BIOLOGICAL OVERVIEW

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. This study shows that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH₂-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN (Terada, 2003).

In animal cells, microtubules are organized from the centrosome/microtubule-organizing center (MTOC), composed of a pair of centrioles and the surrounding pericentriolar material. Individual microtubules are nucleated from an ~25-nm γ-tubulin–containing ring complex (γ-TuRC). At the onset of M phase, the centrosome becomes 'mature' and organizes more microtubules, which is accompanied with an increased level of γ-tubulin accumulation at each spindle pole. One of the molecules that has been implicated in the mechanism of centrosome maturation is Aurora-A, a mitosis-specific Ser/Thr kinase located at mitotic poles and spindle microtubules. The kinase, originally identified as a gene product important in spindle assembly and function in Drosophila, has recently been shown to be in the Ran-signaling pathway and to play an important role in efficient transmission of Ran-GTP gradient established by the condensed chromosomes for the control of spindle assembly and dynamics. Aurora-A binds to spindle components, such as TACC/XMAP215 and TPX2. Although possible functions of those molecules and their interaction with Aurora-A in bipolar spindle formation have been elucidated, mechanisms of how Aurora-A stimulates the recruitment of γ-tubulin to the centrosome at spindle poles have not yet been evaluated. To address this question, centrosomal proteins were sought that interact with Aurora-A and regulate the process of microtubule nucleation onto the centrosome (Terada, 2003).

By screening of a Drosophila two-hybrid library, two clones were isolated encoding a molecule capable of interaction with Aurora-A. The sequence corresponds to the COOH-terminal domain of centrosomin (CNN), a core component of the centrosome important for assembly of mitotic centrosomes in Drosophila (Heuer, 1995; Megraw, 1999). Although the truncated polypeptide covered by clone CNN-C1 appears to be sufficient for interaction with Aurora-A, the binding intensity was weaker than CNN-C. Endogenous Aurora-A, but not Aurora-B, immunoprecipitates with HA-tagged CNN expressed in S2 cells. Specificity of the COOH-terminal domain of CNN for interaction with Aurora-A was further confirmed by in vitro binding assays (Terada, 2003).

To investigate the role of protein interaction in the centrosome, S2 cells were prepared from which Aurora-A or CNN was depleted by RNA interference (RNAi). In cells lacking Aurora-A, not only CNN, but also γ-tubulin, were absent at each spindle pole. When CNN was depleted, neither γ-tubulin nor Aurora-A was seen at the spindle pole. In cells with partially depleted Aurora-A or CNN, comparable amounts of γ-tubulin and CNN or Aurora-A were detected at each pole. Besides γ-tubulin, other centrosome proteins, CP190 and CP60, become dislocated from the spindle poles in RNAi cells. Therefore, it is concluded that CNN and Aurora-A are mutually dependent for localization at spindle poles, which is required for proper targeting of other centrosomal proteins to the centrosome. This is consistent with previous observations (Barbosa, 2000) that the centrosomal association of CNN is not dependent on the presence of γ-tubulin/γ-TuRC (Terada, 2003).

To confirm the role of CNN in recruiting γ-tubulin, protein interaction was analyzed in vitro. Nickel beads conjugated with His-tagged CNN were mixed with cell extracts prepared from colcemid-treated S2 cells. γ-Tubulin was specifically sedimented by the full and NH₂-terminal sequence, but not the COOH-terminal sequence of CNN. Because neither in vitro binding assays nor two-hybrid screens demonstrated direct binding between two molecules, CNN may interact with a γ-tubulin complex, rather than γ-tubulin directly. Further, HA-tagged CNN was expressed in S2 cells. Exogenous proteins caused formation of γ-tubulin–containing cytoplasmic aggregates capable of microtubule formation and association with microtubule asters. These results clearly indicate that the NH₂-terminal domain of CNN interacts with γ-tubulin/γ-TuRC and plays an important role in assembly of MTOCs (Terada, 2003).

γ-Tubulin/γ-TuRC–mediated microtubule assembly is believed to be common among species. Thus, it is highly likely that an Aurora-A–binding molecule(s) equivalent to CNN is functioning in a variety of organisms. Although Drosophila CNN was unable to associate with mammalian Aurora-A in transfected mammalian cells as well as by two-hybrid screens, the NH₂-terminal domain of CNN still interacts with γ-tubulin/γ-TuRC in mammalian cells as in S2 cells. To analyze a possible role of CNN–γ-tubulin interaction in initiation of microtubule assembly, Drosophila CNN was overexpressed in mammalian cells. HA-tagged CNN induces cytoplasmic foci in various sizes and numbers. Significantly, the pattern of microtubule distribution is profoundly affected as a result of microtubule association with virtually every dot containing CNN. These sites can initiate microtubule formation as evidently shown in cells where short microtubules are assembled during brief recovery from nocodazole treatment. All cells overexpressing CNN induced microtubule-organizing sites, which were associated with centrosome proteins, such as pericentrin and Cep135. Particularly prominent was γ-tubulin, which was probably recruited from a large cytoplasmic pool. In support of this view, GFP-tagged exogenous γ-tubulin became colocalized with HA-CNN to participate in the formation of microtubule-nucleating sites. This was in striking contrast with cells expressing γ-tubulin alone, where cytoplasmic aggregates induced by γ-tubulin expression could not contribute to microtubule formation. These results suggest that microtubules are directly nucleated from the CNN aggregates through the mechanism mediated by γ-tubulin/γ-TuRC (Terada, 2003).

To confirm the microtubule-nucleating activity of the CNN aggregates, microtubules were polymerized in vitro by incubating isolated GFP-tagged CNN dots with X-rhodamine–conjugated brain tubulin. There was always a dot positive in GFP fluorescence at the center of the microtubule asters. Although variable numbers of microtubules emanated from the center, more microtubules tended to polymerize onto the GFP dots in larger sizes. The process of aster formation was monitored by time-lapse microscopy. A fluorescence image taken 10 min after mounting the sample on a microscopic stage revealed several microtubules growing from a GFP-positive site. As time progressed, more microtubules appeared to emanate from the center, indicating that microtubules were formed by direct polymerization onto the CNN-containing foci, rather than that preformed microtubules were gathered around the center (Terada, 2003).

Microtubules are nucleated from the pericentriolar material that surrounds the centrioles of the centrosome. To compare ultrastructure of microtubule-initiating sites induced by CNN with that of the pericentriolar material/centrosome, CHO cells expressing GFP-tagged CNN were examined by EM. Two microtubule asters were seen formed in cells that were briefly extracted before fixation. Located at each focal point of microtubule asters was an electron-dense particle in various sizes and shapes. Unlike the pericentriolar material, which has been described as an ill-defined amorphous cloud, the entire structure induced by CNN was well delineated by electron-dense materials to which microtubules were attached. In favorable sections, microtubules could be seen penetrating to the interior region of the aggregates. Neither centrioles nor centrosomal substructures, such as satellites, appendages, and CHO cell–specific virus particles, were generally seen at the site induced by CNN expression. Because CNN is a coiled-coil structural protein (Heuer, 1995), the dense particles likely represent the aggregated form of overexpressed CNN proteins (Terada, 2003).

Multiple centrosomes/MTOCs have been detected in cells in which the mechanism of centrosome duplication coupled with the cell cycle control becomes deregulated. In the case of CNN-containing MTOCs, their number and size formed during relatively short periods (8–12 h) varied greatly according to the level of protein expression. Moreover, no centrioles were found at ectopic MTOCs by EM and immunostaining with centriole-specific centrin-2 antibodies. Therefore, it is plausible that CNN expression causes the formation of protein aggregates that acquire the microtubule-nucleating capacity by recruiting γ-tubulin/γ-TuRC. This unique property of CNN to generate microtubule-nucleating sites by interacting with γ-tubulin/γ-TuRC suggested CNN may function as an adaptor for connecting γ-tubulin to the centrosome (Terada, 2003).

By expressing truncated polypeptides, it was concluded that CNN's ability to interact with γ-tubulin/γ-TuRC and induce ectopic microtubule-nucleating sites resides in the NH₂-terminal sequence of CNN from which the Aurora-A–binding domain is omitted. In contrast, cytoplasmic aggregates formed in cells expressing the COOH-terminal domain failed to initiate microtubule formation in both S2 and mammalian cells. These results lead to the conclusion that CNN consists of two functionally distinct subdomains: the Aurora-A–binding site is at the COOH terminus capable of formation of the protein complex to be recruited to the spindle pole, and the NH₂-terminal sequence is involved in assembling centrosomes/MTOCs by recruiting γ-tubulin/γ-TuRC. Although no CNN homologues have yet been identified outside Drosophila, Aurora-A would likely be involved in the control of microtubule nucleation through its association with the COOH terminus of a CNN-related molecule(s) in mammalian cells (Terada, 2003). Control of mitotic spindle assembly onto the centrosome could be achieved by several mechanisms, including nucleation of individual microtubules onto γ-tubulin–containing protein complexes, stimulation of microtubule nucleation and stabilization of polymerized microtubules by MAPs, and recruitment of minus ends of preexisting microtubules by the action of motor activity to the centrosome. Aurora-A binds not only CNN but also the D-TACC/MSPS/XMAP215 complex. These components appear to be required for microtubule assembly on mitotic centrosomes/poles controlled through the distinct mechanisms from that of γ-tubulin recruitment. Therefore, it is reasonable that Aurora-A plays a role in regulating the overall process of centrosome maturation by orchestrating multiple pathways of microtubule assembly during mitosis. It is worth mentioning that individual mechanisms of microtubule assembly may show a distinct requirement for protein phosphorylation and the Aurora-A kinase activity; although both Aurora-A and CNN are still able to locate at the centrosome, D-TACC/MSPS complex failed to be recruited to spindle poles in the absence of enzymatic activity of Aurora-A kinase (Terada, 2003).

Aurora kinases are highly expressed in cells derived from many human tumor cell types, which frequently contain multiple centrosomes. Because defects in the number, structures, and function of centrosomes are closely associated with the genetic instability in transformed cells, Aurora-A might be involved in tumorigenesis by inducing abnormal numbers of MTOCs as a result of inappropriate distribution of CNN-like molecule(s) (Terada, 2003).

Centrosomin represses dendrite branching by orienting microtubule nucleation

Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization (Yalgin, 2015).

Neurons primarily receive inputs through their dendrite arbors. The shape and complexity of the dendrite arbor, which is elaborated during differentiation, enables the neuron to properly cover its receptive field and establishes the positions of inputs into the arbor. Disruptions to dendritic branching can precipitate intellectual disability and psychiatric disorders (Yalgin, 2015).

Arbor morphology is regulated for each neuron class to support its structural and functional requirements3; it is genetically encoded, being linked to class specification by transcriptional programs. For example, in Drosophila, the single unbranched dendrite of external sensory neurons is specified over an alternative multipolar dendritic arborization (da) neuron fate by the Prdm transcription factor Hamlet. Similarly, the proneural transcription factor Ngn2 regulates multiple aspects of pyramidal neuron development in the mammalian cortex, including the specification of a characteristic apical dendrite, whereas Cux1, Cux2 and SatB2 link dendrite development to cortical layer-specific developmental program (Yalgin, 2015).

Dendrite development is controlled in a neuron class-specific manner to create differences in arbor morphology and complexity. Class-specific dendrite targeting is regulated via the activity of transmembrane adhesion proteins. For example, in C. elegans, class-specific expression patterns of the transcription factors MEC-3, AHR-1 and ZAG-1 regulate the morphology of mechanosensory neurons, and MEC-3 promotes differential expression of the Claudin-like membrane protein HPO-30 to enable lateral branch stabilization. Drosophila da neurons exist in four classes, of which class I neurons express Abrupt (Ab), which defines their simple arbor shape, and class IV express Knot and Cut, which together promote the complex morphology of this class. The EGF-repeat factor Ten-m is co-regulated by both Knot and Ab to control the direction of branch outgrowth in both class I and IV neurons (Yalgin, 2015).

Contrasting activities of Knot, Cut and Ab in da neurons emphasize that altering dendrite branching is fundamental for regulating arbor complexity. Knot and Cut promote branch formation; conversely, Ab represses branch formation. Little is understood about how modulatory control over branching is achieved (Yalgin, 2015).

Microtubules polymerize via the addition of Tubulin dimers, primarily at the plus end. In axons, microtubules polymerize in an anterograde direction, providing a protrusive force for outgrowth. Microtubule polymerization also drives axon branch formation, as precursors only transform into branches after microtubule invasion. Mature dendrites have a predominantly minus-ends-out microtubule array, nevertheless recent studies have identified that anterograde microtubule polymerization events can initiate or extend branches, or modulate the size of dendritic spines. In addition, the re-initiation of dendrite growth and branch formation following injury uses upregulation of microtubule polymerization and its polarization in the anterograde direction (Yalgin, 2015).

This study examined whether class-specific transcription factors regulate branch promotion and repression by controlling microtubule organization during arbor development. In da sensory neurons, microtubule nucleation and polarity can be assayed in vivo using transgenic markers. Using genetic manipulation of class I and class IV da neurons, this study found that Ab controls class-specific differences in the localization of microtubule minus-end-directed markers in the da neuron arbor. By assaying Ab-mediated changes in the expression of a set of candidate microtubule regulators and using chromatin immunoprecipitation (ChIP), this study identified Cnn (Centrosomin) as an effector of Ab action. Cnn-centered control mechanisms, analogous to those that cluster microtubule nucleation events to create the mitotic spindle, are used in growing dendrites to regulate branching and to create class-specific arbor complexity (Yalgin, 2015).

Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization (Yalgin, 2015).

Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization (Yalgin, 2015).

Evidence that a positive feedback loop drives centrosome maturation in fly embryos

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. This study shows that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. These findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos (Alvarez-Rodrigo, 2019).

Centrosomes play an important part in many aspects of cell organisation, and they form when a mother centriole recruits pericentriolar material (PCM) around itself. The PCM contains several hundred proteins, allowing the centrosome to function as a major microtubule (MT) organising centre, and also as an important coordination centre and signalling hub. Centrosome dysfunction has been linked to several human diseases and developmental disorders, including cancer, microcephaly and dwarfism (Alvarez-Rodrigo, 2019).

During interphase, the mother centriole recruits a small amount of PCM that is highly organised. As cells prepare to enter mitosis, however, the PCM expands dramatically around the mother centriole in a process termed centrosome maturation. Electron microscopy (EM) studies suggest that centrioles organise an extensive 'scaffold' structure during mitosis that surrounds the mother centriole and recruits other PCM components such as the γ-tubulin ring complex (γ-TuRC) (Alvarez-Rodrigo, 2019).

In the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, a relatively simple pathway seems to govern the assembly of this mitotic PCM scaffold. The conserved centriole/centrosome protein Spd-2/SPD-2 (fly/worm nomenclature) cooperates with a large, predominantly predicted-coiled-coil, protein (Cnn in flies, SPD-5 in worms) to form a scaffold whose assembly is stimulated by the phosphorylation of Cnn/SPD-5 by the mitotic protein kinase Polo/PLK-1. Mitotic centrosome maturation is abolished in the absence of this pathway, and some aspects of Cnn and SPD-5 scaffold assembly have recently been reconstituted in vitro. Vertebrate homologues of Spd-2 (Cep192), Cnn (Cdk5Rap2/Cep215) and Polo (Plk1) also have important roles in mitotic centrosome assembly, indicating that elements of this pathway are likely to be conserved in higher metazoans. In vertebrate cells another centriole and PCM protein, Pericentrin, also has an important role in mitotic centrosome assembly that is dependent upon its phosphorylation by Plk1. Pericentrin can interact with Cep215/Cnn, but in flies the Pericentrin-like-protein (Plp) has a clear, but relatively minor, role in mitotic PCM assembly when compared to Spd-2 and Cnn (Alvarez-Rodrigo, 2019).

Although most of the main players in mitotic centrosome-scaffold assembly appear to have been identified, several fundamental aspects of the assembly process remain mysterious. Cells entering mitosis, for example, contain two mother centrioles that assemble two mitotic centrosomes of equal size. It is unclear how this is achieved, as even a slight difference in the initial size of the two growing centrosomes would be expected to lead to asymmetric centrosome growth-as the larger centrosome would more efficiently compete for scaffolding subunits. The centrioles in fly embryos appear to overcome this problem by constructing the PCM scaffold from the 'inside-out': Spd-2 and Cnn are only incorporated into the scaffold close to the mother centriole, and they then flux outwards to form an expanded scaffold around the mother centriole. In this way, the growing PCM scaffold could ultimately attain a consistent steady-state size-where incorporation around the mother centriole is balanced by loss of the scaffold at the centrosome periphery-irrespective of any initial size difference in the PCM prior to mitosis (Alvarez-Rodrigo, 2019).

A potential problem with this 'inside-out' mode of assembly is that the rate of centrosome growth is limited by the very small size of the centriole. Mathematical modelling indicates that the incorporation of a crucial PCM scaffolding component only around the mother centriole cannot easily account for the high rates of mitotic centrosome growth observed experimentally. To overcome this problem, it has been proposed that centrosome growth is 'autocatalytic', with the centriole initially recruiting a key scaffolding component that can subsequently promote its own recruitment. It has been proposed that Spd-2 and Cnn could form a positive feedback loop that might serve such an autocatalytic function: Spd-2 helps recruit Cnn into the scaffold, and Cnn then helps to maintain Spd-2 within the scaffold, thus allowing higher levels of Spd-2 to accumulate around the mother centriole, which in turn drives higher rates of Cnn incorporation (Alvarez-Rodrigo, 2019).

In worms and vertebrates, SPD-2/Cep192 can help recruit PLK1/Plk1 to centrosomes and Cep192 also activates Plk1 in vertebrates, in part through recruiting and activating Aurora A, another mitotic protein kinase implicated in centrosome maturation. It is suspected, therefore, that in flies Spd-2 might recruit Polo into the centrosome-scaffold to phosphorylate Cnn and so help to generate a positive feedback loop that drives the expansion of the mitotic PCM. In flies, however, no interaction between Polo and Spd-2 has been reported. Indeed, an extensive Y2H screen for interactions between key centriole and centrosome proteins identified interactions between Spd-2 and the mitotic kinases Aurora A and Nek2, and between Polo and the centriole proteins Sas-4, Ana1 and Ana2, but not between Polo and Spd-2. A possible explanation for this result is that Polo/Plk1 is believed to be largely recruited to its many different locations in the cell, including centrosomes, through its Polo-Box-Domain (PBD), which binds to phosphorylated S-S/T(p) motifs. Perhaps any such Polo binding sites in fly Spd-2 were simply not phosphorylated in the Y2H experiments. In support of this possibility, phosphorylated S-S/T(p) motifs in SPD-2/Cep192 have previously been shown to help recruit PLK1/Plk1 to centrosomes in worms, frogs and humans (Alvarez-Rodrigo, 2019).

This study examined the potential role of Spd-2 in recruiting Polo to centrosomes in Drosophila embryos. Like Cnn, Spd-2 is largely unphosphorylated in the cytosol, but is highly phosphorylated at centrosomes, where Spd-2 and Polo extensively co-localise within the pericentriolar scaffold. A Spd-2 fragment containing 19 S-S/T motifs exhibits enhanced binding to the PBD in vitro when it has been phosphorylated by Plk1, but no enhancement is seen if these S-S/T motifs are mutated to T-S/T-a mutation that strongly perturbs PBD binding. This study expressed forms of Spd-2 in vivo in which either all 34 S-S/T motifs, or the 16 most conserved S-S/T motifs, have been mutated to T-S/T to perturb PBD-binding. These mutant Spd-2 proteins are still recruited to mother centrioles, as are Polo and Cnn, and these proteins assemble a PCM scaffold around the mother centriole. Strikingly, however, this PCM scaffold can no longer expand outwards, and centrosome maturation fails. These observations provide strong support for the hypothesis that Spd-2, Polo and Cnn cooperate to form a positive feedback loop that is required to drive the rapid expansion of the mitotic PCM in fly embryos (Alvarez-Rodrigo, 2019).

It was previously proposed that three proteins -- Spd-2, Polo and Cnn -- together form a scaffold that expands around the mother centriole to recruit other PCM components to the mitotic centrosome. The data presented in this study suggests that these three proteins cooperate to form a positive feedback loop that drives the dramatic expansion of the mitotic PCM scaffold in fly embryos (Alvarez-Rodrigo, 2019).

The following model is proposed (see Polo and Cnn appear to form a positive feedback loop that drives the expansion of the mitotic PCM scaffold). In interphase cells, Spd-2, Polo and Cnn are recruited around the surface of the mother centriole, but Polo is inactive and Spd-2 and Cnn are not phosphorylated-so no scaffold is assembled. As cells prepare to enter mitosis, centrosomal Spd-2 becomes phosphorylated. In vitro data suggests that Polo is involved in this phosphorylation (via a 'self-priming and binding' mechanism), but other mitotic kinases may also be involved. Phosphorylation allows Spd-2 to form a scaffold that fluxes outwards and that can recruit both Polo (via phosphorylated S-S/T(p) motifs) and Cnn . The active Polo phosphorylates Cnn, allowing it to also form a scaffold. The Spd-2 scaffold is inherently unstable, so it can only accumulate around the mother centriole if it is stabilised by the Cnn scaffold. The Cnn scaffold therefore allows the Spd-2 scaffold to expand outward, increasing Spd-2 levels within the PCM scaffold and allowing Spd-2 to recruit more Cnn and more Polo into the scaffold. This is a classical positive feedback loop in which the Output (the PCM scaffold in toto) directly increases the Input (the Spd-2 scaffold) (Alvarez-Rodrigo, 2019).

If Spd-2 cannot efficiently recruit Polo, as appears to be the case with the Spd-2-ALL and Spd-2-CONS mutants, it can still recruit Cnn, and this is, at least initially, phosphorylated by the pool of Polo that is still present around the mother centriole. The data suggests that this centriolar pool of Polo is not recruited by Spd-2 (at least not via the PBD), and it is suspected that S-S/T(p) motifs in other centriole proteins, such as Sas-4, normally recruit Polo to centrioles. As a result, mutant Spd-2 proteins can still support the assembly of a 'mini-scaffold' around the mother centriole, and this can recruit some PCM and organise some MTs. The mutant Spd-2 scaffold that fluxes outwards from the mother centriole, however, cannot recruit Polo. Therefore the Cnn recruited by the expanding Spd-2 network cannot be phosphorylated, and it cannot form a scaffold to support the expanding Spd-2 network. As a result, the expanding mitotic PCM scaffold rapidly dissipates into the cytosol (Alvarez-Rodrigo, 2019).

Although this mechanism is autocatalytic-as the expanding Spd-2 scaffold allows Polo and Cnn to be recruited into the PCM at an increasing rate-crucially, the mother centriole remains the only source of Spd-2. This potentially explains the conundrum of how mitotic PCM growth is autocatalytic, but at the same time requires the mother centriole. This requirement for centrioles can also potentially explain how two spatially separated centrosomes usually grow their mitotic PCM to the same size, as PCM size may ultimately be determined by how much Spd-2 can be provided by the centrioles, rather than how much PCM was present in the centrosome when maturation was initiated (Alvarez-Rodrigo, 2019).

A key feature of this proposed mechanism is that Cnn cannot recruit itself or Spd-2 or Polo into the scaffold (although it helps to maintain the Spd-2 scaffold recruited by the centriole). If it could do so, mitotic PCM growth would no longer be constrained by the centriole as Cnn could catalyse its own recruitment. Interestingly, although Spd-2 and Cnn are of similar size in flies (1146aa and 1148aa, respectively) Spd-2 has >5X more conserved potential PBD-binding S-S/T motifs than Cnn. Moreover, a similar ratio of conserved sites is found when comparing human Cep192 (1941aa) to human Cep215/Cdk5Rap2 (1893aa), even though the human and fly homologues of both proteins share only limited amino acid identity. Perhaps, these two protein families have evolved to ensure that phosphorylated Spd-2/Cep192 can efficiently recruit Polo/Plk1, whereas phosphorylated Cnn/Cep215 cannot (Alvarez-Rodrigo, 2019).

The data indicates that multiple S-S/T(p) motifs in Spd-2 may be involved in Polo recruitment to the PCM. When only the most conserved motifs are mutated, other motifs in Spd-2 appear to be able to help recruit Polo, as evidenced by the additive effect of the Spd-2-ALL mutant compared to the Spd-2-CONS mutant. This mechanism of multi-site phosphorylation and recruitment could help amplify the maturation process (as the additional Polo recruited would allow Cnn to be phosphorylated at a higher rate) and so contribute to the establishment of the positive feedback loop (Alvarez-Rodrigo, 2019).

Another important feature of this proposed mechanism is that Spd-2 is incorporated into the mitotic PCM at the centriole surface and then fluxes outwards. This Spd-2-flux has so far only been observed in Drosophila embryos and mitotic brain cells. In fly embryos, Cnn also fluxes outwards but, unlike Spd-2, this flux requires MTs and is only observed in embryos. In C. elegans embryos, SPD-5 behaves like Cnn in somatic cells: it does not flux outwards and is incorporated isotropically throughout the volume of the PCM. Moreover, a very recent study found no evidence for an outward centrosomal flux of SPD-2 in worm embryos. Clearly, it will be important to determine whether Spd-2/Cep192 homologues flux outwards in other species and, if so, whether this flux provides the primary mechanism by which the mother centriole influences the growth of the expanding mitotic PCM (Alvarez-Rodrigo, 2019).

In vertebrates, Cep192 serves as a scaffold for Plk1 and also Aurora A-another mitotic protein kinase that plays an important part in centrosome maturation in many species. There appears to be a complex interplay between Cep192, Plk-1 and Aurora A in vertebrates, with Cep192 acting as a scaffold that allows these two important regulators of mitosis to influence each other's activity and centrosomal localisation. Spd-2 clearly plays an important part in recruiting Aurora A to centrosomes in fly cells-although it is unclear if this is direct, as fly and worm Spd-2/SPD-2 both lack the N-terminal region in vertebrate Cep192 that recruits Aurora A. How Aurora A might influence the assembly of the Spd-2, Polo/PLK-1 and Cnn/SPD-5 scaffold remains to be determined, although in worms AIR-1 (the Aurora A homologue) is required to initiate centrosome maturation, but is not required for subsequent PCM growth (Alvarez-Rodrigo, 2019).

Finally, there has been great interest recently in the idea that many non-membrane bound organelles like the centrosome may assemble as 'condensates' formed by liquid-liquid phase separation. In support of this possibility for the centrosome, purified recombinant SPD-5 can assemble into condensates in vitro that have transient liquid-like properties, although they rapidly harden into a more viscous gel- or solid-like phase. Moreover, a mathematical model that describes centrosome maturation in the early worm embryo treats the centrosome as a liquid, and it is from this model that the importance of autocatalysis was first recognised. In vivo, however, the Cnn and SPD-5 scaffolds do not appear to be very liquid-like and fragments of Cnn can assemble into micron-scale assemblies in vitro that are clearly solid- or very viscous-gel-like. The current data suggests that the incorporation of Spd-2 into the PCM only at the surface of the centriole, coupled to an amplifying Spd-2/Polo/Cnn positive feedback loop, could provide an 'autocatalytic' mechanism that functions within the conceptual framework of a non-liquid-like scaffold that emanates from the mother centriole (Alvarez-Rodrigo, 2019).

GENE STRUCTURE

cDNA clone length - 4335 bp (isoform A)

Bases in 5' UTR - 127

Exons - 6

Bases in 3' UTR - 761

PROTEIN STRUCTURE

Amino Acids - 1148 (cnn-PA)

Structural Domains

The expression pattern of cnn suggested homeotic regulation, i.e., there were higher levels of expression in the thoracic region of the CNS compared to the abdominal region. The 6.8 kb SalI fragment hybridizes to two major species of polyadenylated RNA on a northern blot of sizes 4.8 and 5 kb. To characterize the gene further this fragment was used as a probe to isolate a cDNA clone from an embryonic library. A single cDNA of 4,332 bp in size was isolated that recognizes the same species of mRNA on a northern blot as the 6.8 kb genomic fragment. Assuming a poly A tail addition of 400 bases, the size of this cDNA is in close agreement to the sizes of polyadenylated RNA species observed on a northern blot (4.8 and 5 kb) and thus appears to be near full length. Both strands of the entire 4,332 bp cDNA were sequenced by a combination of nested primers and exonuclease III treatment of both strands. Within the entire 4,332 bp, a single long open reading frame (ORF) of 3,102 bp was found. The cDNA sequence therefore contains 129 bp of 5' non-translated sequence, 1,101 bp of 3' non-translated sequence and a polyadenylation signal sequence AATAAA is found at its most 3' end. The long ORF encodes a potential polypeptide of 1,034 amino acids in length. The most striking structural features of the protein are the three putative leucine zipper motifs. The first putative leucine zipper is located from amino acids 100-126 and is bounded by several proline residues, the second is located at amino acids 525-551 and the third is from amino acids 946-972. All three zippers are within predicted coiled-coil regions. Helical wheel plots of the three zipper regions show that all contain a continuous spine of hydrophobicity greater than six helical turns and the opposite faces are rich in charged amino acid residues for the formation of salt bridges. No other protein motifs were identified, although the protein does contain a number of protein kinase recognition sites. These include 2 sites for cAMP-dependent kinase, 2 sites for Ca2+- dependent kinase II, 2 sites for GSK3 kinase, 2 sites for protein kinase C and one site for tyrosine kinase. The region of CNN extending from amino acids 85-970 shows limited homology with coiled-coil domains of myosin heavy chain from several species. This region of CNN contains long stretches of alpha helices interrupted by small regions that lack heptad periodicity, often containing proline residues which are known to disrupt alpha helices. There are no other significant homologies to known proteins in the databases (Heuer, 1995)

EVOLUTIONARY HOMOLOGS

Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase. From an insertional mutagenesis screen a novel gene, mod20+, was identified, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. Mod20p is required for the localization of components of the gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin. It is concluded that Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells (Sawin, 2004).

From an insertional mutagenesis screen, a novel gene, mto2+, was isolated involved in microtubule organization in fission yeast. mto2Delta strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2Delta defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the gamma-tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2Delta mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to "satellite" particles on interphase microtubules. In mto1Delta mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. In mto2Delta mutants, cytoplasmic forms of the gamma-tubulin complex are mislocalized, and the gamma-tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the gamma-tubulin complex (Samejima, 2005).

date revised: 2 January 2023

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