A molecular mechanism of mitotic centrosome assembly in Drosophila

1Centrosomes help regulate many cell processes, including cell shape, cell polarity, and cell division, and centrosome defects have been implicated in several human pathologies. Centrosomes are the major microtubule (MT)-organising centres (MTOCs) in many animal cells. They form when centrioles assemble a matrix of pericentriolar material (PCM) around themselves. Several hundred proteins are concentrated in the PCM, including many MT-organising proteins, cell-cycle regulators, and checkpoint and signalling proteins; thus, the centrosome appears to function as an important co-ordination centre in the cell. Although centrioles usually organize only small amounts of PCM in interphase cells, the PCM expands dramatically as cells prepare to enter mitosis—a process termed centrosome maturation.

2Many proteins have been implicated in mitotic PCM assembly. These include (1) centriole-associated proteins, such as ‘Asl/Cep152’ and ‘Sas-4/CPAP’, (2) proteins that have a centriole-associated fraction and a fraction that spreads out into the PCM, such as ‘Pericentrin/D-PLP’ and ‘DSpd-2/Cep192’, (3) proteins that reside in the PCM, such as ‘Cnn/Cdk5Rap2’, ‘DGp71WD/NEDD1’, and ‘γ-tubulin’, and (4) mitotic protein kinases, such as ‘Polo/Plk1’ and ‘Aurora A’. In recent super-resolution microscopy studies, several of these proteins appeared to be highly organized around interphase centrioles, but the organisation of proteins within the extended mitotic PCM was much less apparent.

3It has long been thought that the mitotic PCM is assembled on an underlying scaffold structure. We recently showed that Drosophila Centrosomin (Cnn) can form such a scaffold around centrioles and that this scaffold is assembled from the inside out: Cnn molecules continuously incorporate into the scaffold around the centrioles and the scaffold then fluxes slowly outward, away from the centrioles. This inside out assembly mechanism could be important, as it potentially allows the assembly of the mitotic PCM to be regulated by the centrioles.

4Many PCM proteins, however, can be recruited to mitotic centrosomes in the absence of Cnn, albeit at reduced levels, suggesting that at least one other protein must be able to form a scaffold around centrioles that can recruit other PCM components. We reasoned that such a scaffold might also be assembled from the inside out. To identify such a protein(s), we analyzed the dynamic behaviour of the eight Drosophila centrosomal proteins that, in addition to Cnn, have been most strongly implicated in mitotic PCM recruitment: Asl, Sas-4, D-PLP, DSpd-2, γ-tubulin, DGp71WD, Polo, and Aurora A. We found that only DSpd-2 behaves like Cnn, as it incorporates into the PCM close to the centrioles and then spreads slowly outward to form a scaffold-like structure that recruits other PCM components. Importantly, in the absence of either DSpd-2 or Cnn, PCM recruitment is diminished, but in the absence of both proteins, it is abolished. We show that Asl appears to initiate the recruitment of DSpd-2 and Cnn exclusively to the mother centrioles; DSpd-2 then helps to recruit more Cnn, while Cnn helps to maintain DSpd-2 within the PCM, thus creating a positive feedback loop that promotes the scaffold assembly. Thus, mitotic PCM assembly appears to be a surprisingly simple process in flies: Asl initiates the recruitment of Spd-2 and Cnn to mother centrioles, and these proteins then assemble into scaffolds that spread out from the mother centriole and form a platform upon which most, if not all, other PCM proteins ultimately assemble.

5We used spinning disk confocal microscopy to perform fluorescence recovery after photobleaching (FRAP) experiments in combination with radial-profiling to quantify the spatio-temporal dynamics of various GFP-fusion proteins in Drosophila syncytial embryos. We selected the nine proteins, including Cnn, which have been most strongly implicated in the PCM recruitment in flies: Asl-GFP, AurA-GFP, GFP-Cnn, DGp71WD-GFP, D-PLP-GFP, DSas-4-GFP, DSpd-2-GFP, γ-tubulin-GFP, and Polo-GFP. We assessed the expression level of these fusion proteins relative to their endogenous proteins by Western blotting (Figure 1—figure supplement 1).

6Prior to photobleaching, the fusion proteins displayed different centrosomal distributions (Figure 1A). Asl-GFP and DSas-4-GFP are known to be closely associated with centrioles, and their fusions were tightly localized in the centre of the PCM; they exhibited a fluorescence intensity profile similar to that of sub-resolution (170 nm) beads (Figure 1A), indicating that their true distribution was below the resolution of our microscope system. The other proteins were all distributed more broadly throughout the PCM to varying extents.

7After photobleaching, the Asl-GFP, Sas-4-GFP, AurA-GFP, DGp71WD-GFP, γ-tubulin-GFP, and Polo-GFP fluorescence all recovered at different rates, but each protein appeared to recover evenly throughout the domain that it originally occupied (Figure 1B,D; Figure 1—figure supplement 2; Video 1A,B). This even recovery was confirmed when the recovery profiles were normalized so that their peak fluorescence intensity at each time-point equalled one; this showed that, at all time-points, the shape of each normalized recovery curve closely matched the shape of its respective pre-bleached profile (Figure 1E, Figure 1—figure supplement 2; Video 1C). We conclude that most PCM proteins are recruited to centrosomes by binding sites that are already distributed throughout the PCM volume that each protein occupies (Figure 1H). These observations strongly support the idea that most PCM proteins are recruited by an underlying scaffold structure.

8We note that the dynamics of D-PLP-GFP are complicated by the presence of two distinct centrosomal fractions of D-PLP: one fraction tightly localized at the centriole that turns over slowly, and another that localizes to the PCM and turns over rapidly (Figure 1—figure supplement 2K,L). Nevertheless, it was clear from recovery profiles that the PCM fraction of D-PLP-GFP did not initially recover centrally and then move outwards, but rather recovered throughout the PCM volume.

9Unlike the other PCM components we tested, DSpd-2-GFP fluorescence initially recovered only in the central region of the PCM and then gradually began to recover in the more peripheral regions (Figure 1C,F,G; Video 1D–F). This suggested that, like Cnn (; Figure 1—figure supplement 2M,N), DSpd-2 is recruited to binding sites that are only located close to the centrioles; once released from these sites, it then spreads slowly outward into the more peripheral regions of the PCM (Figure 1I).

10A comparison of DSpd-2-GFP recovery kinetics in the central and peripheral regions strongly supported this interpretation (Figure 2A–C). The central region of DSpd-2-GFP fluorescence exhibited a typical, logarithmic-shaped, FRAP recovery curve, with a fast initial recovery rate that gradually slowed over time (green line, Figure 2B). In contrast, the peripheral regions exhibited an unusual FRAP recovery curve, with a slow initial recovery rate that gradually increased over time (solid red line, Figure 2B); this was best seen when the peripheral recovery curve was normalized so that the pre-bleached signal equalled one (dotted red line, Figure 2B). These unusual recovery dynamics at the periphery of the PCM can most easily be explained if DSpd-2-GFP molecules are constantly moving from the centre of the centrosome to the periphery: as the number of fluorescent molecules in the centre increases with time after photobleaching, so the number of fluorescent molecules moving from the centre into the periphery gradually increases, explaining why the peripheral recovery rate speeds up over time.

11We previously used super-resolution structured illumination microscopy (3D-SIM) in living fly embryos to show that the GFP-Cnn scaffold radiates away from the centrioles and forms large projections that extend outwards along the centrosomal MTs (; Figure 3A). DSpd-2-GFP had a similar distribution with several spoke-like projections extending from a central ring (Figure 3C), although, in agreement with our radial profiling data, much less DSpd-2 extended into the more peripheral regions of the PCM (Figure 3C). Two-colour 3D-SIM in living embryos confirmed that DSpd-2-GFP and mCherry-Cnn extensively overlapped in the more central regions of the PCM (Figure 3E, Figure 3—figure supplement 1) and revealed that the more peripheral Cnn projections usually contained small amounts of DSpd-2 (arrowheads, Figure 3E and Figure 3—figure supplement 1B–D).

12Unsurprisingly, the GFP–Cnn projections (Figure 3A) were no longer visible when the MTs were depolymerized with colchicine, and the GFP–Cnn scaffold became more compact and amorphous in appearance (Figure 3B). The small amounts of DSpd-2 extending into the peripheral PCM (Figure 3C) were also lost when the MTs were depolymerized (Figure 3D), but the more central DSpd-2-GFP region retained a clear organisation, with several spoke-like projections of DSpd-2-GFP emanating outward into the PCM from a central ring (Figure 3D). We conclude that DSpd-2-GFP is part of a scaffold-like structure that forms around the centrioles and that retains some macromolecular structural integrity even in the absence of MTs.

13In our 3D-SIM images, we noticed that DSpd-2 and, to a lesser extent, Cnn both seemed to emanate from a single toroidal structure that we presume contains the mother centriole (arrows, Figure 3A,C). To test if this structure was really the source of the centrosomal DSpd-2 and Cnn, we modified our 3D-SIM system to enable us (to our knowledge for the first time) to combine 3D-SIM with FRAP (‘Materials and methods’). This analysis revealed that after photobleaching DSpd-2-GFP fluorescence initially recovered in a toroidal pattern (t = 30 s, Figure 4A) and then moved slowly outwards on dynamic projections (t = 120 s to t = 450 s, Figure 4A; Video 2A). The initial toroidal distribution was very similar to that previously reported for Asl, one of the several proteins that form a toroid specifically around the mother centriole (compare the recovering DSpd-2-GFP signal at t = 30 s in Figure 4A to the unbleached Asl-GFP signal shown in the inset; also compare the average profile of Asl-GFP to the average initial recovery profile of DSpd-2-GFP, Figure 4C). Thus, DSpd-2-GFP is initially incorporated into the PCM by binding sites that are tightly concentrated around only one of the two centrioles—almost certainly the mother—and the spatial distribution of these sites extensively overlaps with the distribution of Asl.

14Interestingly, when we performed 3D-SIM FRAP with GFP-Cnn, the fluorescence initially recovered in the central region of the PCM (t = 60 s, Figure 4B), but this region was significantly broader than the toroidal region in which DSpd-2-GFP initially recovered (see t = 30 s, Figure 4A). Moreover, centrosomal GFP-Cnn fluorescence recovered more slowly than that of DSpd-2-GFP (compare Figure 1F to Figure 1—figure supplement 2M). Thus, although the centrosomal binding sites for DSpd-2 and Cnn appear to be concentrated around the mother centriole, they do not precisely overlap and they recruit DSpd-2 and Cnn at different rates, strongly suggesting that DSpd-2 and Cnn are not incorporated into the PCM together as part of the same complex.

15We wondered how DSpd-2 might be recruited to the toroidal structure surrounding the mother centriole. The centriole proteins DSas-4, Asl, and D-PLP all localize in a toroid pattern around mother centrioles and have all been implicated in PCM recruitment, suggesting that they could help recruit DSpd-2. Because DSas-4 and Asl mutants lack centrioles, we tested this possibility by inhibiting the function of these proteins in embryos using antibody-injection. Anti-Asl antibodies bound to centrosomes and reduced the rate of DSpd-2-GFP incorporation into the PCM by ∼75%, whereas anti-DSas-4 and anti-D-PLP antibodies had little effect (Figure 5)—although these antibodies bound to centrioles/centrosomes and at least partially disrupted the function of their cognate protein (;; unpublished data). These observations are consistent with the previous finding that Asl is required for the centrosomal localization of DSpd-2. Moreover, our yeast two-hybrid (Y2H) analysis revealed several strong direct interactions between Asl and DSpd-2 (Figure 6, Figure 6—source data 1). Although we cannot be certain that Asl and DSpd-2 interact directly in vivo, collectively our data indicate that Asl has an important, and potentially direct, role in recruiting DSpd-2 to mother centrioles in fly embryos.

16We next addressed how Cnn might be recruited to mother centrioles. We previously showed that anti-DSpd-2 or anti-Asl antibodies, but not anti-D-PLP or anti-Sas-4 antibodies, inhibit the rate of Cnn incorporation into the PCM by ∼75% and ∼55%, respectively, suggesting that both DSpd-2 and Asl may have a role in recruiting Cnn to centrosomes. We noticed, however, that the distribution of DSpd-2-GFP, but not Asl-GFP, around the mother centriole closely matched the distribution of the initial binding sites for GFP-Cnn, as revealed by our 3D-SIM FRAP experiments (compare the recovering GFP-Cnn signal at t = 60 s in Figure 4B to the unbleached DSpd-2-GFP signal at t = −30 s in Figure 4A; also compare the average profile of DSpd-2-GFP to the average initial recovery profile of GFP-Cnn, Figure 4D). This suggests that DSpd-2 may provide the major centrosomal binding site for Cnn in embryos.

17In support of this possibility, we found that the amount of Cnn localized to centrosomes in eggs was reduced by ∼80% in the absence of DSpd-2 (Figure 7) consistent with previous reports that the centrosomal localization of Cnn is perturbed in dspd-2 mutant brain cells. Moreover, our Y2H analysis revealed several strong direct interactions between DSpd-2 and Cnn but only one, much weaker, direct interaction between Cnn and Asl (Figure 6, Figure 6—source data 1). Although we cannot be certain that Cnn and DSpd-2 interact directly in vivo, collectively our data indicate that DSpd-2 has an important, and potentially direct, role in recruiting Cnn to mother centrioles in fly embryos, while Asl appears to have a more minor role.

18To investigate whether Cnn has a role in localizing DSpd-2 to centrosomes, we examined the distribution and dynamics of DSpd-2-GFP in cnn mutant embryos. Although there was a dramatic reduction in the amount of DSpd-2-GFP associated with centrosomes in the absence of Cnn (Figure 8A,B), FRAP experiments revealed that the initial rate of DSpd-2-GFP incorporation was unperturbed (Figure 8C–E). The localization of Asl-GFP (which appears to recruit DSpd-2) was also largely unperturbed in cnn mutant embryos (Figure 8F,G). The residual DSpd-2-GFP appeared to be more tightly associated with the centrioles in the absence of Cnn (Figure 8B), while the PCM fraction of the protein moved rapidly away from the centrioles in small ‘flares’ (Video 4). These observations suggest that Cnn is not required for the initial incorporation of DSpd-2 into the PCM, but is required for the proper maintenance of DSpd-2 within the PCM.

19The experiments described above are consistent with the idea that DSpd-2 and Cnn form molecular scaffolds during mitosis that initially assemble around the mother centriole and then move slowly outward. Both proteins have been implicated in PCM recruitment, so we reasoned that such scaffolds might provide a platform on which other PCM proteins assemble during mitosis.

20To test this idea, we analyzed larval brain cells where centrioles organize almost no PCM or MTs during interphase but mature to organize large amounts of both during mitosis (;; Figure 9A). As shown previously, centrosome maturation in mitotic brain cells that lacked either Cnn or DSpd-2 was perturbed, but not abolished, and mitotic centrosomes organized levels of PCM that were significantly above those observed in interphase cells (Figure 9B,C,F,G). Remarkably, however, centrosome maturation appeared to be abolished in the mitotic brain cells lacking both Cnn and DSpd-2 (Figure 9D,H). This effect was specific to the loss of both Cnn and DSpd-2, as centrosomes in mitotic cells lacking either Cnn or DSpd-2 that also lacked the abundant PCM protein D-TACC could still partially mature (Figure 9—figure supplement 1). Thus, most PCM proteins appear to rely on Cnn and DSpd-2 for their recruitment to mitotic centrosomes.

21To test whether centrioles in cells lacking both Cnn and DSpd-2 were really unable to form mitotic MTOCs, we analyzed MT behaviour. In fixed prophase cells (when centrosomal MT asters are most prominent), centrosomal asters were detectable in 100% of WT cells (Figure 9I; n = 11), 55% of cells lacking Cnn (Figure 9J; n = 58), 57% of cells lacking DSpd-2 (Figure 9K; n = 14), but in 0% of cells lacking both proteins (Figure 9L; n = 15). In live mitotic brain cells co-expressing the centriolar marker GFP-PACT and the MT marker Jupiter-mCherry (Figure 9—figure supplement 2; Video 5), centrosomes with MT asters were detectable in almost all cells lacking either Cnn (n = 18/18) or DSpd-2 (n = 24/25), but in almost no cells lacking both proteins (n = 1/24). We conclude that cells lacking both DSpd-2 and Cnn are unable to assemble centrosomal MTOCs during mitosis.

22Several hundred proteins are recruited to the PCM that expands around the centrioles during centrosome maturation in mitosis, but how so many proteins are organized into a functional mitotic centrosome has remained mysterious. Remarkably, we show here that the assembly of the mitotic PCM in flies appears to depend on just two proteins, Cnn and DSpd-2. Both proteins appear to form scaffolds that initially assemble around the mother centriole and then spread outward, forming a dynamic platform upon which most, if not all, other PCM proteins ultimately assemble. DSpd-2 and Cnn partially depend on each other for their centrosomal localization, and both proteins are required to ensure robust centrosome maturation. In the absence of one of these proteins, reduced levels of the other protein still localize around the centrioles and can support the partial assembly of the mitotic PCM. In the absence of both the proteins, mitotic PCM assembly appears to be abolished (Figure 9).

23How are DSpd-2 and Cnn recruited to mother centrioles? Our results strongly suggest that in fly embryos Asl initially helps recruit DSpd-2 to centrioles and DSpd-2 then helps to recruit Cnn. Cnn does not appear to be required to recruit either Asl or DSpd-2 to centrosomes, but it is required to properly maintain DSpd-2 within the PCM. We speculate that this interaction between DSpd-2 and Cnn creates a positive feedback loop that drives the dramatic expansion of the PCM scaffold around mother centrioles during mitosis (Figure 10A). Although, we have identified direct interactions between Asl and DSpd-2 and between DSpd-2 and Cnn by Y2H, and the endogenous proteins can all co-immunoprecipitate with one another in fly embryo extracts, we stress that we cannot be certain that these interactions are direct in vivo.

24The requirement for Asl to initiate the mitotic recruitment of DSpd-2 and Cnn probably explains why these proteins are specifically recruited to mother centrioles. We recently showed that although Asl is essential for centriole duplication, it is not incorporated into daughter centrioles until they have passed through mitosis and matured into new mother centrioles, and Asl/Cep152 proteins mainly localize to mother centrioles in several species. The PCM appears to be preferentially associated with mother centrioles in many systems. Our findings provide a potential explanation for why this is so, and raise the intriguing possibility that all mitotic PCM may be organized exclusively by mother centrioles.

25Although DSpd-2 seems to be the major recruiter of centrosomal Cnn in embryos, there must be an alternative recruiter, as the centrosomal localization of Cnn is not abolished in the absence of DSpd-2 (Figure 7, Figure 9C). Asl is an attractive candidate as anti-Asl antibodies perturb Cnn recruitment to centrioles (although this could be an indirect consequence of their effect on DSpd-2 recruitment), and Asl and Cnn interact in our Y2H analysis. Moreover, human Cep152/Asl has a role in the centrosomal recruitment of human Cdk5Rap2/Cnn. Interestingly, in flies this alternative pathway appears to be stronger in larval brain cells than in eggs/embryos: in the absence of DSpd-2, Cnn levels are reduced by only ∼35% in brains (Figure 9C) but by ∼80% in eggs (Figure 7C). Thus, the detail of mitotic PCM assembly pathway may vary between different cell types even in the same species.

26Our data suggest that after DSpd-2 and Cnn have been recruited to centrioles, they rapidly assemble into scaffolds that then move slowly away from the centrioles. For Cnn, there is strong data indicating that scaffold assembly is regulated by phosphorylation. Cnn contains a phospho-regulated multimerization (PReM) domain that is phosphorylated by Polo/Plk1 in vitro and at centrosomes during mitosis in vivo. Mimicking phosphorylation allows the PReM domain to multimerize in vitro and Cnn to spontaneously assemble into cytosolic scaffolds in vivo that can organize MTs. Conversely, ablating phosphorylation does not interfere with Cnn recruitment to centrioles, but inhibits Cnn scaffold assembly. We speculate that, like Cnn, DSpd-2 can assemble into a scaffold and that this assembly is regulated in vivo so that it only occurs around mother centrioles. It remains unclear, however, whether DSpd-2 itself can form a scaffold, or whether it requires other proteins to do so.

27It is striking that both DSpd-2 and Cnn exhibit an unusual dynamic behaviour at centrosomes. Both proteins incorporate into the PCM from the inside out, and are in constant flux, as the molecules that move slowly outward away from the centrioles are replaced by newly incorporated molecules close to the centriole surface (see Figure 1I). This inside out assembly is likely to have important consequences, as it means that the events close to the centriole surface, rather than at the periphery of the PCM, can ultimately regulate mitotic PCM assembly. This may be particularly important in cells where centrioles organise centrosomes of different sizes, as is the case in certain asymmetrically dividing stem/progenitor cells. Fly neural stem cells, for example, use centrosome size asymmetry to ensure robust asymmetric division, and there is strong evidence that new and old mother centrioles differentially regulate the rate of Cnn incorporation in these cells. Moreover, mutations in human Cdk5Rap2/Cnn have been implicated in microcephaly, a pathology linked to a failure in neural progenitor cell proliferation, although the precise reason for this is unclear.

28Although DSpd-2 and Cnn have a major role in centrosome maturation, we stress that other PCM components are likely to make important contributions. Pericentrin, for example, has been implicated in PCM recruitment in several systems, and the fly homologue, D-PLP, forms ordered fibrils in cultured S2 cells that extend away from the centriole wall and support PCM assembly in interphase. These centriolar fibrils, however, cannot explain how centrioles organize such a vastly expanded PCM matrix during mitosis, and D-PLP appears to have an important, but more minor, role in mitotic PCM assembly in vivo. Nevertheless, proteins like D-PLP will certainly help recruit other PCM proteins and help form structural links within the PCM, thus strengthening the mitotic PCM matrix. The important distinction is that, in flies at least, most proteins, including the PCM fraction of D-PLP, are recruited into the PCM by an underlying PCM scaffold, whereas DSpd-2 and Cnn appear to form this scaffold.

29Homologues of Asl, DSpd-2, and Cnn have been implicated in PCM assembly in many species, suggesting that the mechanism of mitotic PCM recruitment we identify here in flies may be conserved through evolution. To our knowledge, however, no PCM component has yet been shown to assemble from the inside out and to flux away from the centrioles in any other system. Nevertheless, although the precise molecular details will likely vary from cell type to cell type and from species to species, we suspect that this unusual dynamic behaviour of an underlying mitotic PCM scaffold will prove to be a general feature of mitotic centrosome assembly in many systems.

30P-element-mediated transformation vectors were made by introducing a full-length DGp71WD or D-PLP cDNA into the Ubq-GFPCT Gateway vector. Transgenic lines were generated either by Genetic Services, Inc., Cambridge, MA, Bestgene, Inc., Chino Hills, CA or the Fly Facility in the Department of Genetics, Cambridge, UK. Other GFP, RFP, and mCherry fusions have been described previously: GFP-Cnn, DSpd-2-GFP, Aur-A-GFP, RFP-PACT, Polo-GFP, Jupiter-mCherry, γ-tubulin-GFP, DSas-4-GFP, and Asl-GFP.

31To examine the dynamics of DSpd-2-GFP and Asl-GFP at centrosomes, using both standard spinning disc confocal imaging and 3D-SIM imaging, we analyzed embryos from mothers expressing two copies of Ubq-DSpd-2-GFP in a DSpd-2Z35711/DSpd-2Df(3L)st-j7 hemizygous mutant background or two copies of Asl-GFP (expressed from its endogenous promoter) in an aslmecd mutant background. For analysing the dynamics of γ-tubulin-GFP, AurA-GFP, DGp71WD-GFP, Polo-GFP, DSas-4-GFP, and D-PLP-GFP at centrosomes, we analyzed embryos from mothers expressing either two copies of γ-tubulin-GFP (expressed under the ncd promoter), Ubq-AurA-GFP, Ubq-DGp71WD-GFP, Polo-GFP (expressed under its endogenous promoter), DSas-4-GFP (expressed under its endogenous promoter) or one copy of Ubq-D-PLP-GFP in a WT background. For analysing the dynamics of GFP-Cnn at centrosomes using 3D-SIM imaging, we analyzed embryos from mothers expressing two copies of Ubq-GFP-Cnn in a cnnf04547/cnnHK21 hemizygous mutant background. For comparing the dynamics of DSpd-2-GFP in a WT background to the dynamics of DSpd-2-GFP in the absence of Cnn, we analyzed embryos from mothers expressing one copy of pUbq-DSpd-2-GFP in a WT background and embryos from mothers expressing one copy of pUbq-DSpd-2-GFP in a cnnHK21/cnndf(2R)BSC306 hemizygous mutant background. For comparing the localization of Asl-GFP in a WT background to the localization of Asl-GFP in the absence of Cnn, we analyzed embryos from mothers expressing one copy of pUbq-Asl-GFP in a WT background and embryos from mothers expressing one copy of pUbq-Asl-GFP in a cnnf04547/cnnHK21 hemizygous mutant background.

32For examining PCM recruitment in larval brain cells lacking Cnn, DSpd-2 or Cnn, and DSpd-2, we analyzed cnnf04547/cnnHK21 hemizygous mutants, dspd-2Z35711/dspd-2Df(3L)st-j7 hemizygous mutant, or cnnf04547/cnnHK21; dspd-2Z35711/dspd-2Df(3L)st-j7 double hemizygous mutant larval brains, respectively.

33For examining the behaviour of MTs in living larval brain cells, we analyzed brains expressing one copy of Ubq-GFP-PACT and one copy of Jupiter-mCherry (expressed under its endogenous promoter) in either a WT, cnnf04547/cnnHK21 hemizygous mutant, dspd-2Z35711/dspd-2Df(3L)st-j7 hemizygous mutant, or cnnf04547/cnnHK21; dspd-2Z35711/dspd-2Df(3L)st-j7 double hemizygous mutant background.

34For immunofluorescence analysis, we used the following antibodies: rabbit anti-Cnn (1:1000), rabbit anti-DSpd-2 (1:500), mouse anti-γ-tubulin (1:500; GTU88, Sigma-Aldrich, St. Louis, MO), rabbit anti-D-TACC (1:500), rabbit anti-DGp71WD (1:500), rabbit anti-AurA (1:500), mouse anti-α-tubulin (1:1000; DM1α; Sigma-Aldrich), guinea-pig anti-Asl (1:500) (this study), and anti-PhosphoHistoneH3 (mouse, 1:2000, AbCam, UK or rabbit, 1:500, Cell Signalling Technology, Danvers, MA). Secondary antibodies were from Molecular Probes (Invitrogen, Carlsbad, CA): Alexa Fluor 488, 568, and 647 (all used at 1:1000).

35For antibody injection experiments, we used rabbit anti-Asl (aa665–995), rabbit anti-DSas-4 (aa1–260), and rabbit anti-D-PLP (aa683–974) affinity purified antibodies. We also tested rabbit anti-D-PLP antibodies raised against aa1805–2137, which are predicted to also recognise the N-terminus of the short D-PLP isoform (aa8–350), and found that these antibodies, like the rabbit anti-D-PLP (aa683–974) antibodies, did not significantly perturb the DSpd-2-GFP incorporation (data not shown).

36FRAP experiments were carried out as described previously. Photobleaching was carried out in S phase, which is when mitotic PCM is most actively recruited in these rapidly cycling embryos. We used ImageJ to calculate the fluorescence profile of each centrosome at each time-point. We first scaled the images so that each pixel was split into 25 (5 × 5) pixels in order to increase the resolution of our radial profiling. We then calculated the centre of mass of the centrosome by thresholding the image and running the ‘analyze particles’ (centre of mass) macro on the most central Z plane of the centrosome. We then centred concentric rings (spaced at 0.028 μm and spanning across 3.02 μm) on this centre and measured the average fluorescence around each ring (radial profiling). After subtracting the average cytosolic signal and normalising, so the peak intensity of the pre-bleached image was equal to 1, we mirrored the profiles to show a full symmetric centrosomal profile. For each time-point, an average distribution from at least 10 centrosomes was calculated.

37For analysing DSpd-2-GFP recovery in the centre and periphery of the PCM, we bleached centrosomes at the start of S-phase and then again 3 minutes later (still in S-phase); we measured the fluorescence recovery using radial profiling, as described above. The central PCM measurements were calculated as the average fluorescence intensity of 5 measurements taken between 0.028 μm and 0.14 μm, from the centre of the centrosome. The peripheral PCM measurements were calculated as the average fluorescence intensity of 5 measurements taken between 0.62 mm and 0.73 μm from the centre of the centrosome. Ten centrosomes from 10 embryos were analyzed; values were averaged to produce each data point. The cytosolic signal was subtracted before plotting the recovery graphs. To examine AurA-GFP and Polo-GFP recovery in the centre and periphery of the PCM, the original AurA-GFP and Polo-GFP FRAP data were re-analyzed by measuring the recovery in the same regions as for the DSpd-2-GFP analysis.

38We fixed embryos in methanol, homogenized 50 embryos per genotype in 100 μl sample buffer, and ran either 5 μl or 10 μl on NuPAGE 3–8% Tris-acetate pre-cast gels (Life Technologies, Carlsbad, CA). The proteins were transferred onto nitrocellulose membrane and loading was initially checked using Ponceau staining. The membrane was then blocked and probed with antibodies against the protein in question and against the GFP.

39Living embryos were imaged at 21°C on a DeltaVision OMX V3 Blaze microscope (GE Healthcare, UK) equipped with a 60x/1.42 oil UPlanSApo objective (Olympus), 405 nm and 488 nm and 593 nm diode lasers and sCMOS cameras (PCO). 3D-SIM image stacks were acquired with 5 phases 3 angles per image plane and 0.125μm z-distance between sections. The raw data was computationally reconstructed with SoftWoRx 6.0 (Applied Precision) using Wiener filter settings 0.002 and channel specifically measured optical transfer functions to generate a super-resolution 3D image stack with a lateral (x-y) resolution of 100-130 nm (wavelength-dependent) and an axial (z) resolution of ∼300 nm. For two colour images, Images from the different color channels were registered with alignment parameter obtained from calibration measurements with 0.2 μm diameter TetraSpeck beads (Life Technologies) using the OMX Editor software. Images were processed using SoftWorx software (GE Healthcare). Images shown are maximum intensity projections of several z-slices. When analysing the effect of MT de-polymerisation, embryos were first injected with 1 mM colchicine solution and imaged 20–60 min later.

40To perform 3D-SIM FRAP, we utilized the software development kit (SDK) from GE Healthcare. This allowed us to create a custom acquisition sequence that first acquired a single Z-stack in 3D-SIM, then performed single or multiple spot photobleaching (using the standard OMX galvo scanner TIRF/photo-kinetics module), and then performed time lapse imaging in 3D-SIM mode.

41The centrosomal profiles were calculated in a similar way to that described above, except that the concentric rings for Asl-GFP, DSpd-2-GFP, and GFP-Cnn were spaced at 0.0055 μm, 0.011 μm, and 0.0109 μm and spanned across 1.86 μm, 3.28 μm, and 3.28 μm, respectively. For generating the average 3D-SIM profiles for Asl-GFP, DSpd-2-GFP, and GFP-Cnn, we averaged profiles from 11, 24, and 15 centrosomes, respectively.

42Affinity-purified antibodies were covalently coupled to Texas Red, as described previously. Antibodies were injected at the start of a mitotic cycle, and embryos were observed on the Spinning Disk confocal system described above. Centrosomes were bleached in pairs: one centrosome located close to the injection site (experimental) and one centrosome located far from the injection site (control). Between 2 and 3 centrosome pairs were bleached per embryo, with an average of 7 embryos injected for each antibody, and the data were collated. The average initial rate of DSpd-2-GFP incorporation at control and experimental centrosomes was compared using a paired Student's t test.

43Bait and prey fragments were cloned, introduced into yeast, and tested for interactions as described previously. For the baits, fragments encoding the N-terminal, middle, and C-terminal thirds of the proteins were cloned, along with fragments encoding the N-terminal two-thirds, C-terminal two-thirds, and the full-length protein. For the preys, smaller ∼200 aa fragments and larger combinations of these fragments, including the full-length protein, were cloned.

44For the analysis of centrosomal fluorescence levels of PCM components, third instar larval brains were dissected and incubated in 100 mM colchicine in Schneider's medium, Sigma-Aldrich for 1 hr at 25°C. Colchicine treatment de-polymerizes the MTs and prevents centrosome ‘rocketing’ in cnn mutants, allowing a more accurate quantification of PCM recruitment. The brains were then fixed in paraformaldehyde containing 100 mM PIPES, 1 mM MgSO4, and 2 mM EGTA pH 6.95 for 5 min at room temperature, washed in PBS and then 45% and 60% acetic acid, squashed under a coverslip, post-fixed in methanol, washed in PBT, and then stained with the appropriate antibodies. Images were collected on an Olympus FV1000 scanning confocal microscope using a 60×, 1.4 NA oil objective and maximum intensity projections were made. For each brain at least five images containing multiple cells in both mitosis (as shown by positive Phospho-Histone H3 staining) and interphase were collected. At least fivebrains were imaged for each mutant and staining combination and an average of 40 mitotic and 37 interphase centrosomes were measured per brain. Centrosome fluorescence was calculated by measuring the total fluorescence in a boxed region around the centrosome and subtracting the local cytoplasmic background fluorescence. The average value of all the centrosomes from a single brain was used for each data point. The average value of these data points for mitotic and interphase cells were compared using a Mann–Whitney test. For the analysis of MT asters, the cells were fixed and stained as above, but were not pre-treated with colchicine. A cell was scored as positive if at least 1 centrosome had detectable astral MTs.

45Third instar larval brains were dissected and either semi-squashed under a coverslip or mounted whole in Schneider's medium and then imaged on the Perkin Elmer Spinning Disk confocal system described above. Cells were filmed progressing from interphase/prophase through mitosis. A cell was scored as positive if at least 1 centrosome had detectable astral MTs.