pericentrin-like protein
DEVELOPMENTAL BIOLOGY

The D-PLP protein is associated with centrioles

Antibodies were raised against two regions of D-PLP. Anti-D-PLP antibodies should recognize both D-PLP-S (small isoform) and D-PLP-L (large isoform), whereas anti-D-PLP-L antibodies should recognize only D-PLP-L. In Western blots of third instar larval brains, both antibodies recognized a cluster of 2–4 high mol wt bands in the ~300–450-kD range, and the anti-D-PLP antibodies also recognized a band of ~180 kD. These proteins appear to be products of the d-plp gene, since their abundance is altered in the brains of d-plp mutant larvae (Martinez-Campos, 2004).

Both anti-D-PLP and anti-D-PLP-L antibodies stained centrosomes as a very small dot that was located in the middle of the PCM. The GFP-PACT fusion protein also localizes to centrosomes as a very small dot that colocalizes with the endogenous D-PLP. This suggests that D-PLP and GFP-PACT are primarily associated with centrioles, rather than with the PCM (Martinez-Campos, 2004).

This possibility was tested in three ways. (1) The localization of D-PLP and GFP-PACT was examined in Drosophila oocytes. In fly oogenesis, a cystoblast undergoes four rounds of incomplete cell division to generate a cyst of 16 cells that remain interconnected by intercellular bridges. EM analyses have shown that the centrioles in the 16-cell cyst lack any associated PCM, and they all migrate into the developing oocyte where they become clustered at the posterior pole. Although several immunofluorescence analyses have failed to detect any PCM components associated with these centrioles, a commercially available ascites fluid containing the anti-gamma-tubulin mAb GTU88 has recently been shown to stain oocyte centrioles. In this study, a specific batch of this ascites fluid (GTU88*) stained oocyte centrioles, but several other batches of GTU88 ascites fluid (from the same commercial supplier) did not, and several other anti-gamma-tubulin antibodies also did not stain oocyte centrioles. Thus, GTU88* appears to contain a contaminating antibody that recognizes centrioles in oocytes (and in all other Drosophila tissues tested). Oocyte centrioles were stained by GTU88*, by both D-PLP and D-PLP-L antibodies, and by GFP-PACT, but they were not stained by antibodies raised against any other PCM markers tested (gamma-tubulin, CNN, D-TACC, Msps, CP190, or CP60) (Martinez-Campos, 2004).

(2) The localization of D-PLP and GFP-PACT was examined in Drosophila larval brain cells. None of the known PCM markers tested stained the centrosomes during interphase in these cells. However, the GTU88* antibody stained one or two dots (presumably the centrioles) in each cell during interphase, and these dots were also stained with D-PLP or D-PLP-L antibodies and by GFP-PACT. Interestingly, microtubules were not detectably concentrated around these centrioles during interphase, suggesting that interphase centrosomes do not organize microtubules in these cells (Martinez-Campos, 2004).

(3) The localization of D-PLP and GFP-PACT was examined in spermatocytes. These cells have very large centrioles and only a small amount of PCM. Spermatocyte centrioles were stained with GTU88*, but surprisingly, they were not stained by D-PLP or D-PLP-L antibodies. It is suspected that this is due to antibody penetration problems, since in living spermatocytes GFP-PACT strongly localizes to the centrioles during meiosis (Rebollo, 2004) and to the centrioles at the base of the mature sperm. However, in fixed preparations of testes expressing GFP-PACT, anti-GFP antibodies did not stain the centrioles, even though the centriolar fluorescence of GFP-PACT was still detectable. Thus, GFP-PACT cannot be detected in spermatocyte centrioles with anti-GFP antibodies. This probably explains why anti-D-PLP antibodies cannot detect D-PLP in these unusually large centrioles. Together, these data suggest that D-PLP and GFP-PACT are associated with centrioles in Drosophila (Martinez-Campos, 2004).

To test whether GFP-PACT is stably associated with centrioles, a FRAP analysis was performed in living embryos. A small area of an embryo was photobleached and the recovery of fluorescence was monitored over time by time-lapse confocal microscopy. In all 14 embryos observed, some GFP-PACT fluorescence recovered rapidly (T1/2 ~2 min) in a relatively broad area around the centrioles. However, the very bright, dot-like fluorescence of GFP-PACT never recovered if the embryos remained in interphase. However, the very bright, dot-like fluorescence of the centrioles invariably recovered as soon as the centrioles replicated. Thus, it is concluded that there are two populations of GFP-PACT associated with centrosomes: a fraction that is stably associated with centrioles, and a fraction that is associated with the PCM and is in rapid exchange with a cytoplasmic pool (Martinez-Campos, 2004).

The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte

Molecular motors transport the axis-determining mRNAs oskar, bicoid and gurken along microtubules (MTs) in the Drosophila oocyte. However, it remains unclear how the underlying MT network is organized and how this transport takes place. A centriole-containing centrosome has been detected close to the oocyte nucleus. Remarkably, the centrosomal components, gamma-tubulin and Drosophila Pericentrin-like protein also strongly accumulate at the periphery of this nucleus. MT polymerization after cold-induced disassembly in wild type and in gurken mutants suggests that in the oocyte the centrosome-nucleus complex is an active center of MT polymerization. The MT network comprises two perpendicular MT subsets that undergo dynamic rearrangements during oogenesis. This MT reorganization parallels the successive steps in localization of gurken and oskar mRNAs. It is proposed that in addition to a highly polarized microtubule scaffold specified by the cortex oocyte, the repositioning of the nucleus and its tightly associated centrosome could control MT reorganization and, hence, oocyte polarization (Januschke, 2006).

Both the nature and the localization of the MTOC beyond stage 6 of Drosophila oogenesis have not yet been clarified. Up to stage 6, gamma-tubulin has been shown to closely associate with the nucleus at the posterior of the oocyte. In addition, electron microscopy studies have demonstrated the presence of centrioles close to the oocyte nucleus up to stage 4. Thus, until stage 6, the centrosome associates with the nucleus at the posterior of the oocyte. In Drosophila females, meiosis takes place in the absence of centrosomes. It has therefore been speculated that, at stage 6, centrosome organization changes, involving the disappearance of centrioles and the generation of MTs from a diffuse organizing center. To better understand this process, the distribution of gamma-tubulin in the oocyte was re-investigated. Before repolarization of the MT cytoskeleton, it was found that gammaTub23C and gammaTub37C localize in a layer around the nucleus, with an enrichment at the posterior pole of the oocyte. This is in agreement with the location of the MTOC at this stage. After repolarization of the MT cytoskeleton, both gamma-tubulin isoforms remain located in a perinuclear manner. Interestingly, gammaTub37C, but not gammaTub23C, labels a small body close to the oocyte nucleus. In addition, gammaTub37C and gammaTub23C also exhibit differential expression patterns in embryos: gammaTub37C is located with the centrosomes of mitotic cells, whereas gammaTub23C is not. Thus, gamma-tubulin is distributed in close association with the nucleus periphery and possibly on a centrosome-like structure. Pericentrin/AKAP450 is another major component of the centrosome. Green fluorescence protein (GFP) fusion of the C-terminal part of Pericentrin/AKAP450 and its Drosophila homolog pericentrin-like protein (D-PLP) have been shown to localize to the centrosomes respectively in cultured human cells (Keryer, 2003), Drosophila embryos and spermatocytes (Martinez-Campos, 2004; Rebollo, 2004). Using the UAS/Gal4 system, GFP-cter-D-PLP was specifically expressed in the germline and a bright dot was detected in the vicinity of the nucleus before and after nuclear migration. GFP-cter-D-PLP was also detected in all germline nuclei, as has been observed previously (Martinez-Campos, 2004). From stage 7 onward, the bright dot remained in the immediate vicinity of the oocyte nucleus (<1 µm distance). Furthermore, both GFP-cter-D-PLP and gammaTub37C co-localize to this discrete body, indicating that this structure could correspond to a centrosome. In G2 centriole, tubulin is highly polyglutamylated. The ID5 antibody labels basal bodies and centrioles in several species. Using this antibody, a dot was detected close to the nucleus throughout oogenesis that remained detectable up to stage 10A. This suggests that the dot represents a centriole-containing centrosome. Indeed, using electron microscopy, two to possibly four centrioles were clearly detected closely associated with the nucleus in stage 9 oocytes. This demonstrates the existence of centrioles associated with the nucleus at least up to stage 9. MT fibers emanating from those centrioles could not be unambiguously detected. Then the link between centrosome and nucleus was examined using colchicine. In flies fed with colchicine, MTs in the germline were completely depolymerized, and the oocyte nucleus was mispositioned. In the oocyte, it was observed that the nucleus and the centrosome were significantly separated, the distance between them increasing during oocyte growth. In a few cases, it was noticed that the nucleus could reach the anterior cortex without the centrosome; however, a centrosome was never observed at the anterior without the nucleus. It is concluded that the close localization of the centriole-containing centrosome to the nucleus depends on MTs (Januschke, 2006).

The structure of the MT network during mid-oogenesis is dynamic. At stage 7, MTs are visible as a mesh at the anterior cortex. Later, at stage 10, MT bundles are observed that promote cytoplasmic streaming. In-between MT distribution has been described as an AP gradient. However, high-resolution images of oocyte MTs are lacking. Therefore, a protocol frequently used to increase the detection of the MT cytoskeleton in cell culture was modified for the Drosophila egg chamber to characterize MT organization in the oocyte during the crucial period in which bcd, grk and osk mRNAs are localized. MTs were detected throughout oogenesis using alpha-Tubulin but also with a Kinesin heavy chain antibody (alpha-Khc), which revealed the MT array and its complexity in unprecedented definition. It was noticed that the range of detected details was increased and more reproducible with alpha-Khc antibody. To control the specificity of Khc detection, germline and follicle cell mutant clones were generated homozygous for khc7.288. In such mutant cells, no Khc was detected, indicating that the detection is specific. Labeling with antibodies directed against aromatic C-terminal amino acid residues (Tyr or Phe) of alpha-tubulin and against Khc largely overlapped. This confirmed that the structures revealed by Khc were MTs. A Khc fraction was also detected at the posterior of the oocyte. That Khc accumulates along MTs may be due to permeabilization before fixation, which could cause rigor binding of Khc to MTs. This detection procedure may also permit the extraction of a soluble pool of Khc and reveal the remaining fraction distributed along the MTs. With this detection procedure, Khc revealed by Kinesin-ßgal exhibited a more restricted distribution compared with alpha-Khc antibody. This is probably due to the substitution of the C-terminal part of Khc by the ß-galactosidase in the reporter construct, impairing the recycling of the chimeric Kinesin motor leading to its accumulation exclusively at the posterior. With this detection method, the MT minus-end marker, Nod-ßgal, was detected in the antero-dorsal corner above the oocyte nucleus as well as in the opposite antero-ventral corner. Moreover, localized determinants such as Osk and Grk were correctly positioned in the oocyte (Januschke, 2006).

To confirm that the detection method does not alter MT organization, MT distribution was analyzed in follicle cells, which should be sensitive to the extraction procedure, since they are more directly exposed than the oocyte. MT distribution in different follicle cell types was unchanged, when comparing living and fixed egg chambers. The main body follicle cell MTs seemed unchanged. Main body follicle cell MTs have been shown to be highly stable, and might therefore reflect the sensitivity of the protocol with limitations. Nevertheless, stretched follicle cells showed strikingly similar MT patterns in living and fixed conditions as well. Apicalbasal polarity was not affected in follicle cells, as demonstrated by the correct apical localization of atypical protein kinase C. Importantly, the MT distribution of living egg chambers expressing GFP-alpha-Tubulin at stage 7 and stage 9 was similar to the one observed using anti-alpha-Tubulin and Khc antibodies. Therefore it seems that the fixation conditions preserve the wild-type MT organization and that Khc can be suitable to label bulk MTs (Januschke, 2006).

When fixed wild-type oocytes were analyzed by confocal microscopy, MT organization in the oocyte appeared unchanged from stage 2 to stage 6. With stage 7, MT organization was modified and two MT subsets became apparent. This organization was more evident at stage 8. A first subset consisted of cortical MTs oriented along the dorso-ventral (DV) axis parallel to the oocyte nurse cell border, and juxtaposed to the lateral cortices, wrapping the oocyte from stage 7 to 9. At least some MT bundles of this subset could be traced back to the oocyte nucleus. The DV orientation of MT bundles, depicted as black fibers in the schematic representations, was highly reproducible for all stages and persisted throughout mid-oogenesis (Januschke, 2006).

A second MT subset was present in the center of the oocyte. Although there was some variability in the patterns observed, it was found that each developmental stage showed a characteristic MT distribution. During stage 6, MTs from this subset were cortical and extended from the nucleus at the posterior to the anterior cortex, compact bundles of MTs formed a circle-like structure resembling a diaphragm. This subset was formed by long MT bundles that extended (once or more) along the entire cortex. By stage 8, the oocyte had considerably grown and individual MT bundles were therefore easier to track. MT bundles emanated from the anterior and the nucleus to point toward the posterior. MTs extended again along the entire cortex, after which they turned to the central cytoplasm. This, in turn, generated free MT (plus) ends in the center of the oocyte. By stage 9, the central MT network was clearly oriented along the oocyte AP axis. One or two thick MT bundles extended from the anterior, pointing toward the posterior pole. These bundles formed a structure resembling a horseshoe, with its open side facing the posterior. Importantly, both subsets could also be detected in living egg chambers, as shown for the DV subset and the AP subset. Thus, MTs show strong rearrangements throughout mid-oogenesis, which results in two perpendicular MT arrays reflecting the two axes of the oocyte (Januschke, 2006). An ex-vivo assay was developed to localize MT nucleation sites by dissecting ovaries and placing them on ice for 30 minutes. This treatment resulted in complete depolymerization of MTs. When allowed to recover at 25°C for 30 minutes, MT distribution could be re-established to the wild-type situation, in which both the cortical and the central subsets of MTs were detectable. gamma-Tubulin distribution was not affected by cold-induced MT depolymerization. When short periods of regrowth were analyzed, MT nucleation appeared limited to the close vicinity of the nucleus and was often asymmetric, suggesting a centrosome-associated nucleation activity. MT regrowth appeared to be stepwise, since after 15 minutes only the DV cortical subset was established. MTs clustered around the oocyte nucleus and aligned along the cortex in the DV direction. The cortical location of these fibers was clearly revealed by the presence of Khc-positive dots at either the dorsal or the ventral side. This indicates that the DV MT subset is the first to regrow. The regrowth experiment was repeated using colchicine. After the drug was washed out, MT repolymerization was observed at the oocyte nucleus. Taken together, these results indicate that, at least with the detection method that was used, the oocyte nucleus and its immediate surroundings have the capacity to nucleate MTs (Januschke, 2006).

To test whether the centrosome-nucleus complex could direct the repolarization of the MT network, how MTs distribute in grk mutant oocytes was examined. In this mutant, the nucleus frequently remains at the posterior of the oocyte due to a failure in the signaling cascade that induces the repolarization of the cytoskeleton. In grk mutant oocytes similar in size to wild-type stage 8, the MT distribution was dramatically affected. Specifically, MT organization appeared completely reversed compared with wild type, in which the nucleus is at the anterior and MT plus-ends are located toward the posterior at stage 8. In slightly older oocytes, MTs remain stretched out along the cortex from the posterior toward the anterior, where they fold back to the center of the oocyte. MT ends in the center are most probably plus-ends, since the pool of Khc (localized at the posterior of wild-type oocytes, co-localizes with Kinesin-ßGal to the center of the oocyte, between the flanking MT ends. Interestingly, MT distribution in grk oocytes was strikingly similar to MTs of wild-type egg chambers before the migration of the oocyte nucleus. Likewise, the centrosome, as revealed by gamma-tubulin, which is found at the posterior of stage 6 wild-type oocytes, stays at the posterior in grk mutants. Thus, in grk mutants, distribution of MT and MTOC seemed similar to their distribution in wild-type stage 6 (Januschke, 2006).

grk mutant oocytes, having mispositioned nuclei, provide an ideal basis to test the MT nucleating capacity of the centrosome-nucleus complex using the cold-shock assay. After cold-shock treatment of grk oocytes, complete MT depolymerization was checked for. As in the wild type, during the initial period of recovery at 25°C, MT polymerization took place only in the immediate vicinity of the mispositioned oocyte nucleus. Therefore, as in wild-type oocytes, MT nucleation is often asymmetric and restricted to the area surrounding the nucleus. This result strengthens the possibility that the centrosome-nucleus complex is an active MTOC (Januschke, 2006).

Thus, in the Drosophila oocyte a centriole-containing centrosome is present in close association with the nucleus, which itself is covered by PCM components until late in oogenesis. In addition, MTs can nucleate from this centrosome-nucleus complex. The MTs appear to form two orthogonal MT populations that develop through several steps during mid-oogenesis. It is proposed that the migration of the nucleus in the oocyte could control the reorganization of the MT network (Januschke, 2006).

In region 2 of the germarium, nurse cell centrosomes migrate toward the oocyte. Later, in region 3, these centriole-containing centrosomes become located as an aggregate between the oocyte nucleus and the follicle cell border. Pericentriolar material closely associated with the oocyte nucleus can be clearly detected until stage 6 with several centrosomal markers, such as gamma-tubulin, Centrosomin and D-Tacc. From stage 4 onward, the fate of the centriole cluster has been unknown. This study shows that both gammaTub37C and gammaTub23C are localized in a perinuclear manner throughout oogenesis. gammaTub37C highlights a discrete body close to the nucleus. This body is similarly detected by the centrosomal marker D-PLP and by a specific antibody for polyglutamylated Tubulin, which detects centrioles. Consistent with this, two to possibly four centrioles were detected in the immediate vicinity of the nucleus in stage 9 oocytes. This result demonstrates that at least until stage 9, a centriole-containing centrosome is present in the oocyte. Currently, it is not known whether they are still present at the onset of meiosis I during stage 13, since it has previously been proposed that the meiotic spindle is achieved without centrosomes. During skeletal muscle morphogenesis, myotube centrosomes dissociate from their nuclei, centrioles disappear and the centrosomal matrix is redistributed to the nucleus periphery. Similarly, during oogenesis, centrioles from nurse cell centrosomes may disappear. However, their pericentriolar material may relocate to the oocyte nucleus periphery. This would explain the specific enrichment of the oocyte nucleus with perinuclear MTOC material. The only centrosome remaining associated with a nucleus is that of the oocyte. Furthermore, the structure of this centrosome remains intact. It is concluded that the four centrioles found close to the nucleus in stage 9 may correspond to the initial oocyte centrosome in the duplication phase observed in G2 (Januschke, 2006).

Asymmetric inheritance of mother versus daughter centrosome in stem cell division

Adult stem cells often divide asymmetrically to produce one self-renewed stem cell and one differentiating cell, thus maintaining both populations. The asymmetric outcome of stem cell divisions can be specified by an oriented spindle and local self-renewal signals from the stem cell niche. Developmentally programmed asymmetric behavior and inheritance of mother and daughter centrosomes underlies the stereotyped spindle orientation and asymmetric outcome of stem cell divisions in the Drosophila male germ line. The mother centrosome remains anchored near the niche while the daughter centrosome migrates to the opposite side of the cell before spindle formation (Yamashita, 2007).

Adult stem cells maintain populations of highly differentiated but short-lived cells throughout the life of the organism. To maintain the critical balance between stem cell and differentiating cell populations, stem cells have a potential to divide asymmetrically, producing one stem and one differentiating cell. The asymmetric outcome of stem cell divisions can be specified by regulated spindle orientation, such that the two daughter cells are placed in different microenvironments that either specify stem cell identity (stem cell niche) or allow differentiation (Yamashita, 2007).

Drosophila male germline stem cells (GSCs) are maintained through attachment to somatic hub cells, which constitute the stem cell niche. Hub cells secrete the signaling ligand Unpaired, which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the neighboring germ cells to specify stem cell identity. Drosophila male GSCs normally divide asymmetrically, producing one stem cell, which remains attached to the hub, and one gonialblast, which initiates differentiation. This stereotyped asymmetric outcome is controlled by the orientation of the mitotic spindle in GSCs: The spindle lies perpendicular to the hub so that one daughter cell inherits the attachment to the hub, whereas the other is displaced away (Yamashita, 2007).

The stereotyped orientation of the mitotic spindle is set up by the positioning of centrosomes during interphase. GSCs remain oriented toward the niche throughout the cell cycle. In G1 phase, the single centrosome is located near the interface with the hub. When the duplicated centrosomes separate in G2 phase, one stays next to the hub, whereas the other migrates to the opposite side of the cell. Centrosomes in the GSCs separate unusually early in interphase, rather than at the G2-prophase transition, so it is common to see GSCs with fully separated centrosomes without a spindle (Yamashita, 2007).

Differences between the mother and daughter centrosomes underlie the stereotyped behavior of the centrosomes in Drosophila male GSCs. The mother centrosome normally remains anchored to the hub-GSC interface and is inherited by the GSC, whereas the daughter centrosome moves away from the hub and is inherited by the cell that commits to differentiation. Mother and daughter centrosomes were differentially labeled by transient expression of green fluorescent protein-pericentrin/AKAP450 C-terminus (GFP-PACT) from the Drosophila pericentrin-like protein under heat shock-Gal4 control. The PACT domain, which is necessary and sufficient for centriolar localization, is incorporated into centrioles only during centrosome duplication and does not exchange with the cytoplasmic pool. Both the mother and daughter centrosomes are labeled by GFP-PACT in the first cell cycle after heat shock. In the second cell cycle, the daughter centrosome retains GFP-PACT, whereas the mother centrosome is not labeled, thus distinguishing the mother and daughter centrosomes. After a short burst of GFP-PACT expression induced by a 2.5-hour heat shock, 20% to 30% of the GSCs had GFP-labeled centrosomes, indicating the duplication of centrosomes during the window of GFP-PACT expression. By 12 hours after heat shock, >90% of the labeled GSCs had two GFP-positive centrosomes, indicating that they had progressed to the G2 phase of the first cell cycle after GFP-PACT incorporation (Yamashita, 2007).

By 18 to 24 hours after heat shock, the number of GSCs with two GFP-positive centrosomes had decreased, whereas the number of GSCs with one GFP-positive and one GFP-negative centrosome had increased, suggesting progression into the second cell cycle. Generally, the centrosome distal to the hub was labeled, whereas the centrosome proximal to the hub was GFP-negative, indicating that the daughter centrosomes migrate away from the hub-GSC interface during asymmetric GSC divisions (Yamashita, 2007).

Labeling the mother rather than the daughter centrosomes confirmed that the male GSCs in the niche preferentially retain mother centrosomes over time. Centrioles assembled during early embryogenesis were labeled using the NGT40 Gal4 driver to drive the expression of GFP-PACT in blastoderm-stage embryos, shutting off after germband extension. In the first cell cycle after the depletion of the cytoplasmic pool of GFP-PACT in the GSCs, both the mother and daughter centrosomes should be labeled. In subsequent cell cycles, only the mother centrosomes should be labeled (Yamashita, 2007).

In most GSCs in the second or later cell cycle after the depletion of cytoplasmic GFP-PACT, the labeled centrosome was positioned next to the hub-GSC interface, and the unlabeled centrosome had moved away from the hub. The frequency of GSCs that had the proximal, but not distal, centrosome labeled remained constant over time for 10 days (L3 larvae to day-3 adults), suggesting that the mother centrosomes are reliably retained by the GSCs, even through multiple rounds of GSC divisions. Some GSCs maintained cytoplasmic GFP-PACT, especially in L3 larvae, suggesting that the GFP-PACT had not yet been diluted out. Some GSCs were also observed with two labeled centrosomes, suggesting that they are in the first cell cycle after the depletion of cytoplasmic GFP-PACT (Yamashita, 2007).

The mother centrosomes in GSCs appeared to maintain robust interphase microtubule arrays. Ultrastructural analysis of the GSCs revealed that the centrosome proximal to the hub was commonly associated with many microtubules throughout the cell cycle. Nineteen centrosomes in GSCs were scored in serial sections of the apical tips of five wild-type testes. Eleven centrosomes were localized close to the adherens junctions between the hub and the GSCs. Nine of these proximal centrosomes appeared to be in interphase cells, based on nuclear morphology and microtubule arrangement. Typically, these interphase centrosomes proximal to the hub were associated with numerous microtubules. In some samples, microtubules appeared to extend from the centrosome toward the adherens junctions. The other two proximal centrosomes appeared to be in cells in mitotic prophase, based on their robust microtubule arrays containing bundled microtubules running parallel to or piercing the nuclear surface (Yamashita, 2007).

In contrast, of the five distal centrosomes in the apparently interphase cells that were scored, four had few associated microtubules. The remaining three distal centrosomes appeared to be in cells in mitotic prophase, based on microtubule arrays containing bundled microtubules. Thus, the mother centrosomes may maintain interphase microtubule arrays that anchor them to the hub-GSC interface, whereas the daughter centrosomes may initially have few associated microtubules and be free to move, establishing a robust microtubule array only later in the cell cycle (Yamashita, 2007).

Consistent with the idea that astral microtubules anchor the mother centrosomes to the hub-GSC interface, mother versus daughter-centrosome positioning was randomized in GSCs that were homozygous mutant for centrosomin (cnn), an integral centrosomal protein required to anchor astral microtubules to centrosomes. Analysis of mother and daughter centrosomes after transient expression of GFP-PACT revealed that, for cnn homozygous mutant GSCs where one of the two centrosomes was positioned next to the hub, it was essentially random whether the mother or the daughter centrosome stayed next to the hub. In addition, in >25% of total labeled GSCs, neither of the two centrosomes was next to the hub (Yamashita, 2007).

These results indicate that the two centrosomes in Drosophila male GSCs have different characters and fates. The mother centrosome stays next to the junction with the niche and is inherited by the cell that self-renews stem cell fate. Thus, GSCs can maintain an old centriole assembled many cell generations earlier. In contrast, the daughter centrosome migrates away from the niche and is inherited by the cell that will initiate differentiation. It is postulated that the mother centrosomes in male GSCs may remain anchored to the GSC-niche interface throughout the cell cycle by attachment to astral microtubules connected to the adherens junction, whereas the daughter centrosomes may initially have few associated microtubules and thus can move away from the niche. Microtubule-dependent differential segregation of mother and daughter spindle-pole bodies (equivalent to centrosomes in higher organisms) is observed in budding yeast (Pereira, 2001). In cultured vertebrate cells, the centrioles mature slowly over the cell cycle, and the mother centrosomes (containing a mature centriole) attach astral microtubules more effectively and are more stationary than daughter centrosomes in interphase (Piel, 2000). The unusually early separation of centrosomes in interphase male GSCs may provide a way to move the daughter centrosome out of range of the stabilizing influence of the adherens junction complex before it becomes competent to hold a robust microtubule array (Yamashita, 2007).

Developmentally programmed anchoring of the mother centrosome may provide a key mechanism to ensure the stereotyped orientation of the mitotic spindle and thus the reliably asymmetric outcome of the male GSC divisions. Although it is tempting to speculate that determinants associated with the mother or daughter centrosome may play a role in specifying stem cell or differentiating-cell fates, such determinants are yet to be identified. Rather, the asymmetric inheritance of mother and daughter centrosomes in male GSCs may be a consequence of the cytoskeletal mechanisms that are imposed as part of the stem cell program to anchor one centrosome next to the niche throughout the interphase, ensuring a properly oriented spindle (Yamashita, 2007).

Effects of Mutation or Deletion

To investigate the function of D-PLP, the databases were searched for mutations in the d-plp gene. The lethal line l(3)s2172 contains a P-element inserted within an intron of d-plp. When homozygous, this mutant is lethal, but when transheterozygous with Df(3L)BrdR15, a deficiency that uncovers the d-plp gene, the mutant flies were viable. Thus, there is at least one lethal mutation on the l(3)s2172 chromosome that is not associated with the P-element. Standard meiotic recombination was used to separate the P-insertion from the other lethal mutation. This 'cleaned up' recombinant chromosome is referred to as d-plp2172 (Martinez-Campos, 2004).

Flies that were homozygous for the d-plp2172 chromosome were viable, but they exhibited a severe 'uncoordinated' phenotype and died shortly after eclosion because they immediately got stuck in the food. In Western blots of homozygous third instar larval brain extracts, the levels of D-PLP-L were reduced in mutant brains by >95%, but D-PLP-S was present at normal levels. Therefore, an ethylmethane sulphate mutagenesis screen was performed to generate new d-plp alleles. ~5,000 mutagenized chromosomes were screened for lethality or for an uncoordinated phenotype when transheterozygous with d-plp2172. 17 new alleles of d-plp were generated, all of which were viable but uncoordinated when transheterozygous with either d-plp2172 or Df(3L)BrdR15. Western blotting revealed that all 17 lines had at least reduced levels of D-PLP-L, and five lines had undetectable levels of both D-PLP-L and D-PLP-S. In immunofluorescence analyses of mutant larval brains, no D-PLP protein was detectable at the centrioles of these five mutant lines with either the D-PLP or D-PLP-L antibodies. Thus, these five alleles appear to be protein nulls (Martinez-Campos, 2004).

The d-plp mutant phenotype was examined in detail in both homozygous d-plp2172 animals and in animals that were transheterozygous for d-plp5 (a putative protein null) and the Df(3L)BrdR15 deficiency (for simplicity these flies are referred to as d-plp5 mutants). d-plp2172 and d-plp5 mutants had very similar phenotypes, suggesting that D-PLP-S is largely nonfunctional in the processes described in this study (Martinez-Campos, 2004).

The distribution of gamma-tubulin was examined in immunostained preparations of wild-type (WT) and d-plp mutant brains. There is very little (if any) PCM associated with the centrioles of these cells in interphase. However, as WT cells entered mitosis, the levels of gamma-tubulin at the centrosome increased dramatically, and the gamma-tubulin formed particles in the cytoplasm that were initially recruited to a relatively broad region around the D-PLP-stained centrioles. In d-plp mutant cells, the centrioles were no longer detectable with anti-D-PLP-L antibodies, and the gamma-tubulin particles were either weakly accumulated around the centrosomes (centrosome position was confirmed by following the localization of GFP-PACT in d-plp mutants) or were dispersed throughout the cytoplasm (Martinez-Campos, 2004).

In WT cells, D-PLP remains tightly associated with centrioles from metaphase to telophase, and gamma-tubulin also becomes tightly associated with the centrosomes during this period. In d-plp mutant metaphase cells, gamma-tubulin is often only weakly concentrated at centrosomes or is abnormally dispersed in the cytoplasm. Quantification of these phenotypes revealed that the defect in gamma-tubulin recruitment is similar in both d-plp5 and d-plp2172 mutants, suggesting that D-PLP-L plays the major role in recruiting gamma-tubulin to centrosomes. Moreover, the recruitment of gamma-tubulin to centrosomes is most aberrant in early mitosis and improves dramatically as cells progress through cell division. This suggests that there is a D-PLP–independent mechanism that can eventually recruit gamma-tubulin to the centrosomes/spindle poles during mitosis (Martinez-Campos, 2004).

To test whether D-PLP is required to recruit any other proteins to the centrosome during mitosis, WT and d-plp mutant cells were stained with several other centrosomal markers. The centrosomal recruitment of all the proteins tested (CNN, D-TACC, CP60, Msps and CP190) was disrupted in d-plp2172 and d-plp5 mutant cells. Thus, in larval brain cells, D-PLP is required for the efficient recruitment of several proteins to the PCM (Martinez-Campos, 2004).

Since D-PLP is associated with centrioles, the failure to efficiently recruit PCM components in d-plp mutants could be due to a failure to properly form centrioles. To determine if centriole replication is disrupted in d-plp mutant cells, the distribution of the centrioles was examined using the GTU88* centriolar marker in d-plp2172 and d-plp5 mutant brains. The size and distribution of centrioles in WT and mutant interphase brain cells was indistinguishable, and a similar result was obtained using GFP-PACT as a centriolar marker in living preparations of WT or mutant brains (note that the expression of GFP-PACT did not alter any aspect of the d-plp mutant phenotype, nor did it have any detectable dominant-negative affect). Thus, centriole replication appears to occur normally in d-plp mutant brain cells. The same is also true in d-plp mutant testes (Martinez-Campos, 2004).

Surprisingly, the arrangement of microtubules in d-plp mutant cells was not grossly perturbed, and anaphase cells usually had a normal arrangement of microtubules and chromosomes, even when centrosomal markers were not tightly concentrated at centrosomes. Most prophase and metaphase mutant cells also had a normal arrangement of microtubules and chromosomes, although there was a modest increase in the number of cells that had at least one clearly disorganized spindle pole or that were more severely disorganized. In mutant cells that also expressed GFP-PACT as a centriolar marker, the centrioles were always at the center of a microtubule aster in prophase cells, and the centrioles were invariably located at the poles of the mitotic spindle from metaphase to anaphase. Thus, the centrioles appear to function as microtubule-organizing centers in d-plp mutant cells even though the recruitment of the PCM is inefficient. This suggests that most d-plp mutant cells assemble their spindles in a centrosome-dependent manner (Martinez-Campos, 2004).

Compared with controls, mutant cells did not have higher levels of either polyploidy/aneuploidy during metaphase or lagging chromosomes during anaphase (unpublished data). In addition, the mitotic index was no higher in mutant cells than in control cells. Thus, although the recruitment of several proteins to centrosomes was impaired in mutant cells, this did not lead to dramatic defects in mitosis (Martinez-Campos, 2004).

The uncoordinated phenotype seen in d-plp mutant flies is often associated with defects in mechanosensory transduction. Type I sensory organs contain neurons with specially modified cilia that transduce proprioceptive and auditory stimuli, and these cilia are required for normal sensory transduction. It was found that GFP-PACT was concentrated at the centrioles/basal bodies in all of the ciliated sensory neurons that were observed (chordotonal, olfactory, taste, and mechanosensory), so it might be required for proper cilia formation in these cells (Martinez-Campos, 2004).

To assess sensory neuron function, sound-evoked potentials (SEPs) were recorded from Johnston's organ—the large, auditory, chordotonal organ in the second antennal segment. Johnston's organ contains ~200 sensory units, or scolopidia, which respond to sound-induced vibrations in the distal antennal segments. Cilia extending from each of two or three sensory neurons in a scolopidium are attached at their distal tips to a dendritic cap that transmits the vibration to the cilia. To record sound-evoked responses, individual d-plp2172 mutant flies or their heterozygous siblings were exposed to a standard sound stimulus while recording extracellular potentials with electrodes placed in the antenna and head. Heterozygous flies had normal responses to each sound pulse. In contrast, most (16/26) homozygous mutant flies had no sound-evoked responses, and the amplitude of the response was greatly reduced in those flies that did respond (Martinez-Campos, 2004).

To see if this defect in sensory neuron function could be due to defects in the cilia, the cilia were visualized by labeling the plasma membrane of all neurons with the mCD8-GFP marker. In d-plp2172 heterozygous antennae, the ciliary outer segments of the mechanosensory chordotonal neurons in the second antennal segment and of the chemosensory neurons in the third antennal segment were clearly visible. By contrast, in d-plp2172 homozygous mutant antennae the sensory cilia in both the chordotonal organs and the olfactory bristles were absent or severely shortened and kinked. It seems likely that all ciliated sensory neurons, including those innervating tactile and proprioceptive bristles, are similarly affected. Thus, the uncoordinated phenotype of d-plp mutant flies seems to be caused by cilia defects in the type I sensory neurons (Martinez-Campos, 2004).

Apart from the type I sensory neurons, the only other cells in flies that have cilia or flagella are the sperm. Therefore, whether flagella function was abnormal in d-plp mutant sperm was analyzed. During normal spermatogenesis, each spermatogonium goes through four rounds of mitotic division to generate a cyst of 16 primary spermatocytes. These cells contain four large centrioles arranged as two orthogonal pairs. These centrioles are ~10-fold larger and are much more structurally elaborate than those found in any other cells in Drosophila. In d-plp2172 and d-plp5 mutant testes, morphologically normal primary spermatocytes were formed that each contained two pairs of large, orthogonally arranged centrioles, suggesting that centriole replication occurs normally during these premeiotic rounds of cell division in d-plp mutants (Martinez-Campos, 2004).

However, as the spermatocytes matured, the centrioles in mutant spermatocytes often lost their orthogonal arrangement and partially fragmented. This was confirmed in living spermatocytes using GFP-PACT as a centriolar marker. As a result of this fragmentation, mutant spermatocytes often formed multipolar meiosis I spindles, with each spindle pole organized by at least one centriole (or centriole 'fragment'). Thus, D-PLP appears to be essential for maintaining the structural integrity of the large centrioles that form in spermatocytes (Martinez-Campos, 2004).

Although meiosis was highly abnormal in mutant spermatocytes, at least some cells developed into relatively normal-looking sperm. However, the distribution of centrioles and nuclei in the cysts was often disorganized . Moreover, although the mutant sperm contained flagella, these were invariably nonmotile. However, an EM analysis revealed that the mutant sperm tails appeared structurally normal (Martinez-Campos, 2004).

Golgi outposts shape dendrite morphology by functioning as sites of acentrosomal microtubule nucleation in neurons

Microtubule nucleation is essential for proper establishment and maintenance of axons and dendrites. Centrosomes, the primary site of nucleation in most cells, lose their function as microtubule organizing centers during neuronal development. How neurons generate acentrosomal microtubules remains unclear. Drosophila dendritic arborization (da) neurons lack centrosomes and therefore provide a model system to study acentrosomal microtubule nucleation. This study investigated the origin of microtubules within the elaborate dendritic arbor of class IV da neurons. Using a combination of in vivo and in vitro techniques, it was found that Golgi outposts can directly nucleate microtubules throughout the arbor. This acentrosomal nucleation requires gamma-tubulin and CP309, the Drosophila homolog of AKAP450, and contributes to the complex microtubule organization within the arbor and dendrite branch growth and stability. Together, these results identify a direct mechanism for acentrosomal microtubule nucleation within neurons and reveal a function for Golgi outposts in this process (Ori-McKenney, 2012).

Microtubules are organized into dynamic arrays that serve as tracks for directed vesicular transport and are essential for the proper establishment and maintenance of neuronal architecture. The organization and nucleation of microtubules must be highly regulated in order to generate and maintain such complex arrays. Nucleating complexes, in particular, are necessary because spontaneous nucleation of new tubulin polymers is kinetically limiting both in vivo and in vitro. Gamma(Γ)-tubulin is a core component of microtubule organization centers and has a well-established role in nucleating spindle and cytoplasmic microtubules. Previous studies have proposed that in mammalian neurons, microtubules are nucleated by γ-tubulin at the centrosome, released by microtubule severing proteins, and then transported into developing neurites by motor protein. Indeed, injection of antibodies against γ-tubulin or severing proteins inhibited axon outgrowth in neurons cultured for one day in vitro (DIV1) (Ori-McKenney, 2012).

However, proper neuron development and maintenance may not rely entirely on centrosomal sites of microtubule nucleation. Although the centrosome is the primary site of microtubule nucleation at DIV2, it loses its function as a microtubule-organizing center during neuronal development. In mature cultured mammalian neurons (DIV 11-12), γ-tubulin is depleted from the centrosome, and the majority of microtubules emanate from acentrosomal sites. In Drosophila dsas-4 mutants that lack centrioles, organization of eye-disc neurons and axon outgrowth are normal in third-instar larvae. Within the Drosophila peripheral nervous system (PNS), although dendritic arborization neurons contain centrioles, they do not form functional centrosomes, and laser ablation of the centrioles does not perturb microtubule growth or orientation (Nguyen, 2011). These results indicate that acentrosomal generation of microtubules contributes to axon development and neuronal polarity. How and where acentrosomal microtubule nucleation may contribute to the formation and maintenance of the more complex dendrites, and what factors are involved in this nucleation is unknown. Dendritic arborization (da) neurons provide an excellent system for investigating these questions. They are a subtype of multipolar neurons in the PNS of Drosophila melanogaster which produce complex dendritic arrays and do not contain centrosomes. Based on their patterns of dendrite projections, the da neurons have been grouped into four classes (I-IV) with branch complexity and arbor size increasing with class number. Class IV da neurons are ideal for studying acentrosomal microtubule nucleation because they have the most elaborate and dynamic dendritic arbor, raising intriguing questions about the modes of nucleation for its growth and maintenance (Ori-McKenney, 2012).

One potential site of acentrosomal microtubule nucleation within these neurons is the Golgi complex. A number of studies have shown that the Golgi complex can nucleate microtubules in fibroblasts. Although, in these cell types, the Golgi is tightly coupled to the centrosome, it does not require the centrosome for nucleation. It does, however, require γ-tubulin, the centrosomal protein AKAP450, and the microtubule binding proteins CLASPs. When the Golgi is fragmented upon treatment with nocodazole, the dispersed Golgi ministacks can still promote microtubule nucleation, indicating that these individual ministacks contain the necessary machinery for nucleation (Ori-McKenney, 2012 and references therein).

In both mammalian and Drosophila neurons, the Golgi complex exists as Golgi stacks located within the soma and Golgi outposts located within the dendrites. In cultured mammalian hippocampal neurons, these Golgi outposts are predominantly localized in a subset of the primary branches; however, in Drosophila class IV da neurons, the Golgi outposts appear throughout the dendritic arbor, including within the terminal branches (Ye, 2007). The Golgi outposts may provide membrane for a growing dendrite branch, as the dynamics of smaller Golgi outposts are highly correlated with dendrite branching and extension. However, the majority of larger Golgi outposts remains stationary at dendrite branchpoints and could have additional roles beyond membrane supply. It is unknown whether Drosophila Golgi outposts contain nucleation machinery similar to mammalian Golgi stacks. Such machinery could conceivably support microtubule nucleation within the complex and dynamic dendritic arbor. This study identifies a direct mechanism for acentrosomal microtubule nucleation within the dendritic arbor and reveal a role for Golgi outposts in this process. Golgi outposts contain both γ-tubulin and CP309, the Drosophila homolog of AKAP450, both of which are necessary for Golgi outpost-mediated microtubule nucleation. This type of acentrosomal nucleation contributes not only to the generation of microtubules at remote terminal branches, but also to the complex organization of microtubules within all branches of the dendritic arbor. Golgi outposts are therefore important centers of acentrosomal microtubule nucleation, which is necessary to establish and maintain the complexity of the class IV da neuronal arbor (Ori-McKenney, 2012).

This study has addressed how microtubules are organized and nucleated within the complex arbor of class IV da neurons and how essential these processes are for dendrite growth and stability. Microtubule organization within different subsets of branches in da neurons must require many levels of regulation. This study has identified the first direct mechanism for acentrosomal microtubule nucleation within these complex neurons and has uncovered a role for Golgi outposts in this process. The data are consistent with the observation that pericentriolar material is redistributed to the dendrites in mammalian neurons (Ferreira, 1993) and that γ-tubulin is depleted from the centrosome in mature mammalian neurons (Stiess, 2010). This suggests that the Golgi outposts may be one structure involved in the transport of centriole proteins such as γ-tubulin and CP309. This study found that microtubule nucleation from these Golgi outposts correlates with the extension and stability of terminal branches, which is consistent with the observation that EB3 comet entry into dendritic spines accompanies spine enlargement in mammalian neurons (Jaworski, 2009). It is striking that microtubule organization in shorter branches, but not primary branches, mimics the organization in mammalian dendrites, with a mixed microtubule polarity in the secondary branches and a uniform plus end distal polarity in the terminal branches. Kinesin-2 and certain +TIPS are necessary for uniform minus end distal microtubule polarity in the primary dendrites of da neurons. Golgi outpost mediated microtubule nucleation could also contribute to establishing or maintaining this polarity both in the terminal branches and in the primary branches. It will be of interest to identify other factors that may be involved in organizing microtubules in different subsets of branches in the future (Ori-McKenney, 2012).

In vivo and in vitro data support a role for Golgi outposts in nucleating microtubules at specific sites within terminal and primary branches. However, it is noted that not all EB1 comets originate from Golgi outposts, indicating other possible mechanisms of generating microtubules. One potentially important source of microtubules is the severing of existing microtubules by such enzymes as katanin and spastin, both of which are necessary for proper neuronal development. It is likely that both microtubule nucleation and microtubule severing contribute to the formation of new microtubules within the dendritic arbor; however, the current studies suggest that Golgi-mediated nucleation is especially important for the growth and maintenance of the terminal arbor. In γ-tubulin and CP309 mutant neurons, the primary branches contain a similar number of EB1 comets, but only a small fraction of the terminal branches still contain EB1 comets. This result indicates that severing activity or other sources of nucleation may suffice for microtubule generation within the primary branches, but γ-tubulin mediated nucleation is crucial in the terminal branches. As a result, the terminal branch arbor is dramatically reduced by mutations compromising the γ-tubulin nucleation activity at Golgi outposts (Ori-McKenney, 2012).

It is important to note that Golgi outposts are present in the dendrites, but not in the axons of da neurons; thus, this mode of nucleation is dendrite specific and likely contributes to the difference in microtubule arrays in axons and dendrites. While the axon is one long primary branch with uniform microtubule polarity, the dendrite arbor is an intricate array of branches where microtubule polarity depends on branch length. Therefore, this more elaborate branched structure may have evolved a variety of nucleation mechanisms, including Golgi outpost nucleation and microtubule severing. Intriguingly, in da neurons lacking cytoplasmic dynein function, the Golgi outposts are mislocalized to the axon, which appears branched and contains microtubules of mixed polarity (Zheng, 2008). It is speculated that in these mutants, Golgi-mediated microtubule nucleation within the axon is contributing to the mixed microtubule orientation and formation of ectopic dendrite-like branches (Ori-McKenney, 2012).

Only a subpopulation of Golgi outposts could support microtubule nucleation both in vivo and in vitro. The results show that Golgi outpost mediated microtubule nucleation is restricted to stationary outposts and dependent upon γ-tubulin and CP309, but why some outposts contain these proteins while others do not is unknown. γ-tubulin and CP309 could be recruited to the Golgi outposts in the cell body and transported on the structure into the dendrites, or they could be recruited locally from soluble pools throughout the dendritic arbor. Golgi outposts are small enough to be trafficked into terminal branches that are 150-300 nm in diameter, and therefore may provide an excellent vehicle for transporting nucleation machinery to these remote areas of the arbor. It will be interesting to determine how these nucleation factors are recruited to the Golgi outposts (Ori-McKenney, 2012).

It has been previously shown that GM130 can recruit AKAP450 to the Golgi complex, but whether the first coiled-coil domain of the Drosophila AKAP450 homolog, CP309, can also bind GM130 is unknown. Interestingly, this study has observed that predominantly stationary Golgi outposts correlated with EB1 comet formation, indicating that this specific subpopulation may contain γ-tubulin and CP309. What other factors may be necessary to properly position the Golgi outposts at sites such as branchpoints, and how this is achieved will be a fascinating direction for future studies (Ori-McKenney, 2012).

Whether the acentrosomal microtubule nucleation uncovered in this study also occurs in the dendrites of mammalian neurons is a question of great interest. Golgi outpost distribution in cultured hippocampal neurons is significantly different than that in da neurons, and hippocampal neurons do not form as elaborate arbors as da neurons. However, other types of mammalian neurons form much more complex dendritic arbors and may conceivably require acentrosomal nucleation for the growth and perpetuation of the dendrite branches (Ori-McKenney, 2012).

This study provides the first evidence that Golgi outposts can nucleate microtubules at acentrosomal sites in neurons, shedding new light on the longstanding question about the origin of the microtubule polymer in elongated neuronal processes. This source of nucleation contributes to the complex organization of microtubules within all branches of the neuron, but is specifically necessary for terminal branch development. It is thus conclude that acentrosomal microtubule nucleation is essential for dendritic branch growth and overall arbor maintenance of class IV da neurons, and that Golgi outposts are important nucleation centers within the dendritic arbor (Ori-McKenney, 2012).

PLP inhibits the activity of interphase centrosomes to ensure their proper segregation in stem cells

Centrosomes determine the mitotic axis of asymmetrically dividing stem cells. Several studies have shown that the centrosomes of the Drosophila melanogaster central brain neural stem cells are themselves asymmetric, organizing varying levels of pericentriolar material and microtubules. This asymmetry produces one active and one inactive centrosome during interphase. This study identifies pericentrin-like protein (PLP or cp309) as a negative regulator of centrosome maturation and activity. PLP is enriched on the inactive interphase centrosome, where it blocks recruitment of the master regulator of centrosome maturation, Polo kinase. Furthermore, it was found that ectopic Centrobin expression influenced PLP levels on the basal centrosome, suggesting it may normally function to regulate PLP. Finally, it is concluded that, although asymmetric centrosome maturation is not required for asymmetric cell division, it is required for proper centrosome segregation to the two daughter cells (Lerit, 2013).

A model is presented in which PLP functions as a negative regulator of centrosome maturation in interphase NBs by preventing the localization of Polo to the basal centrosome. Loss of PLP leads to the activation of both centrosomes in interphase and greatly reduces their mobility, resulting in the atypical apical positioning and inheritance of both centrosomes. Temperature shift experiments illustrate that plp- mutants are sensitized to increased centrosome segregation and mitotic spindle defects. It is concluded that inhibition of basal/mother centrosome maturation during interphase is critical for proper centrosome partitioning and mitotic spindle organization in neural stem cells (Lerit, 2013).

It is unknown how PLP functions as a negative regulator, but one model may involve an indirect role for PLP. For example, PLP might normally promote early centriole separation to ensure efficient movement of the mother centriole away from the apical cortex to allow for its inactivation. Two experiments were performed to test this model. First, the timing of centriole separation was analyzed in cells exiting mitosis in WT and plp- NBs, and it was found that centriole disengagement and the earliest signs of independent centriole movement are unaffected. Therefore, centrosomes remain apically positioned in early interphase in both WT and plp- NBs. Next, whether apical positioning of centrioles is sufficient for their activation was tested. Early interphase WT NBs were analyzed that contained two apical centrioles (<30o separation) and no correlation was found between centrosome position and activity. Collectively, these observations argue that PLP does not influence early centrosome separation and that centrosome location is not critical for mother centrosome inactivation (Lerit, 2013).

Instead, the alternative hypothesis is favored that PLP directly regulates centrosome activity, which, in turn, influences centrosome position. One possible model is that PLP acts on the centrosome by physically shielding the docking of promaturation factors until mitotic entry. A biochemical modification and/or conformational change would then allow PLP to transition to a positive regulator in mitosis that scaffolds other PCM factors. Another possible direct function for PLP might be to differentially modulate the behavior of proteins at the apical and basal centrosomes. It will be critical to examine how the dynamics of other centrosome proteins are affected by the loss of PLP. Given the importance of interphase centrosome asymmetry in stem cell division, it is believed that an in depth understanding of the interplay between PCM proteins in both interphase and mitosis is a critical area of future research (Lerit, 2013).

The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts

Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates and Drosophila neuroblasts, the neural precursors of the fly's central nervous system, contain molecularly and physically asymmetric centrosomes, established through differences in pericentriolar matrix (PCM) retention. For instance, the daughter centriole maintains PCM and thus microtubule-organizing center (MTOC) activity through Polo-mediated phosphorylation of Centrobin (Cnb). The mother centriole, however, quickly downregulates PCM and moves away from the apical cortex, randomly migrating through the cytoplasm until maturation sets in at prophase. How PCM downregulation is molecularly controlled is currently unknown, but it involves Pericentrin (PCNT)-like protein (plp) to prevent premature Polo localization and thus MTOC activity. This study reports that the centriolar protein bld10, the fly ortholog of Cep135, is required to establish centrosome asymmetry in Drosophila neuroblasts through shedding of Polo from the mother centrosome. bld10 mutants fail to downregulate Polo and PCM, generating two active, improperly positioned MTOCs. Failure to shed Polo and PCM causes spindle alignment and centrosome segregation defects, resulting in neuroblasts incorrectly retaining the older mother centrosome. Since Cep135 is implicated in primary microcephaly, it is speculated that perturbed centrosome asymmetry could contribute to this rare neurodevelopmental disease (Singh, 2014).

In a gene candidate approach to identify molecules required for centrosome asymmetry in Drosophila neuroblasts, this study identified bld10/Cep135 as a potential centrosome dematuration regulator. bld10 is a ubiquitous centriolar protein, localizing to centrioles in Drosophila larval neuroblasts and other cell types. To investigate centrosome asymmetry, live imaging experiments were performed in intact third-instar larval brains, labeling centrosomes with the centriolar markers DSas6::GFP or DSas4::GFP and mCherry::Jupiter. jupiter encodes for a microtubule binding protein, sharing properties with several structural microtubule-associated proteins (MAPs), and is ideally suited to visualize microtubule dynamics and microtubule-organizing center (MTOC) activity. In agreement with previous findings, it was found that wild-type (WT) interphase neuroblasts contained one apical MTOC only. The second MTOC appeared during prophase in close proximity to the basal cortex. By prometaphase, both MTOCs reached maximal activity and intensity. However, in bld10 mutant interphase neuroblasts (bld10c04199/Df(3L)Brd15, two centrosomes of similar size and MTOC activity were observed close together on the apical cortex. The two centrosomes progressively separated from each other until they reached their respective positions on the apical and basal cortex by prometaphase. Thus, in contrast to the wild-type, bld10 mutant neuroblasts show symmetric centrosome behavior. bld10's centrosome asymmetry defect could be rescued with bld10::GFP, and immunohistochemistry experiments confirmed the live imaging results (Singh, 2014).

The bld10c04199 allele is predicted to produce a truncated protein, retaining bld10's N terminus. A new N-terminal deletion allele (bld10ΔN was generated that showed the same centrosome asymmetry phenotype. In addition, neuroblasts were found containing monopolar and multipolar spindles, which are not observed with the bld10c04199 allele. This suggests that bld10c04199 is a separation-of-function allele, specifically disrupting centrosome asymmetry. Unless otherwise noted, all of the experiments described in the following sections were performed with the bld10c04199 allele (Singh, 2014).

The lack of centrosome asymmetry in bld10 mutant neuroblasts could be due to aberrant centriole migration. For example, the mother centriole could either fail to migrate through the cytoplasm or migrates back to the apical cortex to mature. This hypothesis was tested, measuring centriole migration as a function of time, and it was observed that centriolar migration in wild-type and bld10 mutant neuroblasts occur in two distinct phases: (1) centrioles steadily separated from each other, followed by (2) a sudden increase in intercentriolar distance, which peaked when centrioles reached a separation distance of ~4-6 μm in the wild-type and bld10 mutants. Centrioles in bld10 mutants did not require more time to reach this threshold distance and did not return to the apical cortex to mature. It is concluded that bld10's centrosome asymmetry defect is not due to aberrant centriole migration (Singh, 2014).

To get mechanistic insight into the bld10 phenotype, live imaging was used to measure the dynamic localization of three GFP-tagged pericentriolar matrix (PCM) markers: γ-tubulin (γ-Tub), Mini spindles (Msps; CKAP5 in vertebrates) and centrosomin (Cnn; CDK5Rap2 in vertebrates). Wild-type neuroblasts showed robust localization of γ-Tub, Msps, and Cnn to the apical centrosome during interphase. After centrosome splitting, all three PCM markers were downregulated from the basal centrosome (shedding phase) but reaccumulated during prophase (maturation phase. bld10 mutant neuroblasts also correctly localized γ-Tub, Msps, and Cnn to the apical interphase centrosome. However, similar to the MTOC marker Jupiter, γ-Tub, Msps, and Cnn were not downregulated from the separating centriole. Centrosome size was measured and a centrosome asymmetry index was plotted, starting at centrosome splitting until metaphase. Wild-type centrosomes developed a clear size asymmetry during the shedding phase and reduced it during the maturation phase. bld10 mutant centrosomes stayed similar in size, manifested in an asymmetry index below 1.5. Centrosome size and intensity measurements also revealed that in most wild-type neuroblasts, γ-Tub, Msps, and Cnn were removed from the basal centrosome ~15 min after centrosome splitting. Basal wild-type centrosomes were essentially devoid of γ-Tub after that time, whereas bld10 mutants contained equal amounts of this PCM marker. Changes in centrosome size were further compared, and it was found that wild-type apical centrosomes predominantly grew, whereas basal centrosomes increased (maturation phase) and decreased (shedding phase) their size to almost the same extent. bld10 mutant centrosomes were able to enlarge but showed very little size reduction, comparable to apical wild-type centrosomes. It is concluded that bld10 mutant centrosomes are able to mature but fail to downregulate the PCM markers γ-Tub, Msps, and Cnn (Singh, 2014).

The results suggest two possible mechanisms for centrosome asymmetry: (1) bld10 could prevent premature mother centrosome maturation by blocking the precocious accumulation of PCM proteins. (2) Alternatively, bld10 could promote PCM shedding right after centrosomes separate, thereby preventing the basal mother centrosome to prematurely become an MTOC. An in vivo pulse-chase labeling experiment was devised to distinguish between these two possibilities. To this end, Cnn at its endogenous locus was tagged with the photoconvertable fluorescent protein mDendra2. If bld10 blocks premature PCM accumulation, mother centrioles should quickly shed photoconverted Cnn and prematurely reaccumulate unconverted Cnn in bld10 mutants. Vice versa, if PCM shedding is compromised, it should be possible to follow the photoconverted centrosomes from the moment they separate until telophase. It was found that apical wild-type daughter centrioles retained the majority of photoconverted Cnn::mDendra2 from early interphase until prophase (possibly longer), indicating that very little Cnn protein gets exchanged. The basal mother centriole, however, lost photoconverted Cnn::mDendra2 within approximately 10-15 min after centriole separation, confirming that Cnn is shed quickly. Interestingly, bld10 centrioles retained photoconverted Cnn::mDendra2 for at least 45 min after separation. In some cases, one of the centrioles decorated with photoconverted Cnn::mDendra2 was even inherited by the newly formed GMC. It is conclude that (1) on the apical centrosome, Cnn protein turnover is absent or significantly reduced during interphase, that (2) on the basal centrosome, Cnn is shed quickly and replaced with new Cnn when maturation sets in, and that (3) bld10's centrosome asymmetry defect is not due to premature centrosome maturation. Instead, separating basal centrioles fail to shed Cnn in particular and possibly PCM proteins in general (Singh, 2014).

To elucidate the molecular mechanism underlying PCM shedding, the relationship was analyzed between bld10, Centrobin (Cnb), and Pericentrin (PCNT)-like protein (plp). Recently, it was shown that Cnb is necessary and sufficient for PCM retention on the apical daughter neuroblast centrosome. However, gain- and loss-of-function experiments with Cnb did not perturb bld10's localization. Similarly, as in the wild-type, Cnb was localized asymmetrically in bld10 mutants. plp mutants fail to downregulate γ-Tub on the mother centrosome. In plp mutant neuroblasts, the basal centrosome retained Cnn and MTOC activity during interphase. Interestingly, photoconversion experiments showed that similar to bld10, Cnn shedding from the basal centrosome was compromised in plp mutants. However, plp localization is not perturbed in bld10 mutant neuroblasts, and bld10 was normally localized in plp mutants. Knockdown of plp in bld10 mutants did not enhance bld10 PCM shedding phenotype, but due to the occurrence of additional phenotypes (fragmented or multiple centrosomes), the shedding phenotype could also be partially masked. In sum, it is concluded that bld10 is regulating centrosome asymmetry independently of Cnb and that plp is also required to shed Cnn (Singh, 2014).

Since the mitotic kinase Polo has been implicated in PCM retention during interphase, Polo localization dynamics were analyzed in wild-type and bld10 mutant neuroblasts. Recently, it was reported that Polo localizes to the apical centrosome during interphase and is only detectable at the basal centrosome during prophase, when maturation sets in. A Polo::GFP protein trap line was used and it was confirmed that Polo is stably localized to the apical interphase centrosome. Surprisingly, weak Polo was also found on the separating mother centrosome. Subsequently, Polo disappeared from the basal mother centriole within 10 min, comparable to Cnn, γ-Tub, and Msps shedding times. With a genomic Polo::GFP transgene, showing lower fluorescence intensity, Polo was found to be localized on both centrosomes in bld10 mutants. These data suggest that in wild-type neuroblasts, Polo is not just recruited onto the basal mother centrosome by prophase as previously reported, but is also subject to shedding during interphase. Polo is required for PCM retention since in bld10 mutant neuroblasts treated with the Polo inhibitor BI2536, both centrosomes lose MTOC activity. Thus, it is concluded that shedding of Polo is a requirement for the subsequent shedding of Cnn, γ-Tub, and Msps, enabling basal mother centrosome dematuration and the establishment of centrosome asymmetry (Singh, 2014).

Finally, the consequences were analyzed of disrupted centrosome asymmetry. The daughter centriole was labeled with Cnb::YFP ] and centrosome segregation was assayed. It was confirmed that wild-type neuroblasts faithfully retain the Cnb* daughter centriole, whereas the Cnb- centrosome segregates into the GMC. bld10 mutants showed correct asymmetric Cnb localization, but ~45% of bld10 mutant neuroblasts wrongly retained the mother centriole and segregated the daughter centriole into the GMC. Cnb+ centrosomes are usually bigger in wild-type and bld10 mutant neuroblasts, but centrosome segregation is independent of MTOC activity and size since bld10 mutant neuroblasts often retained the smaller centrosome. It is concluded that centrosome asymmetry is required for faithful centrosome segregation (Singh, 2014).

Since bld10 mutant neuroblasts have mispositioned MTOCs in relation to the apical-basal division axis, spindle orientation was analyzed. Immunohistochemistry experiments showed that bld10 mutant neuroblast spindles deviate from the regular orientation range, with isolated cases of extreme misalignment. Metaphase spindles, aligned orthogonally to the apical-basal polarity axis, can induce symmetric neuroblast divisions, resulting in an increase of the neural stem cell pool. However, neuroblast numbers were unchanged in bld10 mutants compared to control brains, and symmetric neuroblast divisions were not found with live imaging. Instead, time-lapse experiments showed that bld10 mutant centrosomes prematurely formed misaligned bipolar spindles. Spindle rotation during metaphase corrected this misalignment. Apical-basal polarity is a prerequisite for correct spindle orientation, but apical and basal polarity markers localized normally in bld10 mutants. Similarly, the spindle orientation regulators, Partner of Inscuteable (Pins; LGN/AGS3 in vertebrates) and the NuMA ortholog Mud, were also correctly localized. It is concluded that controlled PCM shedding and maturation is required for correct centrosome positioning but backup mechanisms exist, correcting for misaligned metaphase spindles (Singh, 2014).

Many cell types, including stem and progenitor cells, contain asymmetric centrosomes and segregate them nonrandomly, suggesting a connection between centrosome asymmetry and cell fate. How centrosome asymmetry is regulated is currently not understood, but centrosome dematuration is a critical step in establishing centrosome asymmetry. It was found that the centriolar protein bld10/Cep135, known as a centriole duplication and elongation factor, is required to establish centrosome asymmetry. On the basis of these data, it is proposed that plp and bld10 induce Polo's removal from the mother centriole, triggering the shedding of PCM proteins such as Cnn, γ-Tub, and Msps. Polo has been reported to be closely associated with centrioles, ideally positioned to phosphorylate both centriolar and PCM proteins. Thus, it is proposed that Polo-mediated phosphorylation of PCM proteins maintains a stable interaction between the centriole and surrounding PCM (Singh, 2014).

How Polo localization is regulated is currently not known, and no direct molecular interaction was detected between bld10 and Polo. Although no centriolar markers were found to be mislocalized inbld10 mutants (at the resolution level of confocal microscopy), it is possible that structural centriole defects, as detected in bld10 mutant spermatocytes and wing disc cells, could affect PCM turnover rates. However, since bld10 is not asymmetrically localized, it is difficult to conceive how such defects specifically compromise the behavior of the mother but not the daughter centrosome (Singh, 2014).

Although perturbed centrosome asymmetry does not seem to undermine neuroblast polarity, the cell cycle, or physical and molecular asymmetric cell division, the possibility cannot be excluded that centrosome asymmetry could have long-term consequences currently beyond the ability to detect. Interestingly, defects in centrosome maturation or mutations in Cep135 can cause neurodevelopmental disorders such as primary microcephaly. It will be interesting to address the question whether lack of Cep135 is causing microcephaly due to compromised centrosome asymmetry and dematuration (Singh, 2014).


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pericentrin-like protein: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 October 2014

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