Protein Interactions

Proper recruitment of γ-Tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires centrosomin motif 1

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 cnnβ1 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 cnnβ1 mutant centrosomes, centrosome separation, a microtubule-dependent process, is severely affected. In addition, the less-understood process of satellite formation is inhibited at cnnβ1 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 cnnβ1 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 cnnβ1 mutants, accumulating at the centrosome periphery in cnnβ1 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 cnnβ1 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 cnnβ1 mutant. Moreover, D-TACC is phosphorylated by Aurora A in cnnβ1 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 cnnβ1 on Aurora A activity toward D-TACC cannot be excluded (Zhang, 2007).

In cnnβ1 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 cnnβ1 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 cnnβ1 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 cnnβ1 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 cnnWT and cnnβ1 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 cnnβ1 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. cnnβ1 mutant embryos produce significantly fewer satellites. Even incipient satellites, which are apparent on cnnWT centrosomes and are present at colchicine-treated centrosomes, were nearly absent at cnnβ1 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 cnnβ1 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 cnnβ1 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 cnnβ1 embryos, the data support the model that centrosomes can organize actin independent of microtubules, but the possibility that cnnβ1 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 cnnβ1 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 cnnβ1 embryos but not in the cnn null, evidence that organization of actin by centrosomes is partially restored to cnnβ1 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).

Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila Centrosomin

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).

Centrocortin cooperates with centrosomin to organize Drosophila embryonic cleavage furrows

In the Drosophila early embryo, the centrosome coordinates assembly of cleavage furrows. Currently, the molecular pathway that links the centrosome and the cortical microfilaments is unknown. In centrosomin (cnn) mutants, in which the centriole forms but the centrosome pericentriolar material (PCM) fails to assemble, actin microfilaments are not organized into furrows at the syncytial cortex. Although CNN is required for centrosome assembly and function, little is known of its molecular activities. This study shows the novel protein Centrocortin (CEN; CG1962), which associates with centrosomes and also with cleavage furrows in early embryos, is required for cleavage furrow assembly. CEN binds to CNN within CNN Motif 2 (CM2), a conserved 60 amino acid domain at CNN's C terminus. The cnnB4 allele, which contains a missense mutation at a highly conserved residue within CM2, blocks the binding of CEN and disrupts cleavage furrow assembly. Together, these findings show that the C terminus of CNN coordinates cleavage furrow formation through binding to CEN, thereby providing a molecular link between the centrosome and cleavage furrow assembly (Kao, 2009).

To address the role of CEN in early embryos, the phenotype of a cen mutant was examined. A piggybac transposon insertion mutation within the coding sequence of cen at amino acid position 290 was available for this analysis. Maternal effect cen mutant embryos, collected from hemizygous cenf04787 mutant mothers [cenf04787 heterozygous with a deficiency chromosome, Df(2L)Fs(2)KetRX32, which deletes the cen locus], contained no detectable CEN protein by western blotting with antibodies directed against either the C or N terminus of CEN. A truncated protein product, predicted by the site of insertion of the transposon to be at least 33 kDa, was also not detected with the antibody directed against the amino terminus of CEN, which was raised against a polypeptide included within this truncation. Moreover, no CEN signal was detected at centrosomes or furrows in cenf04787 embryos upon immunostaining with CEN antibodies (Kao, 2009).

Homozygous and hemizygous cenf04787 mutants were viable and fertile. However, hemizygous cenf04787 females laid eggs that failed to hatch at a significantly higher rate of 8.70% ± 0.50% compared to wild-type females (4.29% ± 0.42%). A cen transgene that expresses a cen cDNA, including the entire coding sequence with the 5'and 3' UTRs, rescued this hatch rate deficiency to the levels seen with wild-type females. Together, these data show that cenf04787 is a cen mutation that reduces its expression to an undetectable level and affects embryonic development maternally (Kao, 2009).

To investigate CEN's role in centrosome and cleavage furrow function, hemizygous maternal cenf04787 mutant embryos, hereafter referred to as cenf04787 embryos, were stained for CNN, α-tubulin, and filamentous actin to examine centrosome and cytoskeleton organization during cleavage. In cenf04787 embryos, mitotic spindles were frequently linked together, a phenotype also characteristic of cnn mutant embryos, as well as an indication of furrow assembly failure. Although the severity of linked spindles is variable among cenf04787embryos, this phenotype is highly penetrant, with 30.77% (16/52) of metaphase cenf04787 embryos showing linked spindles, compared to wild-type embryos. Actin staining showed that furrows form aberrantly, are consistently less robust, and have decreased furrow depth in cenf04787 embryos. Actin organization into pseudocleavage furrows displayed variable degrees of disruption in cenf04787 embryos; however, overall 28% of cenf04787 embryos displayed broken or weak furrows at prophase or metaphase. In addition, the distribution of actin density in furrows was frequently irregular in cenf04787 embryos, and patches of small furrows were common. However, no obvious effects on actin cap formation were observed at interphase. Thus, cen mutant embryos are deficient in mitotic cleavage furrow assembly. Although the furrow defects and linked spindles of cenf04787 embryos are ~28%-31%, the embryo hatch failure rate is ~9%, only 5% higher than that of the wild-type, thereby attesting to the ability of embryos to cope with the furrow defects in cen mutant embryos, with the likely exception of those with very severe defects (Kao, 2009).

On the minority of wild-type spindles in which CEN appeared to split symmetrically between the two centrosomes at mitosis, no effect on furrow assembly was observed. This suggests that the asymmetry of CEN distribution at mitosis may not have any acute effect on furrow assembly. It is proposed that CEN localization at centrosomes may function early in mitosis to initiate a signaling process that is required as furrow assembly proceeds, thus impacting furrow actin assembly at mitotic furrows (Kao, 2009).

Recycling endosomes (REs) have a defined role in the trafficking of actin- and membrane-containing vesicles to organize cleavage furrows. To investigate an impact on RE function in cenf04787 embryos, RE markers Nuf and Rab11, which localize to REs that are distributed in a pericentrosomal pattern, were stained. Localization of neither of these RE components nor the Dystrophin ortholog and furrow membrane protein Dah, which is very sensitive to perturbations of RE activity, was affected by loss of CEN function. Given that the REs activate Rho1 at furrows through RhoGEF2 recruitment to promote actin assembly, it therefore appears that CEN promotes actin assembly at furrows by a Rho-independent pathway (Kao, 2009).

The cleavage furrow defects seen in cenf04787 embryos are probably not due to microtubule assembly defects because the astral microtubules in cenf04787 embryos are comparable to those in the wild-type. Moreover, the pericentrosomal localization of Nuf and Rab11 are dependent upon microtubules; yet their localization appeared to be normal in cenf04787 embryos (Kao, 2009).

The phenotypes seen in cenf04787 embryos are consistent with CEN functioning in conjunction with CNN in the early embryo: mutations in cnn cause defective furrow assembly. However, the defects in these processes in cenf04787 embryos are not as severe as those that occur in cnn mutants, including the hypomorphic cnnB4 mutant. This suggests that CEN may not be the only factor involved in conveying the signal from the CM2 domain of CNN to cleavage furrows. Nevertheless, given that CNNB4 localizes to centrosomes, albeit with an altered PCM pattern, the ability of CEN to localize to centrosomes in cnnB4 embryos was examined. Consistent with the yeast two-hybrid interaction assay, CEN failed to localize detectably at cnnB4 centrosomes and yet was localized variably at furrows during early cortical cycles (10 and 11), but not at the residual patches of furrows that form at later cycles (12 and 13) in cnnB4 embryos. Because the interaction of CEN and CNNB4 is reduced but not abolished, it is possible that some CEN is localized to centrosomes and that this facilitates the inefficient localization to cnnB4 furrows. Alternatively, CEN association with CNN may be required for its activation prior to its action at furrows to promote actin assembly (Kao, 2009).

Together, these data indicate that the conserved domain at the C terminus of CNN is critical for cleavage furrow formation and for recruitment of CEN to centrosomes. However, because furrow defects in the cen mutant are not as severe as cnnB4, CEN is unlikely to be the only factor involved in the regulation of furrow formation by CNN CM2. In summary, CNN CM2 instructs cleavage furrow formation and appears to accomplish this in part through the recruitment of CEN to the centrosome and/or directing it to the cleavage furrow (Kao, 2009).

In conclusion, the CM2 domain of CNN is required for the signaling from the centrosome to instruct cleavage furrow assembly at the embryonic cortical membrane. CM2 accomplishes this through binding with CEN, which is required for efficient cleavage furrow formation. Thus, the CM2 domain of CNN and CEN represent a molecular link between centrosomes and the signals that regulate cleavage furrow assembly (Kao, 2009).

Maelstrom coordinates microtubule organization during Drosophila oogenesis through interaction with components of the MTOC

The establishment of body axes in multicellular organisms requires accurate control of microtubule polarization. Mutations in Drosophila PIWI-interacting RNA (piRNA) pathway genes often disrupt the axes of the oocyte. This results from the activation of the DNA damage checkpoint factor Checkpoint kinase 2 (Chk2) due to transposon derepression. A piRNA pathway gene, maelstrom (mael), is critical for the establishment of oocyte polarity in the developing egg chamber during Drosophila oogenesis. Mael forms complexes with microtubule-organizing center (MTOC) components, including Centrosomin, Mini spindles, and γTubulin. Mael colocalizes with αTubulin and γTubulin to centrosomes in dividing cyst cells and follicle cells. MTOC components mislocalize in mael mutant germarium and egg chambers, leading to centrosome migration defects. During oogenesis, the loss of mael affects oocyte determination and induces egg chamber fusion. Finally, this study shows that the axis specification defects in mael mutants are not suppressed by a mutation in mnk, which encodes a Chk2 homolog. These findings suggest a model in which Mael serves as a platform that nucleates other MTOC components to form a functional MTOC in early oocyte development, which is independent of Chk2 activation and DNA damage signaling (Sato, 2011).

In this study, it was shown that Mael is an MTOC component and that dynamic organization of MTs does not occur in developing mael oocytes, which correlates with mislocalization of other MTOC components. It was also observed that loss of mael affects the number and position of the oocytes in egg chambers and induces fusion of egg chambers. These results indicate that Mael specifically regulates MTOC formation, and thereby plays a key role in coordinating dynamic MT organization during Drosophila oogenesis (Sato, 2011).

Initial polarization of the oocyte during the oocyte specification phase in the germarium requires replacement of the fusome by a polarized MT network, which correlates with the formation of the MTOC. Mael is concentrated in the centrosomal region and is colocalized with αTub and γTub during cyst cell divisions. γTub does not migrate to a developing oocyte in mael germariums, suggesting that Mael is required for the migration of centrioles from the cytoplasm of cysts to pro-oocytes in the germarium. Currently, the detailed mechanism by which Mael functions in MT organization is not clear. The simplest hypothesis is that Mael might serve as a platform that nucleates other MTOC components to form a functional MTOC. A previous report has shown that weaker mutant alleles of γTub affect the number of nurse cells and oocytes within the egg chamber. These γTub mutant defects are very similar to those found in mael mutants in this study. γTub is involved in the nucleation of MTs and is present in the centrosomes and MTOCs in many different systems. It was hypothesized that reduced activity of γTub could activate the oocyte determination program in one of the nurse cells by ectopically presenting MTOC material. The findings that γTub does not accumulate at centrosomes in the mael germarium and is ectopically expressed in the mael egg chamber suggest that Mael regulates localization of γTub at centrosomes through its complex formation and is thereby involved in properly organizing or positioning the MTOC (Sato, 2011).

PIWI proteins function in transposon silencing via association with piRNAs and maintain genome integrity during germline development. Recent studies have suggested that PIWI proteins in sea urchin (Seawi) and Xenopus (Xiwi) can interact with the MTs of the meiotic spindle, while fly ovarioles with mutations in any of several piRNA pathway genes, including spn-E, aub, and armi, have disorganized MTs. This raises the possibility for either a functional role of PIWI proteins in the machinery that impacts on MT organization (in addition to transposon silencing), a role of the MT cytoskeleton in piRNA generation, or both. The current findings further corroborate a link between components of the piRNA pathway and proper MT organization. Although it was found that Mael forms a complex with MTOC components, components of the piRNA pathway in this complex could not be identified. This is in contrast to observations of the mouse Mael homolog, which functions in the piRNA pathway (similar to fly Mael) and interacts with mouse PIWI proteins in the testes (Costa, 2006). Mouse Mael in the testes is almost exclusively cytoplasmic with accumulation at nuage (Soper, 2008). In contrast, fly Mael is located in both the nucleus and the cytoplasm in the ovary and is known to shuttle between them. Thus, one possibility is that in fly ovaries, there may exist nuclear Mael complexes involved in both piRNA generation and transposon silencing, which are distinct from the cytoplasmic complex containing MTOC components that were identified in this study (Sato, 2011).

Female flies with mutations in several genes in the piRNA pathway often lay eggs with axis patterning defects because of MT cytoskeletal changes that result in the mislocalization of bic, grk, and osk mRNAs within the egg chamber. These defects have been linked to the Chk2 DNA damage checkpoint that may be activated by increased retrotransposon transcript levels in mutants defective in piRNA biogenesis. However, because a mutation in mnk does not suppress the mislocalization of Osk and Grk in the mael oocyte, the axis specification defect of mael oocytes does not appear to be triggered by the activation of germline-specific DNA breaks and damage signaling through Chk2. In addition, a mutation in the mei-W68 locus, which encodes the Drosophila Spo11 homolog and induces meiotic double-strand breaks in chromosomes, cannot suppress the axis specification defect of mael oocytes. Therefore, these results suggest that the axis specification defects of mael oocytes are not a secondary consequence of DNA damage signaling. However, it has been shown that in mael mutant ovaries, Vas is post-translationally modified. These results together imply that, acting not only through Chk2, the functions of Mael in MT organization are in parallel with its function in piRNA generation and transposon silencing. There are mutants—including zuc and spn-E that are piRNA pathway genes; their axis defects cannot be rescued by mnk mutations. Vas also appears modified in these mutants, although the relationship with activated checkpoint-modified Vas is unclear (Sato, 2011).

Given that Mael is a new component of the MTOC in the Drosophila ovary, identification of a domain within Mael that is responsible for binding to other MTOC components could aid in understanding how Mael nucleates and regulates MTOC formation. Because Mael contains an evolutionarily highly conserved domain of unknown function, termed the Mael domain (Zhang, 2008), determination of its crystal structure should prove valuable in elucidating mechanisms of both MTOC formation and piRNA generation processes (Sato, 2011).

The centrosome-specific phosphorylation of Cnn by Polo/Plk1 drives Cnn scaffold assembly and centrosome maturation

Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis - a process termed centrosome maturation. How centrosomes mature remains mysterious. This study identified a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. It is concluded that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies (Conduit, 2014).

As cells enter mitosis, centrosomes mature, and the amount of PCM recruited around the centrioles dramatically increases. Although many proteins have been implicated in this process, little is known about how they organize a functional mitotic centrosome. Previous studies have hinted at the existence of a PCM scaffold, but its molecular nature has remained elusive. The current data suggest that Cnn is phosphorylated specifically at centrosomes during mitosis, and this phosphorylation allows Cnn to assemble into a scaffold around the centrioles. Perturbing Cnn phosphorylation prevents efficient scaffold assembly and efficient mitotic PCM recruitment, demonstrating that the phosphorylated Cnn scaffold plays an important part in centrosome maturation in flies (Conduit, 2014).

This study demonstrates unambiguously that the Cnn scaffold is in constant flux: as the Cnn scaffold spreads slowly outward, it is continuously replenished by new phosphorylated Cnn that assembles around the centrioles; in this way, the Cnn scaffold is built from the inside out. This inside-out assembly mechanism has important implications, because it potentially explains how centrioles can influence the size of the PCM and organize centrosomes of different sizes within the same cell - as seems to occur in several asymmetrically dividing stem/progenitor cells (Conduit, 2014).

How does Cnn assemble into a scaffold structure? Cnn contains a PReM domain that contains a LZ and ten Ser/Thr residues that are highly conserved in Drosophila species. Mutating the LZ or the ten Ser/Thr residues to Ala strongly inhibits Cnn scaffold assembly in vivo, while mutating these ten Ser/Thr residues to phosphomimicking residues promotes spontaneous Cnn scaffold assembly in the cytosol, independently of centrosomes. Moreover, whereas the WT PReM domain predominantly forms dimers via the LZ in vitro, replacing the ten Ser/Thr residues with phosphomimicking residues allows the PReM domain to assemble into higher-order multimers in an LZ-dependent manner. Modeling suggests that the arrangement of hydrophobic and hydrophilic residues within the LZ could allow multiple LZs to associate laterally to form such multimeric structures. It is speculated, therefore, that these stable multimers formed by the phosphomimicking mutant PReM domains in vitro may be the fundamental building blocks of the phosphorylated Cnn scaffold in vivo. How these multimers assemble into a larger macromolecular scaffold is unclear, but Y2H analysis indicates that multiple regions of Cnn can self-interact and so could potentially participate in such a process (Conduit, 2014).

How is Cnn scaffold assembly regulated so that it only occurs during mitosis? Polo/Plk1 is a key regulator of PCM assembly in many systems and it is activated in human cells during the G2/M transition. In flies, knocking down Polo in cultured fly cells abolishes Cnn phosphorylation and strongly perturbs Cnn's centrosomal localization. This study shows that recombinant human Plk1 can phosphorylate the PReM domain of Cnn in vitro and that at least six of the putative phosphorylation sites within the PReM domain conform to a Polo/Plk1 recognition motif. Moreover, abolishing these putative phosphorylation sites prevents Cnn phosphorylation in vitro and Cnn scaffold formation in vivo, whereas mutating these sites to phosphomimicking residues promotes multimerization in vitro and spontaneous scaffold formation in vivo. Thus, it seems likely that Polo is activated during mitosis in fly cells and directly phosphorylates Cnn to initiate Cnn scaffold assembly, although the possibility cannot be excluded that Polo activates an unknown kinase that then phosphorylates Cnn (Conduit, 2014).

How is Cnn scaffold assembly regulated so that it only occurs around the centrioles? The data strongly indicate that Cnn is normally phosphorylated exclusively at centrosomes, and Polo is highly concentrated at centrioles throughout the cell cycle. While it remains formally possible that Cnn is phosphorylated in the cytosol and phosphorylated Cnn is then rapidly sequestered at centrosomes, this is thought unlikely for two reasons: (1) phosphomimetic Cnn is not rapidly transported to centrosomes, but rather spontaneously assembles into scaffolds in the cytoplasm, and (2) in mitotic extracts of brain cells that lack centrosomes, phosphorylated Cnn cannot be detected. It is interesting that the phosphorylation of at least six of the ten conserved Ser/Thr residues within thePReMdomain appears to be required for efficient scaffold assembly. The potential advantages of regulation by multisite phosphorylation in allowing switch-like transitions are well documented. Thus, it seems likely that the requirement for multisite phosphorylation helps ensure that Cnn normally only efficiently forms a scaffold around the centrioles, where there is a high concentration of both the kinase and its substrate. Cnn is a large protein that contains several predicted coiledcoil regions, supporting the idea that it can act as a molecular scaffold onto which other PCM proteins can assemble. Proteins related to Cnn have been identified in species ranging from yeasts to humans, and many of these proteins have been implicated in centrosome or MT organizing center assembly; they are also usually large proteins with several predicted coiled-coil domains, and some family members have been shown to interact directly with several other PCM components, including the γlTuRC, Aurora A, and Pericentrin. Althoug no obvious PReM domain has been identified in vertebrate Cnn family members, many of these proteins have regions that might fulfill the minimal requirements for a PReMlike domain - a potential coiled-coil interaction domain, and a region containing multiple potential phosphorylation sites. It is therefore suspected that Cnn-like proteins will contribute to PCM scaffold formation in many systems (Conduit, 2014).

centrosomin: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation

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