Centrosomes are microtubule-organizing centers and play a dominant role in assembly of the microtubule spindle apparatus at mitosis. Although the individual binding steps in centrosome maturation are largely unknown, Centrosomin (Cnn) is an essential mitotic centrosome component required for assembly of all other known pericentriolar matrix (PCM) proteins to achieve microtubule-organizing activity at mitosis in Drosophila. A conserved motif (Motif 1) has been identified near the amino terminus of Cnn that is essential for its function in vivo. Motif 1 has a higher degree of sequence conservation (40% identity/49% similarity) between Cnn and human CDK5RAP2 and is present in all homologues from S. pombe to human. Cnn Motif 1 is necessary for proper recruitment of γ-tubulin, D-TACC (the homolog of vertebrate transforming acidic coiled-coil proteins [TACC]), and Minispindles (Msps) to embryonic centrosomes but is not required for assembly of other centrosome components including Aurora A kinase and CP60. Centrosome separation and centrosomal satellite formation are severely disrupted in Cnn Motif 1 mutant embryos. However, actin organization into pseudocleavage furrows, though aberrant, remains partially intact. These data show that Motif 1 is necessary for some but not all of the activities conferred on centrosome function by intact Cnn (Zhang, 2007).

Previous studies showed that Cnn is required for centrosome assembly/maturation, for microtubule assembly from the centrosome at mitosis, and to organize actin into pseudocleavage furrows in the early embryo. It is shown here that Motif 1 of Cnn is required for specific and essential aspects of centrosome function. Centrosomes assembled in cnn^β1 embryos recruit some PCM components and are partially proficient to organize actin into pseudocleavage furrows, but do not properly recruit or maintain proteins with an established role in microtubule assembly: γ-tubulin, D-TACC, and Msps. Thus, although astral microtubules are produced at cnn^β1 mutant centrosomes, centrosome separation, a microtubule-dependent process, is severely affected. In addition, the less-understood process of satellite formation is inhibited at cnn^β1 centrosomes (Zhang, 2007).

Microtubule assembly at centrosomes is regulated by nucleation, where γ-Tub plays a key role, and by microtubule growth, which depends on a host of factors including Aurora A, D-TACC, and Msps, that promote stability. How these proteins are assembled and regulated is still largely unknown. This study shows that Cnn Motif 1 controls assembly of PCM proteins that are required for MTOC activity at centrosomes (Zhang, 2007).

γ-Tub is an essential component of MTOCs in eukaryotes for microtubule assembly. In cnn null mutant neuroblasts, imaginal disk cells, and cells depleted of Cnn by RNAi, neither γ-Tub nor astral microtubules are detected at centrosomes. However, in contrast to the above cell types, a Cnn-independent pool of γ-Tub is at the centrosome remnant in cnn null mutant early embryonic spindle poles. The small, sharp signal for γ-Tub at cnn null spindle poles implicates a centriolar pool of γ-Tub that is unique to the rapid divisions of early embryos. The level of γ-Tub at cnn^β1 mutant centrosomes is similar to the cnn null mutant, indicating that Motif 1 is required for recruitment of the Cnn-dependent pool of γ-Tub to the PCM in embryos. Drosophila Cnn and the S. pombe homolog Mto1p have been reported to coIP with γ-Tub, but a direct interaction with γ-Tub or any of the γ-TuRC proteins has not been demonstrated (Zhang, 2007).

D-TACC and Msps, and their counterparts in Xenopus (TACC3/maskin and XMAP215) and C. elegans (TAC-1 and ZYG-9) are direct binding partners required for centrosome-dependent growth of long microtubules. Mutation or depletion of D-TACC or its homologues does not affect γ-Tub localization to centrosomes, but rather appears to function with Msps in the stability of microtubules that are nucleated by γ-Tub. D-TACC and Msps are partially recruited to centrosomes in cnn null and cnn^β1 mutants, accumulating at the centrosome periphery in cnn^β1 embryos. This incomplete assembly suggests that recruitment of D-TACC and Msps to centrosomes normally involves at least two steps and that Motif 1 of Cnn is required for a secondary step in the process subsequent to docking of D-TACC at the periphery of the centrosome. Thus, Cnn Motif 1 may be required for a later phase of recruitment to the centrosome or have a role in maintaining D-TACC and Msps once they are recruited (Zhang, 2007).

Aurora A kinase is required to localize D-TACC to centrosomes and directly phosphorylates D-TACC at Ser863 to activate its microtubule-stabilizing activity. The reduced recruitment of Aurora A to cnn null centrosomes further highlights the requirement for Cnn in PCM assembly. However, Aurora A localization did not appear affected in cnn^β1 embryos, indicating that, although Aurora A is necessary to recruit D-TACC/Msps, its localization at centrosomes is not sufficient to accomplish this. Aurora A binds directly to the C-terminal half of Cnn, which remains intact in the cnn^β1 mutant. Moreover, D-TACC is phosphorylated by Aurora A in cnn^β1 embryos; however, this activated pool of D-TACC is exiled to the centrosome periphery with the bulk pool of centrosomal D-TACC. This indicates that Motif 1 of Cnn is required for anchoring or maintaining D-TACC at centrosomes subsequent to its regulatory phosphorylation by Aurora A. Alternatively, because the immunofluorescence signal for P-D-TACC was weak and P-D-TACC levels were not quantified, an effect of cnn^β1 on Aurora A activity toward D-TACC cannot be excluded (Zhang, 2007).

In cnn^β1 and cnn null embryos microtubule asters are present, particularly at early cortical cycles (cycles 10 and 11). At later cycles asters are not detected at spindle poles in cnn null embryos, coinciding with centriole loss, which is evident from the absence of Nek2 kinase (a centriolar protein) signal. Centriole displacement from the spindle poles in cnn null embryos leads to centriole loss, resulting in anastral spindle poles (Lucas and Raff, personal communication to Zhang, 2007). By comparison to cnn null embryos, PCM integrity is restored to cnn^β1 mutant centrosomes, enough to retain centrosomes at spindle poles into later cleavage cycles and with retained ability to assemble astral microtubules. Nevertheless, centrosome separation failure indicates that microtubule-dependent processes are impaired at cnn^β1 centrosomes (Zhang, 2007).

Centrosome separation is a microtubule-dependent process that is coordinated by pushing forces from interpolar microtubules and forces supplied by molecular motors that include kinesin-5, kinesin-14 (Ncd), and dynein/Lis1/dynactin. The relative contributions of motor proteins and the pushing forces generated from the assembly of interpolar centrosomal microtubules have not been determined (Zhang, 2007).

A necessary role for microtubules in centrosome separation has been demonstrated using microtubule-depolymerizing drugs in cell culture and in early Drosophila embryos. Interpolar centrosomal microtubules may represent a specialized class of microtubules, an idea supported by the recent discovery of an α-tubulin variant, α4-tubulin, which is associated with faster-growing microtubules and is enriched in interpolar microtubules. α4-tubulin is required for centrosome separation in early embryos. Cnn localized more strongly to interpolar fibers compared with spindle microtubules, suggesting that Cnn Motif 1 may regulate the organization of interpolar centrosomal microtubules to promote centrosome separation. In instances when cnn^β1 centrosomes separated, interpolar fibers formed, suggesting that interpolar fibers are obligatory to centrosome separation. Although the proposal that Motif 1 regulates microtubule assembly to achieve centrosome separation is favored, a role for Motif 1 in regulating molecular motors that are involved in this process cannot be ruled out. However, localization of the kinesin-5/Eg5 family member Klp61F to spindle poles and spindle microtubules was no different in cnn^WT and cnn^β1 embryos (Zhang, 2007).

Consistent with a role for γ-Tub and D-TACC recruitment to centrosomes by Cnn Motif 1 in centrosome separation, depletion or mutation of γ-Tub, γ-TuSC proteins, and D-TACC also perturbed centrosome separation. Thus, γ-Tub at reduced levels and also astral microtubules cannot be detected, embryonic cnn^β1 centrosomes have insufficient or inappropriate microtubule assembly activity to achieve centrosome separation (Zhang, 2007).

It has been shown by live imaging of GFP-Cnn embryos that centrosomal satellites are highly dynamic structures that traffic in a microtubule-dependent and an actin-independent manner. Satellites, or 'flares,' emerge from the PCM and move bidirectionally at speeds of 4–20 µm min^–1 and are produced at highest numbers at telophase/interphase, coincident with the relative intensity of astral microtubules during the cleavage cycle. cnn^β1 mutant embryos produce significantly fewer satellites. Even incipient satellites, which are apparent on cnn^WT centrosomes and are present at colchicine-treated centrosomes, were nearly absent at cnn^β1 centrosomes. Satellite assembly may be an intrinsic function for Motif 1. Alternatively, fewer satellites may arise as a secondary consequence of altered MTOC activity at cnn^β1 centrosomes. Currently, it is not possible to distinguish between these two possibilities (Zhang, 2007).

The organization of actin into pseudocleavage furrows, an activity conveyed by centrosomes, is highly aberrant yet partially restored in cnn^β1 mutant embryos. This is in sharp contrast to cnn null embryos, where no apparent organization of cortical actin occurs. Although some studies have indicated that microtubules are required for cortical actin organization in the early Drosophila embryo, other evidence suggests that centrosomes organize actin and cortical polarity independent of microtubules. Because microtubule-dependent processes are disrupted in cnn^β1 embryos, the data support the model that centrosomes can organize actin independent of microtubules, but the possibility that cnn^β1 centrosomes produce sufficient astral microtubules to coordinate with actin in the assembly of furrows cannot be excluded (Zhang, 2007).

In summary, Motif 1, conserved among Cnn family members, is required for centrosome function in early embryos through the recruitment and anchoring of γ-Tub, D-TACC, and Msps, key factors in MTOC function in all eukaryotes where they have been examined. PCM architecture is partially restored in the cnn^β1 mutant compared with the cnn null, as shown by the normal distribution of CP60 and Aurora A. In addition, conspicuous yet aberrant pseudocleavage furrows assemble in cnn^β1 embryos but not in the cnn null, evidence that organization of actin by centrosomes is partially restored to cnn^β1 mutant centrosomes. This suggests that the activity to direct actin organization into cleavage furrows resides in another domain of Cnn. Identification of the direct binding partner for Cnn Motif 1 will be an important step toward understanding the relationship between Motif 1 and the MTOC functions that it governs (Zhang, 2007).

Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. This study shows that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles 'rocket' around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and only subtle defects are observed in the asymmetric divisions of mutant neuroblasts. It is concluded that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts (Lucas, 2007).

In fixed embryos and somatic cells that lack Cnn, PCM components are barely detectable at the poles of the mitotic spindles. Centrioles are still present in cnn mutant cells, but their function and positioning within the centrosome have not been analyzed. To understand better how Cnn normally recruits PCM components to the centrioles, transgenic Drosophila lines were generated expressing an mRFP-centriolar marker (either mRFP-Fzr or mRFP-PACT), together with one of three PCM markers fused to GFP: Aurora A-GFP, Grip75-GFP (a component of the γ-tubulin ring complex), and GFP- D-TACC. It has previously been shown that Cnn can interact with both the γ-tubulin ring complex and Aurora A, but this study found no evidence for an interaction between Cnn and D-TACC in coimmunoprecipitation experiments (Lucas, 2007).

In wild-type (WT) syncytial embryos, centrioles recruited approximately equal amounts of PCM at all stages of the rapid mitotic cycles, and they remained well centered within the PCM throughout the cell cycle. During interphase, the centrioles are always closely associated with the nuclear envelope, whereas in mitosis, they are always closely associated with the spindle poles. In embryos laid by cnn homozygous females (hereafter, cnn embryos), it was surprising to observe that the centrioles were associated with appreciable amounts of PCM, but they were often not properly centered within it. In video recordings of cnn embryos, the centrioles appeared to be constantly nucleating PCM but seemed unable to maintain their connection to it. The centrioles often exhibited irregular, stochastic movements, leaving a trail of PCM behind them as they moved away. This PCM trail was most easily seen in cnn embryos expressing GFP-D-TACC; this protein was recruited in particularly large amounts to the centrioles, and large clusters of GFP-D-TACC often remained in the cytoplasm for some time after the centrioles had moved away. Smaller amounts of Aurora A-GFP and Grip75-GFP were recruited to the centrioles, and so only small amounts of these proteins remained associated with the centrioles as they moved around the embryo. As a result of this abnormal centriole behavior, the centrioles in cnn embryos often lose their attachment to the nuclear envelope in interphase and to the spindle poles in mitosis. This behavior of the centrioles is referred as 'centriole rocketing' (Lucas, 2007).

Previous studies suggested that centrosomes lacking Cnn fail to function as MTOCs during mitosis. It was therefore examined whether the PCM organized by the centrioles in cnn embryos is capable of nucleating MTs. The centrosomes in cnn embryos organized astral MT arrays but seem unable to maintain their connection with them. When the embryos enter mitosis, many nuclei are not associated with centrioles, and anastral spindles assembled around the mitotic chromatin. Many nuclei, however, were close enough to a centriole for the astral MTs to contribute to spindle assembly. Often, however, these centrioles fail to maintain their position at the spindle pole and either wander around within the spindle or lose their connection to the spindle altogether. It is concluded that the dramatic mitotic defects observed in cnn embryos do not result from a failure of the centrioles to recruit PCM, or of the centrosomes to nucleate astral MTs, but instead result from the failure of the centrioles to maintain a stable connection to the PCM and MTs that they organize (Lucas, 2007).

The centriole rocketing appears to be driven by the asymmetric organization of the PCM and MTs around the centrioles. To test whether the rocketing is MT dependent, the MT-depolymerizing drug colchicine was injected into embryos. In WT embryos in late interphase, the centrioles have already migrated around the nuclei and, when their movement was plotted over time, the centrioles moved regularly across the embryo cortex. This regular movement of the centrosomes and their associated nuclei across the cortex is driven by actin- and myosin-dependent cortical contractions, and it continues after colchicine injection. In contrast, when centriole movement was plotted in cnn embryos in late interphase, the rocketing behavior was observed. The rocketing ceases after colchicine injection, and the centrioles revert to a regular movement across the embryo cortex. Thus, centriole rocketing in cnn embryos depends on intact MTs (Lucas, 2007).

The injection of colchicine into cnn embryos also enables the centrioles to remain associated with the PCM, suggesting that it is the MT-dependent rocketing of the centrioles that ultimately breaks the link between the centrioles and the PCM in cnn embryos. Intriguingly, however, the injection of colchicine into cnn embryos does not correct the positioning defect of the centrioles within the PCM: whereas the centrioles were usually (>90%) well centered within the PCM in colchicine-injected WT embryos, they were very rarely centered within the PCM in colchicine-injected cnn embryos (<10%) and were usually positioned at the very edge of the PCM (Lucas, 2007).

This last observation was unexpected, and no other perturbation to the centrosome is known that results in this very specific displacement of the centrioles from the center of the PCM. This observation may have important implications for understanding how Cnn functions to maintain the link between the centrioles and the PCM. One interesting possibility is that the MT-dependent centriole rocketing observed in cnn embryos may be mechanistically related to the actin-dependent rocketing of certain pathogenic bacteria. These bacteria are coated with proteins that initially stimulate the polymerization of an actin 'cloud' symmetrically around the surface of the bacteria. If the actin surrounding the bacteria is structurally weak, it can 'fracture', allowing the bacteria to move to the edge of the actin cloud and rocketing to begin. Thus, it is proposed that the primary function of Cnn may be to mechanically strengthen the PCM: in the presence of Cnn, the PCM is structurally strong and the centrioles can maintain their position at the center of the PCM; in the absence of Cnn, the PCM is weakened and the centrioles move to the edge of the PCM. This then initiates centriole rocketing, although the exact mechanism of this MT-dependent rocketing remains unclear (Lucas, 2007).

Maintaining the proper connection between the centrioles and the PCM is clearly crucial in syncytial embryos, as a lack of Cnn results in catastrophic failures in mitosis. In contrast, somatic cells that lack Cnn have few mitotic defects, and cnn mutant flies are viable. To test whether Cnn was required to maintain the proper connection between the centrioles and the PCM in somatic cells, third instar larval brain cells were treated with colchicine to depolymerize the MTs and then fixed and stained to examine the distribution of the centrioles and the PCM. It was found that hardly any PCM was detectable around the centrioles in cnn brain cells that had not been treated with colchicine. In cnn cells treated with colchicine, however, considerable amounts of PCM accumulated around the centrioles, but, as in cnn embryos, the centrioles were displaced from the center of the PCM (Lucas, 2007).

To further investigate whether the centrioles in cnn somatic cells behaved in the same way as the centrioles in cnn embryos, living third instar larval NBs expressing the centriole marker DSas-4-mRFP and GFP-α-tubulin were examined. In WT NBs entering mitosis, the centrioles were always centered within astral MT arrays, and the centrioles remained tightly associated with the poles of the spindle throughout mitosis. In contrast, the centrioles in cnn NBs were often not associated with prominent astral MTs and exhibited irregular movements throughout the cell during mitosis. As a consequence, they were often abnormally displaced from the poles of the mitotic spindles. Nevertheless, astral MTs associated with some of the 'rocketing; centrioles could be transiently detected in some cnn NBs. In fixed larval cnn NBs, the centrioles are often randomly positioned around the cell, and it was noticed that 20%-30% of brain cells had either too few or too many centrioles. Taken together, these findings suggest that the centriole behavior is similar in cnn embryos and somatic cells; while these defects do not lead to dramatic errors in somatic cell division, they do lead to errors in centriole segregation. These findings support the hypothesis that centrioles have evolved the ability to recruit PCM to ensure the equal partitioning of the centrioles during cell division, rather than to ensure the efficient assembly of the mitotic spindle (Lucas, 2007).

The Cnn-related protein CDK5RAP2 has been implicated in human microcephaly (Bond, 2005), and several recent studies have shown that centrosomes exhibit an asymmetric behavior during the asymmetric divisions of male germline stem cells (GSCs) and larval neural stem cells (NBs). During interphase in these cells, only one centrosome is initially associated with PCM and MTs, and this centrosome becomes anchored on one side of the cell (near the stem cell niche in GSCs, or near cortically localized cell polarity markers in NBs). When the second centrosome eventually associates with PCM and MTs, it localizes to the opposite side of the cell, thus ensuring that the forming mitotic spindle is correctly oriented relative to these positional cues (Lucas, 2007).

This asymmetric centrosome behavior appears to be important in male GSCs; the asymmetric division of these cells is dramatically perturbed in cnn mutants. Although cnn mutant NBs have defects in aligning their spindles with cortical determinants early in mitosis, it is not clear that this ultimately leads to failures in asymmetric division: early mitotic spindle alignment defects are often corrected in these cells by the time the cells divide. To determine whether cnn mutant NBs ultimately divide asymmetrically, living WT and cnn third instar larval NBs expressing only GFP-α-tubulin were analyzed (Lucas, 2007).

A single, anchored MTOC was usually visible in WT NBs before the entry into mitosis. After nuclear envelope breakdown (NEB), however, both centrosomes nucleated prominent arrays of MTs, and spindle assembly occurred primarily by a centrosomal pathway. As expected, the cells divided asymmetrically to produce a large NB and a small ganglion mother cell (GMC). In most cnn NBs, no prominent MTOC was detectable before NEB, and spindle assembly occurred largely by an acentrosomal pathway. Nonetheless, ~95% of cnn NBs ultimately divided asymmetrically, whereas ~4% divided symmetrically and ~1% failed in cytokinesis. Although this failure rate is modest, it is considerably higher than in WT, as was observed only one symmetric division in >100 WT central brain NBs examined (Lucas, 2007).

This study has shown that mutations in DSas-4, which encodes the Drosophila homologue of the human microcephaly protein CenpJ/CPAP, also lead to defects in the asymmetric divisions of larval NBs. The defects were much more severe in DSas-4 mutants, which completely lack centrioles/ centrosomes (~15% of NBs divided symmetrically, whereas ~15% failed in cytokinesis). The much milder defects in asymmetric division that were observe in cnn NBs suggest that centrosomes are partially functional as MTOCs in cnn mutant somatic cells. Indeed, relatively well-focused astral MT arrays were frequently observed forming and disassembling in the cytoplasm, and these were often transiently associated with the spindle poles in cnn NBs (Lucas, 2007).

Taken together, these observations on cnn and DSas-4 mutant NBs reveal that, unlike the situation in male GSCs, the asymmetric behavior of the centrosomes is not essential for the accurate asymmetric division of larval NBs. Nevertheless, mutations in the Drosophila homologues of two of the three human centrosomal proteins implicated in microcephaly do lead to relatively subtle defects in NB divisions in flies. Drosophila cnn and DSas-4 mutants do not have small brains, suggesting that flies are able to compensate for defects in these divisions in a way that perhaps humans cannot (Lucas, 2007).