chromosome bows/orbit/mast: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - chromosome bows

Synonyms - Orbit, Mast

Cytological map position - 78C1--2

Function - microtubule-associated protein

Keywords - microtubule organizing center, kinetochore-microtubule interaction, oogenesis, mitosis, axon guidance

Symbol - chb

FlyBase ID: FBgn0021760

Genetic map position - 3-46.6

Classification - microtubule-associated plus end tracking protein, Non-SMC condensin subunit, Mast C-terminus

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene | HomoloGene
Recent literature
Trogden, K. P. and Rogers, S. L. (2015). TOG proteins are spatially regulated by Rac-GSK3β to control interphase microtubule dynamics. PLoS One 10: e0138966. PubMed ID: 26406596
Summary:
Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs) where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and their basic contributions to microtubule dynamics are understood, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. This study have identified a novel protein-protein interaction between the XMAP215 homologue in Drosophila, Mini spindles (Msps), and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. Microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3β in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on these findings, it is proposed that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics.
Moriwaki, T. and Goshima, G. (2016). Five factors can reconstitute all three phases of microtubule polymerization dynamics. J Cell Biol 215: 357-368. PubMed ID: 27799364
Summary:
Cytoplasmic microtubules (MTs) undergo growth, shrinkage, and pausing. However, how MT polymerization cycles are produced and spatiotemporally regulated at a molecular level is unclear, as the entire cycle has not been recapitulated in vitro with defined components. In this study, dynamic MT plus end behavior involving all three phases was reconstituted by mixing tubulin with five Drosophila melanogaster proteins (EB1, XMAP215Msps, Sentin, kinesin-13Klp10A, and CLASPMast/Orbit). When singly mixed with tubulin, CLASPMast/Orbit strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASPMast/Orbit acted as an inducer of pausing. The mitotic kinase Plk1Polo modulated the activity of CLASPMast/Orbit and kinesin-13Klp10A and increased the dynamic instability of MTs, reminiscent of mitotic cells. These results suggest that five conserved proteins constitute the core factors for creating dynamic MTs in cells and that Plk1-dependent phosphorylation is a crucial event for switching from the interphase to mitotic mode.
BIOLOGICAL OVERVIEW

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to chromosomal kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Studies on the function of Orbit/MAST (FlyBase designation: Chromosome bows or Chb for short) in Drosophila and its human homolog CLASP1 (Maiato, 2003a: CLASP stand for CLIP-associated protein), have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizes to the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. CLASP1 is therefore required at kinetochores to regulate the dynamic behavior of attached microtubules (reviewed by Maiato, 2003b).

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, the microtubule-associated protein Orbit/MAST has been identified as a partner of the Abelson (Abl) tyrosine kinase. Identical axon guidance phenotypes are found in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors (Lee, 2004).

In order to establish an intricate yet specific network of neuronal connections, axons are guided from intermediate to final targets by an assortment of attractive and repellent factors. The navigational response to such guidance cues depends on a complex and dynamic cytoskeletal machine within the growth cone that is linked via signaling pathways to receptors at the cell surface. In essence, the growth cone acts as an exquisitely sensitive molecular compass, translating the spatial asymmetry of extracellular cues into polarization of the cytoskeletal elements that determine the directional specificity of cell movement. Two major targets in this cell polarity machine are the microfilament networks that support the growth cone perimeter and the microtubule arrays that build the core of the nascent axon. While a comprehensive understanding of cytoskeletal signaling is still elusive, much progress has been made in identifying pathways and effector molecules that control the rapid and initial response of actin assembly to particular guidance factors. However, very little progress has been made in identifying microtubule-associated proteins (MAPs) that participate in specific pathways (Lee, 2004 and references therein).

The actin networks that propel membrane protrusion are thought to mount the initial response to guidance information. Actin remodeling in the growth cone peripheral (P) domain remains dynamic and unstable, allowing for rapid changes in direction or cell contact. Microtubules are also dynamic in the periphery, and their recruitment and subsequent bundling to establish the central (C) domain of the growth cone represent a hallmark of directional growth. Consequently, as growth cones make guidance decisions in situ and select particular filopodia to define the new direction of movement, it is the consolidation of microtubule structures and concomitant dilation of filopodia that are most predictive of a change in direction. Indeed, perturbation of microtubule dynamics has a dramatic effect on growth cone navigational behavior (Lee, 2004 and references therein).

In the transition zone between the P and C domains of the growth cone, a specialized class of microtubules extend and penetrate into the actin-rich perimeter. Recent studies in growth cones and nonneuronal cells show that such 'pioneer' microtubules enjoy an intimate relationship with specialized actin structures that support individual filopodia and define the region between P and C domains, supporting theoretical models that predict crosstalk between the two polymer networks. Consistent with this idea, pharmacological studies show that growth cone microtubule structures and guidance are dependent on actin dynamics. Interestingly, nonneuronal studies suggest that a reciprocal signaling relationship exists at this cytoskeletal interface and that molecules such as Rho family GTPases act to coordinate the dynamics of both actin and microtubules. Signaling molecules or 'nodes' that coordinate multiple downstream events are common in signal transduction pathways. While Rho family GTPases are well known for this capacity, additional classes of proteins are also likely to serve this function during axon guidance (Lee, 2004 and references therein).

One excellent candidate as an axon guidance signaling node in Drosophila is the Abelson (Abl) protein tyrosine kinase. Abl is required for the accurate guidance of both central and peripheral axon pathways and modulates the function of several axonal receptors. Study of these signaling pathways shows that Abl interacts genetically with a number of intracellular effector proteins, including Enabled, Profilin, Trio, and the cyclase-associated protein (Lee, 2004 and references therein).

So far, the majority of known Abl interactors control aspects of actin assembly. However, a new Abl interactor known to associate with microtubules has been discovered, suggesting a link that might coordinate actin and microtubule dynamics. Genetic analysis has identified a number of MAPs that are necessary for axonogenesis, including Futsch/MAP1b, Short stop, and Pod-1. Although some of these effector proteins are targets for intracellular kinases, none have been shown to act downstream of specific axon guidance factors. While the Semaphorin effector Collapsin response mediator protein (CRMP) has been shown to bind Tubulin heterodimers and influence polymer assembly, the primary role of the CRMP family is thought to involve membrane dynamics (Lee, 2004 and references therein).

A kinase-dependent gain-of-function (GOF) phenotype for Abl in the Drosophila retina has been described that displays sensitive genetic interactions with receptors in the Roundabout (Robo) family (Wills, 2002). Loss-of-function (LOF) studies confirm that Abl plays a complex role in axon guidance at the midline, where Robo receptors mediate the repellent action of Slit. Moreover, there is an endogenous role for Abl in the retina, suggesting that this neural tissue can be used as a tool to identify additional classes of effectors in the Abl pathway. One of the genes derived from an ongoing retinal screen for modifiers of AblGOF is described in this study, the MAP Orbit/MAST (also known as Chromosome Bows [Chb]; Fedorova, 1997; Inoue, 2000 and Lemos, 2000), ortholog of the vertebrate cytoplasmic linker protein (CLIP)-associated proteins (CLASPs; Akhmanova, 2001). Using zygotic null alleles to escape a requirement during oogenesis, Orbit/MAST was found to be necessary for accurate axon guidance at the midline choice point. Phenotypic characterization, genetic interactions, and genetic epistasis experiments suggest that this MAP acts downstream of Abl in the Slit repellent pathway, consistent with its localization to axons and growth cones. Parallel imaging studies in Xenopus growth cones show that vertebrate CLASP identifies a subset of axonal microtubules that extend into the peripheral domain, where actin dynamics are known to influence microtubule behavior. Elevation of CLASP activity in Xenopus neurons has been shown to reduce not only microtubule advance but also growth cone translocation (Lee, 2004).

Genetic analysis reveals a postmitotic requirement for CLASP during the guidance of axons in multiple contexts. At the midline, CLASP is found to be necessary for accurate growth cone orientation away from the source of Slit and for lateral positioning of longitudinal axon fascicles, suggesting a model in which CLASP acts positively downstream of Abl as part of the repellent response initiated by activation of Roundabout receptors. Genetic interaction and epistasis experiments support this model. Moreover, protein localization studies in Drosophila and Xenopus growth cones indicate that this role for CLASP is likely to occur near the leading edge, where guidance cues exert their initial influence on cytoskeletal remodeling. In contrast to CLIP-170, CLASP is enriched at microtubule plus ends within the growth cone itself, suggesting a specialized role in neuronal cell biology. Indeed, CLASP-positive plus ends penetrate the growth cone peripheral domain and track along microfilament bundles into individual filopodia proximal to the site of guidance receptor activation. Taken together, the genetics and cell biology suggest a model in which Slit activation of the Abl kinase leads to a CLASP-dependent inhibition of microtubule extension favoring growth cone advance toward regions of low signaling activity (Lee, 2004).

Growth cone repellents play a major role in patterning neuronal connectivity and restricting regenerative capacity within the CNS, making repellent signaling a high priority for functional dissection. One theme to emerge from many studies of growth cone guidance is that the cell biology of directional navigation requires a coordination of many different subcellular events. This predicts the existence of signaling proteins that can regulate the combined activities of multiple effectors. Receptor-proximal factors fitting this profile have been found in several repellent pathways. For example, the guanine-nucleotide exchange factor Ephexin mediates EphA4-dependent repulsion by activating RhoA and simultaneously inhibiting Rac and Cdc42. The adaptor protein Grb4 displays coordinate interactions with a different cast of players downstream of Ephrin-B, including the kinase Pak1, the Cbl-associated protein (CAP/Ponsin), and the Abl-associated protein-1 (Abi-1), highlighting the diversity of potential effectors (Lee, 2004).

Through genetic analysis in Drosophila, the Abl tyrosine kinase has emerged as another key signaling center that is capable of coordinating multiple outputs (reviewed by Moresco, 2003). Abl is both necessary and sufficient to define axon guidance behavior (e.g., Wills, 1999a; Wills, 1999b; Wills, 2002; Bashaw, 2000 and Hsouna, 2003), suggesting that it acts high in the signaling hierarchy. At the CNS midline, Abl interacts with Enabled and the cyclase-associated protein (Capulet) to control growth cone behavior (Bashaw, 2000 and Wills, 2002). These Abl effectors are actin binding proteins with different types of activity in cytoskeletal dynamics (reviewed by Lee, 2003). Abl is also likely to regulate ß-Catenin/Armadillo function, which may be important for the in vivo response to Slit. While many studies on the cell biology of Abl family kinases have focused on actin effectors and Abl's ability to bind directly to actin polymers, recent work reveals that the Abl-related gene (Arg) associates directly with microtubules as well, thus placing it at the interface between the two cytoskeletal arrays Miller, 2004). Genetic screen and analysis of CLASP function in Drosophila adds a new dimension to this picture, showing that Abl controls both actin and MAPs in parallel (Lee, 2004).

CLASP family proteins fall into an intriguing group of microtubule-associated plus end tracking proteins (+TIPs; reviewed by Carvalho, 2003). While little is known about their function in neurons, and their precise mechanism of action is still mysterious, accumulated evidence suggests that +TIPs act to regulate microtubule stability. For example, dominant-negative experiments with CLIP-170 in nonneuronal cells suggest that this +TIP acts to reduce the frequency of rapid microtubule depolymerization (or 'catastrophe'; Komarova, 2002). While the impact of CLASP family function has not been determined using dynamic assays, overexpression of CLASP in COS cells increased the number of stabilized microtubules (Akhmanova, 2001). Preliminary RNA interference to remove CLASP in Drosophila S2 cells indicates that this function has been conserved (A. Ghose, U.E, and D.V.V., unpublished data cited by Lee, 2004). However, if an increase in stable microtubules comes at the expense of dynamic microtubule segments, then a negative impact on the persistence of growth cone advance might be predicted, based on existing pharmacological data. This provides an attractive model to explain how CLASP can cooperate with Slit, Robo, Robo2, and Abl during midline repulsion (Lee, 2004).

A growing number of MAPs have been shown to localize to plus ends (reviewed by Carvalho, 2003), raising the possibility that +TIP protein complexes coordinate multiple activities. In addition to the CLASP localization in this study, the EB1 family member EB3 displays +TIP behavior within the growth cone (Stepanova, 2003). Interestingly, the +TIPs EB1 and APC associate with Short stop/Kakapo/MACF in Drosophila cells. Short stop is known to bind both actin and microtubule polymers, suggesting a role in mediating interactions between the two polymer networks. Moreover, short stop mutants display an ISNb motor axon phenotype that is nearly identical to orbit/MAST and Abl loss of function. Other MAPs have also been implicated in mediating interaction between microfilaments and microtubules in developing axons, suggesting that this interface will be complex and highly coordinated. Future experiments will address whether other +TIP-associated proteins also contribute to repellent signaling and whether all the +TIP proteins display similar activities in vivo (Lee, 2004).

With a combination of in vivo genetic analysis and dynamic imaging, a working model can be proposed for the role of CLASP in growth cone repellent signaling. Recent work indicates that Abl functions positively to support Slit/Robo-mediated axon repulsion at the midline (Wills, 2002 and Hsouna, 2003). This suggests a model in which the polarity of cytoskeletal advance within the growth cone reflects an asymmetry of Abl kinase activity in response to a graded distribution of Slit. Since multiple lines of genetic evidence show that CLASP cooperates with Abl and acts genetically downstream of the kinase, a model is favored where CLASP helps to induce cytoskeletal events that are needed to impede leading edge advance nearest the source of Slit, thus allowing relative advance at sites most distant from the source. This model predicts that elevation of CLASP activity will have a negative impact on growth cone extension and on microtubule advance. Xenopus overexpression experiments satisfy this prediction (Lee, 2004).

Since the entire growth cone perimeter must remain competent to respond to asymmetrically localized repellents in order to navigate through complex terrain, it is anticipated that CLASP activity rather than its localization will change in response to local kinase activation. Studies of the actin regulatory protein mammalian Enabled (Mena), which localizes to the tips of filopodia, suggest an analogous mechanism downstream of cyclic nucleotide-gated kinases to control filopodium formation. Abl regulates both CLASP and Enabled during axon guidance in Drosophila (Wills, 1999a and Bashaw, 2000), suggesting that independent cytoskeletal effectors must be coordinated to achieve accurate navigational choices. In this regard, it will be interesting to ask how CLASP interacts with key factors like the GTPase Rac1, which appears to be important in midline guidance and interfaces with CLIP-170 in nonneuronal cells. While many assume that the immediate effectors in axon guidance are actin regulators, data from nonneuronal systems suggest that microtubule dynamics can control actin assembly from the inside out (Lee, 2004 and references therein).


GENE STRUCTURE

cDNA clone length - 5959 base pairs

Bases in 5' UTR - 769

Exons - 2

Bases in 3' UTR - 668

PROTEIN STRUCTURE

Amino Acids - 1402

Structural Domains

The sequence of orbit cDNAs has revealed that the gene encodes a protein of 1,492 amino-acids. The first ATG consistent with Drosophila translation initiation consensus is found at position 769 of the 5,959 nucleotide cDNA sequence. A poly(A) addition signal AATAAA lies at position 5,892. The novel protein contains a centrally located highly basic region (pI = 11.0) of 472 amino acids, flanked on both sides by short stretches of acidic residues. Within the basic domain are two consensus sites for phosphorylation by P34cdc2, and two putative GTP-binding motifs. The motif GGGTGTG (residues 544-550) closely resembles the glycine-rich peptide that interacts with the guanine or phosphate groups of the bound GTP in ß-tubulin and in the Escherichia coli FtsZ protein. The sequence NKLD (residues 400-403) corresponds to the NKXD (X for any amino acid residues) consensus motif that can interact with the purine base of the bound nucleotide in the GTPase superfamily. A BLAST search with the Orbit protein sequence has revealed the presence of four closely related proteins from other organisms: two identified by the human putative open reading frame, KIAA0622 and KIAA0627, and two, R107.6 and ZC84.3, predicted from the C. elegans genome sequencing project. The homologies fall into two regions: HR1 lying between residues 290 and 1,068, and HR2 between zresidues 1,093 and 1,271. Since the regions of homology between the five proteins lie in register, it is likely that they are the signature of a family of related proteins. Moreover, the basic domain contained in the HR1 is a common feature of all five proteins, and the consensus sequences for cdc2 phosphorylation are found within or in the vicinity of this basic region in all except ZC84.3. The NKXD motifs are also conserved in the two human homologs. Basic domains are a characteristic of microtubule-binding proteins and in this context, it is of interest that one of the conserved motifs of HR1 (residues 326-350) shows considerable similarity to the sequence involved in the binding of human MAP4 to microtubules. Furthermore, another conserved sequence within the basic domain (residues 479-506) has similarity to a motif in Stu1, a MAP identified from budding yeast (Inoue, 2000).

Mast shares significant identity with proteins encoded by two human cDNAs (KIAA0622 and KIAA0627), three putative proteins in C. elegans (C07H6.3, R107.6 and ZC84.3) and also limited identity with Stu1p from S. cerevisiae (Pasqualone, 1994) and its putative homolog in S. pombe, termed SpStu1p. Multiple alignment of the Mast sequence with those most closely related from other species shows that all the proteins share identity throughout their sequence; however, three regions (CR-1, CR-2 and CR-3) are more highly conserved. These results suggest that Mast and its homologs define a new evolutionarily conserved protein family that has been named Stu1-Mast. Database searches also indicate that Mast shares identity with proteins from the dis1-TOG family, especially at the N-terminal half of the protein (amino acids 1-494), where they are 20%-25% identical and 40%-45% similar. Inside this region, there is a small domain of 18 amino acid residues that is highly conserved among these proteins and falls inside the first HEAT repeat of Mast. Phylogenetic analysis including all sequences from the two groups suggests that they are evolutionarily close, but distinct, since they are positioned in different branches of the dendrogram (Lemos, 2000).


chromosome bows/orbit/mast: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 August 2004

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