centrosomin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - centrosomin

Synonyms -

Cytological map position- 50A8--9

Function - cytoskeleton

Keywords - cell cycle, spindle organization and biogenesis, oogenesis, centrioles

Symbol - cnn

FlyBase ID: FBgn0013765

Genetic map position - 2-65

Classification - leucine zippers within predicted coiled-coil regions

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Baumbach, J., Novak, Z.A., Raff, J.W. and Wainman, A. (2015). Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo. PLoS Genet 11: e1005261. PubMed ID: 26020779
Summary:
Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. This study shows that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2-the same proteins that appear to drive mitotic centrosome assembly in flies. This finding was used to ablate aMTOC formation in acentriolar cells, and perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. It was shown that although aMTOCs could nucleate microtubules, these microtubules did not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. However, they were found to be required for robust microtubule array assembly in cells without centrioles that also lacked microtubule nucleation from around the chromatin. Importantly, aMTOCs were also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). The study proposes an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

Conduit, P. T. and Raff, J. W. (2015). Different Drosophila cell types exhibit differences in mitotic centrosome assembly dynamics. Curr Biol 25: R650-651. PubMed ID: 26241137
Summary:
Centrosomes are major microtubule organising centres comprising a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, and we previously showed that two key PCM components, Centrosomin (Cnn) and Spd-2, cooperate to form a scaffold structure around the centrioles that recruits the mitotic PCM in Drosophila; the SPD-5 and SPD-2 proteins appear to play a similar function in C. elegans. In fly syncytial embryos, Cnn and Spd-2 are initially recruited into a central region of the PCM and then flux outwards. This centrosomal flux is potentially important, but it has so far not been reported in any other cell type. This study examine the dynamic behaviour of Cnn and Spd-2 in Drosophila larval brain cells. Spd-2 fluxes outwards from the centrioles in both brains and embryos in a microtubule-independent manner. In contrast, although Cnn is initially incorporated into the region of the PCM occupied by Spd-2 in both brains and embryos, Cnn fluxes outwards along microtubules in embryos, but not in brain cells, where it remains concentrated around the centrosomal Spd-2. Thus, the microtubule-independent centrosomal-flux of Spd-2 occurs in multiple fly cell types, while the microtubule-dependent outward flux of Cnn appears to be restricted to the syncytial embryo.

Eisman, R. C., Phelps, M. A. and Kaufman, T. (2015). An amino-terminal Polo kinase interaction motif acts in the regulation of centrosome formation and reveals a novel function for centrosomin (cnn) in Drosophila. Genetics 201: 685-706. PubMed ID: 26447129
Summary:
The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. A Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis.

Eisman, R. C., Phelps, M. A. and Kaufman, T. C. (2016). The End of a Monolith: Deconstructing the Cnn-Polo interaction. Fly (Austin): [Epub ahead of print]. PubMed ID: 27096551
Summary:
In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. A recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at two residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the two phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic.
Chen, J. V., Buchwalter, R. A., Kao, L. R. and Megraw, T. L. (2017). A splice variant of Centrosomin converts mitochondria to microtubule-organizing centers. Curr Biol 27(13): 1928-1940 e1926. PubMed ID: 28669756
Summary:
Non-centrosomal microtubule organizing centers (MTOCs) direct microtubule (MT) organization to exert diverse cell-type-specific functions. In Drosophila spermatids, the giant mitochondria provide structural platforms for MT reorganization to support elongation of the extremely long sperm. However, the molecular basis for this mitochondrial MTOC and other non-centrosomal MTOCs has not been discerned. This study reports that Drosophila centrosomin (cnn) expresses two major protein variants: the centrosomal form (CnnC) and a non-centrosomal form in testes (CnnT). CnnC is established as essential for functional centrosomes, the major MTOCs in animal cells. This study shows that CnnT is expressed exclusively in testes by alternative splicing and localizes to giant mitochondria in spermatids. In cell culture, CnnT targets to the mitochondrial surface, recruits the MT nucleator γ-tubulin ring complex (gamma-TuRC), and is sufficient to convert mitochondria to MTOCs independent of core pericentriolar proteins that regulate MT assembly at centrosomes. Two separate domains in CnnT were mapped: one that is necessary and sufficient to target it to mitochondria and another that is necessary and sufficient to recruit gamma-TuRCs and nucleate MTs. In elongating spermatids, CnnT forms speckles on the giant mitochondria that are required to recruit gamma-TuRCs to organize MTs and support spermiogenesis. This molecular characterization of the mitochondrial MTOC defines a minimal molecular requirement for MTOC generation and implicates the potent role of Cnn (or its related) proteins in the direct regulation of MT assembly and organization of non-centrosomal MTOCs.
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).


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).


centrosomin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 24 September 2006

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