector (Lee et al., 1988), and used to produce transgenic flies by standard P element-mediated transformation. All asl mutant phenotypes (Bonaccorsi et al., 1998) were rescued by the YFP-Asl fusion expressed under the control of the polyubiquitin promoter. Time-Lapse Recording Dispersed cells were prepared following the clot method (Forer and Pickett-Heaps, 1998), adapted to Drosophila NBs by using Schneider’s Drosophila medium (GIBCO) and MatTek culture dishes. The results obtained using this method were confirmed using an alternative protocol based on the culture of whole-mount brains (Siller et al., 2005). To document the behavior of asters, PCM, and centrioles, we recorded 77, 11, and 26 movies, respectively. The Pins phenotype is documented in 10 movies. The results reported in the manuscript were observed in all ventral ganglion and central brain NBs recorded, without exception, regardless of the culture method used. All recordings were performed acquiring six to ten confocal Z sections for each time point to ensure full coverage of cell depth. We used a Leica TCS SP2 confocal system using a 63× glycerol immersion objective lens (NA=1.3). ACKNOWLEDGMENTS We are grateful to W. Chia, J. Raff, A. Guichet, and the Bloomington Stock Center for contributing reagents and fly strains. Work in our laboratory is supported by EU and Spanish MEC grants COMBIO, MADCD, ONCASYM, SAF2003, BFU2006, and Consolider-Ingenio2010 CENTROSOME_3D CSD2006-23. REFERENCES Barros, T.P., Kinoshita, K., Hyman, A.A., and Raff, J.W. (2005). Aurora A activates D-TACC-Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules. J. Cell Biol. 170, 1039–1046. Basto, R., Lau, J., Vinogradova, T., Gardiol, A., Woods, C.G., Khodjakov, A., and Raff, J.W. (2006). Flies without centrioles. Cell 125, 1375–1386. Betschinger, J., and Knoblich, J.A. (2004). Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates. Curr. Biol. 14, R674–R685. Betschinger, J., Mechtler, K., and Knoblich, J.A. (2006). Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells. Cell 124, 1241–1253. Blagden, S.P., and Glover, D.M. (2003). Polar expeditions—provisioning the centrosome for mitosis. Nat. Cell Biol. 5, 505–511. Bonaccorsi, S., Giansanti, M.G., and Gatti, M. (1998). Spindle self-organization and cytokinesis during male meiosis in asterless mutants of Drosophila melanogaster. J. Cell Biol. 142, 751– 761. Bowman, S.K., Neumuller, R.A., Novatchkova, M., Du, Q., and Knoblich, J.A. (2006). The Drosophila NuMA homolog Mud regulates spindle orientation in asymmetric cell division. Dev. Cell 10, 731–742. Brown, N.H., and Kafatos, F.C. (1988). Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203, 425–437. Forer, A., and Pickett-Heaps, J.D. (1998). Cytochalasin D and latrunculin affect chromosome behaviour during meiosis in crane-fly spermatocytes. Chromosome Res. 6, 533–549. Hirata, J., Nakagoshi, H., Nabeshima, Y., and Matsuzaki, F. (1995). Asymmetric segregation of the homeodomain protein Prospero during Drosophila development. Nature 377, 627–630. Ikeshima-Kataoka, H., Skeath, J.B., Nabeshima, Y., Doe, C.Q., and Matsuzaki, F. (1997). Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions. Nature 390, 625–629. Ito, K., Urban, J., and Technau, G.M. (1995). Distribution, classification and development of Drosophila glial cells in the late embryonic and early larval ventral nerve cord. Rouxs Arch. Dev. Biol. 204, 284–307. Izumi, Y., Ohta, N., Itoh-Furuya, A., Fuse, N., and Matsuzaki, F. (2004). Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division. J. Cell Biol. 164, 729–738. Izumi, Y., Ohta, N., Hisata, K., Raabe, T., and Matsuzaki, F. (2006). Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization. Nat. Cell Biol. 8, 586–593. Kaltschmidt, J.A., and Brand, A.H. (2002). Asymmetric cell division: microtubule dynamics and spindle asymmetry. J. Cell Sci. 115, 2257–2264. Kaltschmidt, J.A., Davidson, C.M., Brown, N.H., and Brand, A.H. (2000). Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system. Nat. Cell Biol. 2, 7–12. Kawamura, K. (1960). Studies on cytokinesis in neuroblasts of the grasshopper, Chortophaga viridifasciata (De Geer). I. Formation and behavior of the mitotic apparatus. Exp. Cell Res. 21, 1–18. Knoblich, J.A., Jan, L.Y., and Jan, Y.N. (1995). Asymmetric segregation of Numb and Prospero during cell division. Nature 377, 624–627. Kuchinke, U., Grawe, F., and Knust, E. (1998). Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka. Curr. Biol. 8, 1357–1365. La Terra, S., English, C.N., Hergert, P., McEwen, B.F., Sluder, G., and Khodjakov, A. (2005). The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturation. J. Cell Biol. 168, 713–722. Lee, H.S., Simon, J.A., and Lis, J.T. (1988). Structure and expression of ubiquitin genes of Drosophila melanogaster. Mol. Cell. Biol. 8, 4727–4735. Lee, C.Y., Wilkinson, B.D., Siegrist, S.E., Wharton, R.P., and Doe, C.Q. (2006). Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal. Dev. Cell 10, 441–449. Lu, B., Rothenberg, M., Jan, L.Y., and Jan, Y.N. (1998). Partner of Numb colocalizes with Numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors. Cell 95, 225–235. Magnuson, T., and Epstein, C.J. (1984). Oligosyndactyly: a lethal mutation in the mouse that results in mitotic arrest very early in development. Cell 38, 823–833. Marshall, W.F., Vucica, Y., and Rosenbaum, J.L. (2001). Kinetics and regulation of de novo centriole assembly. Implications for the mechanism of centriole duplication. Curr. Biol. 11, 308–317. Martinez-Campos, M., Basto, R., Baker, J., Kernan, M., and Raff, J.W. (2004). The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis. J. Cell Biol. 165, 673–683. Megraw, T.L., Kilaru, S., Turner, F.R., and Kaufman, T.C. (2002). The centrosome is a dynamic structure that ejects PCM flares. J. Cell Sci. 115, 4707–4718. Palazzo, R.E., Vaisberg, E., Cole, R.W., and Rieder, C.L. (1992). Centriole duplication in lysates of Spisula solidissima oocytes. Science 256, 219–221. Petronczki, M., and Knoblich, J.A. (2001). DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3, 43–49. Piel, M., Meyer, P., Khodjakov, A., Rieder, C.L., and Bornens, M. (2000). The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. J. Cell Biol. 149, 317–330. Figure S1. YFP-Asl Is a Centriolar Marker Detail of a centrosome on one side of a metaphase plate (blue; DAPI) showing the centriolar marker YFP-Asl (yellow) within a cloud of pericentriolar material revealed by the antigammaTUB antibody GTU-88. Scale bar: 2.5µm. Figure S2. The Two Centrosomes of the Symmetrically Dividing GMC Behave Similarly Selected frames from a time-lapse recording of cultured NBs and GMCs expressing either YFPAsl, (A) or GFP-Cnn (B). A) The accumulated green and blue tracings highlight the movement of the apical and basal centrosomes, of the NB, 36´prior to NEB (0). The cyan and magenta lines (21´to 25´) represent the accumulated trajectories of centrosomes in a GMC derived from the NB. Both GMC centrosomes behave equally, are highly motile and move throughout the cell. B) Recruitment of GFP-Cnn signal (arrows) start to be detectable shortly before mitosis in a GMC. A magnified view of the dividing GMC (0 to 47´) shows that the rate of GFP-Cnn recruitment increases synchronously in both centrosomes during mitosis (6´, 20´) and as the cytokinesis furrow (47´, double arrowhead) cleaves the cell in two. The arrows in the DIC panels highlight the position of the centrosomes as inferred from the fluorescence images. Time is shown in min. relative to NEB (0); scale bar: 5µm.