chromosome bows/orbit/mast

Yeast homologs of Orbit/Mast

A cold-sensitive allele of TUB2 has been isolated, the sole gene encoding beta-tubulin in S. cerevisiae; the mutation confers a specific defect in spindle microtubule function. At 14 degrees C, tub2-406 cells lack a normal bipolar spindle but do assemble functional cytoplasmic microtubules. In an attempt to identify proteins that are important for spindle assembly, a screen was carried out for suppressors of the cold-sensitivity of tub2-406 and four alleles were obtained of a novel gene, STU1. Genetic interactions between stu1 alleles and alleles of TUB1 and TUB2 suggest that Stu1p specifically interacts with microtubules. STU1 is essential for growth; disruption of STU1 causes defects in spindle assembly that are similar to those produced by the tub2-406 mutation. The nucleotide sequence of the STU1 gene predicts a protein product of 174 kD with no significant similarity to known proteins. An epitope-tagged Stulp colocalizes with microtubules in the mitotic spindle of yeast. These results demonstrate that Stulp is an essential component of the yeast mitotic spindle (Pasqualone, 1994).

A mutant allele of the DAM1 gene has been identified in a screen for mutations that are lethal in combination with the mps1-1 mutation. MPS1 encodes an essential protein kinase that is required for duplication of the spindle pole body and for the spindle assembly checkpoint. Mutations in six different genes were found to be lethal in combination with mps1-1, of which only DAM1 was novel. The remaining genes encode a checkpoint protein, Bub1p, and four chaperone proteins, Sti1p, Hsc82p, Cdc37p, and Ydj1p. DAM1 is an essential gene that encodes a protein recently described as a member of a microtubule binding complex. Cells harboring the dam1-1 mutation fail to maintain spindle integrity during anaphase at the restrictive temperature. Consistent with this phenotype, DAM1 displays genetic interactions with STU1, CIN8, and KAR3, genes encoding proteins involved in spindle function. A Dam1p-Myc fusion protein expressed at endogenous levels and localized by immunofluorescence microscopy, appears to be evenly distributed along short mitotic spindles but is found at the spindle poles at later times in mitosis (Jones, 1999).

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Evidence is provided that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p is a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and the nature of this interaction has been examined. Stu1p interacts specifically with beta-tubulin and the domains required for this interaction on both Stu1p and beta-tubulin have been identified. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles (Yin, 2002).

Stu1p performs a role that is essential for mitotic spindle function. It is necessary for complete spindle pole body (SPB) separation during assembly of the bipolar spindle. After the spindle is assembled, its activity is also necessary to prevent spindle collapse. Therefore, it is reasonable to conclude that Stu1p aids in producing a pole-separating force (Yin, 2002).

The phenotype of stu1-5 resembles that of cin8 and kip1 mutations. Cin8p and Kip1p are BimC kinesin-related proteins that localize to the yeast spindle. Like other BimC family members, Cin8p and Kip1p are believed to cross-link overlapping polar microtubules and slide them past one another to generate an outward force on the spindle. In the absence of Cin8p and Kip1p, SPBs fail to separate and the SPBs of preformed spindles collapse back to the side-by-side configuration. The facts that Stu1p and Cin8p/Kip1p are both required for spindle pole separation and that a single extra copy of CIN8 can suppress a stu1 mutation suggests that these proteins have overlapping roles in the cell. This view is also supported by the observation that cells containing the stu1-5 mutation and a deletion of cin8 are inviable. Although the sequence of Stu1p does not indicate that it is a motor protein, its ability to bind microtubules and its localization to the midregion of anaphase spindles are consistent with Stu1p playing a structural role in the spindle through its interaction with antiparallel interpolar microtubules. Stu1p self-association may produce homodimers that are capable of cross-linking these antiparallel microtubules. Stu1p is the first example of a nonmotor microtubule-binding protein that is necessary for bipolar spindle formation in yeast (Yin, 2002).

A phenotypic comparison can also be made between stu1-5 yeast and mast/orbit Drosophila mutants (Inoue, 2000; Lemos, 2000). The mast/orbit mutations lead to high levels of cells with polyploid chromosomes in the larval CNS. Such chromosomes are frequently associated with multipolar spindles. In addition, these mutations cause the appearance of circular mitotic figures in which the major chromosomes are arranged in a circle with their centromeres inward and arms oriented toward the periphery. In general, these cells contain a reduced number of microtubule-organizing centers relative to their chromosome content, but their microtubule-organizing centers frequently contain multiple centrosomes. These results suggest that mast/orbit cells cannot separate centrosomes properly or cannot maintain centrosome separation once it has occurred. This model is supported by the observation that the mast/orbit phenotype closely resembles the phenotype caused by mutations in KLP61F, a Drosophila kinesin-like protein in the BimC family, which is necessary to maintain spindle pole separation. Thus, it appears likely that Stu1p and Mast/Orbit play similar roles in spindle assembly and maintenance in yeast and Drosophila, respectively (Yin, 2002).

Stu1p binds to microtubules in vitro and the domains on both Stu1p and ß-tubulin necessary for this interaction have been identified. Stu1p binds to microtubules with an apparent Kd of 0.32 µM, a value that is approximately two times greater than that obtained for the neuronal microtubule-associated protein tau by use of the same assay. In vitro binding assays using truncations of Stu1p have localized the microtubule-binding region to a 256-amino-acid sequence in the amino-terminal half of the protein. This region is highly basic and contains a 103-amino-acid serine-rich stretch. Two of the Stu1p homologs, the Drosophila Orbit/Mast and the human CLASP proteins, have also been shown to bind microtubules (Inoue, 2000; Lemos, 2000; Akhmanova, 2001). Although the specific regions of these proteins that mediate their interactions with microtubules have not been defined, they do contain a sequence that is similar to the microtubule-binding domain of the microtubule-associated protein MAP4. This sequence is located just inside the amino-terminal half of these proteins, as is the microtubule-binding region of Stu1p (Yin, 2002).

Stu1p binds to ß-tubulin, but not alpha-tubulin, in the two-hybrid assay. To map the Stu1p-binding site on ß-tubulin, an approach was used that is similar to one previously used for mapping binding sites of other proteins on alpha-tubulin and actin. Use was made of a set of clustered charge-to-alanine mutations in ß-tubulin and the three-dimensional structure of yeast ß-tubulin was constructed by modeling its sequence onto the mammalian ß-tubulin structure. It was reasoned that the mutations in ß-tubulin that disrupt the interaction with Stu1p in the two-hybrid assay would identify side chains that make up the interacting surface. Four tub2 alleles, comprising eight amino acid substitutions, specifically disrupt Stu1p binding. These residues form a patch on the surface of ß-tubulin that is exposed to the cytoplasm when tubulin is assembled into a microtubule and most likely define the domain on tubulin that mediates the interaction between Stu1p and microtubules. Two of the tub2 alleles (tub2-455 and tub2-456) that disrupt the interaction with Stu1p are recessive lethal mutations in yeast. Their lethality could result from their inability to interact productively with Stu1p (Yin, 2002).

STU1 was initially identified by allele-specific suppression of a cold-sensitive tub2 mutation. tub2 mutations can also suppress the temperature sensitivity of stu1-5. Such mutual suppression has been taken as strong evidence of a direct in vivo interaction. This type of suppression is likely to occur in that an alteration in one protein causes a decrease in binding affinity, which is restored by a compensating alteration in the second protein. tub2 suppressor mutations change residues predicted to lie in internal regions of ß-tubulin or on the inside of the microtubule, which is inconsistent with their direct participation in the Stu1p-binding interface. Thus, if these mutations increase the affinity of ß-tubulin for Stu1-5p, they must do so by longer-range actions, such as altering the conformation of ß-tubulin in a way that exposes novel residues or changes the orientation of existing surface residues. Such conformational changes have been proposed to account for the suppression of actin alleles by mutations in Sac6p. A second mechanism by which suppression could occur is that the tub2 suppressors change the properties of microtubules so that they do not require the wild-type level of Stu1p activity. For example, an alteration in ß-tubulin may increase its affinity for another protein whose activity overlaps with that of Stu1p (Yin, 2002).

Mammalian homologs of Orbit/Mast

CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Two CLIP-associated proteins (CLASPs) have been isolated that are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. Evidence suggests that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. It is proposed that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues (Akhamanova, 2000).

The role of plus end-tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removes endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150Glued. To distinguish between these possibilities, rescue experiments were performed. Normal MT dynamics are restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued head domain only partially restored MT dynamics. It is concluded that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin (Komarova, 2002).

One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto plus ends of microtubules that are actively gaining or losing tubulin subunits. CLASP1, a microtubule-associated protein, localizes preferentially near the plus ends of growing spindle microtubules and is also a component of a kinetochore region that has been termed the outer corona. A truncated form of CLASP1 lacking the kinetochore binding domain behaves as a dominant negative, leading to the formation of radial arrays of microtubule bundles that are highly resistant to depolymerization. Microinjection of CLASP1-specific antibodies suppresses microtubule dynamics at kinetochores and throughout the spindle, resulting in the formation of monopolar asters with chromosomes buried in the interior. Incubation with microtubule-stabilizing drugs rescues the kinetochore association with microtubule plus ends at the periphery of the asters. The data suggest that CLASP1 is required at kinetochores for attached microtubules to exhibit normal dynamic behavior (Maiato, 2003a).

Abelson phosphorylation of CLASP2 modulates its association with microtubules and actin

The Abelson (Abl) non-receptor tyrosine kinase regulates the cytoskeleton during multiple stages of neural development, from neurulation, to the articulation of axons and dendrites, to synapse formation and maintenance. It was previously shown that Abl is genetically linked to the microtubule (MT) plus end tracking protein (+TIP) Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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