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© The Rockefeller University Press,
0021-9525/2000//5 $5.00
The Journal of Cell Biology, Volume 149, Number 1,
, 2000 5-6
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Microtubules in orbit
cwalczak{at}indiana.edu
© 2000 The Rockefeller University Press
The faithful segregation of DNA during mitosis is critical to the life of a cell. A complex molecular machine called the mitotic spindle mediates this task. The spindle is made up of microtubule-binding proteins and microtubules on which the chromosomes are segregated. A complete understanding of the formation of the bipolar spindle and the mechanism of chromosome movement on the spindle remains elusive. Many key players in this process have been identified, and we are beginning to understand the various classes of proteins and their functions. However, not surprisingly, new players with potentially novel modes of action continue to be discovered. In this issue, Inoue and colleagues describe a novel microtubule-associated protein encoded by the Drosophila gene orbit that influences the structure of the spindle and binds microtubules in a GTP-dependent manner (Inoue et al. 2000).
Microtubules are dynamic polymers made up of 
,β-tubulin heterodimers. Microtubules exhibit a phenomenon called dynamic instability, wherein they coexist in states of growth and shrinkage (reviewed in Desai and Mitchison 1997). Many different proteins have been discovered that bind to microtubules (reviewed in Kreis and Vale 1999), including microtubule stabilizers and destabilizers, microtubule severing proteins, and motor proteins. It is the balanced interplay between these microtubule-binding proteins that enables the dramatic, coordinated alterations of microtubules necessary for spindle formation and chromosome movements during mitosis. One specialized group of binding proteins called microtubule-associated proteins (MAPs) enhances the stability of microtubules (reviewed in Cassimeris 1999). Although MAPs were initially characterized in neurons, several non-neuronal MAPs important in regulating cellular microtubule dynamics have been identified. These MAPs appear to be highly regulated during interphase and mitosis; upon inactivation a MAP is no longer able to bind to microtubules, and its stabilizing activity is reduced.
Because of the importance of microtubule stability in the spindle, it is not surprising that multiple MAPs exist that are essential during mitosis. Inoue and colleagues report the discovery of the Drosophila gene orbit, which encodes a novel MAP (Inoue et al. 2000). The orbit mutation was isolated in a screen for maternal effect lethal genes. Characterization of spindle and DNA morphology in the syncytial embryo revealed free centrosomes, multipolar spindles, and curved or bent spindles (Fig. 1 a), phenotypes that are consistent with defects in chromosome segregation. Somatic cell defects were also observed in the larval central nervous system (CNS), including circular mitotic figures (CMFs), polyploidy and hypercondensed chromosomes, as well as a small number of monopolar spindles with chromosomes in an anaphase-like configuration (Fig. 1 b). In addition, they observed that orbit mutants were delayed in progression through the cell cycle, which is consistent with the hypercondensed chromosomes that were observed.
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The preliminary model put forth by Inoue and colleagues suggests a primary role for Orbit in regulating the function of spindle microtubules. The high percentage of polyploid cells and the defects in spindle structure are consistent with the idea that Orbit provides stability to spindle microtubules that is lost in the mutants. The hypercondensed chromosomes in larval CNS cells suggest that orbit mutants remain blocked in metaphase, which is not surprising if spindle stability is compromised.
It may also be possible that Orbit has a less direct role in centrosome separation during mitosis. Several orbit mutant phenotypes resemble those found in other Drosophila mutants that block spindle pole separation, such as aurora and merry-go-round (Gonzalez et al. 1988; Glover et al. 1995). In addition, the bent spindles and monopolar spindles in orbit mutants are reminiscent of those seen in abnormal spindle (asp) mutants (Gonzalez et al. 1990); the Asp protein has recently been shown to be important for proper spindle pole structure (do Carmo Avides and Glover 1999). Unlike other spindle pole mutants, however, orbit mutants can occasionally form a normal bipolar spindle, suggesting that a defect in centrosome separation is not the primary defect in these mutants. One can easily envision how a decrease in microtubule stability could ultimately lead to spindle collapse, resulting in the appearance of a monopolar spindle. It will be interesting to employ time-lapse microscopy of early embryos as they proceed through the early divisions to determine how the spindles in orbit mutants are assembled and disassembled.
Future experiments aimed at elucidating the mechanism of action of this interesting MAP are numerous. A biochemical analysis of Orbit regulation of microtubule dynamics and an analysis of its GTPase activity will be essential in providing the groundwork for understanding how it functions in the spindle. In addition, generation of transgenes that express constitutively active forms of Orbit may provide insight into how nucleotide hydrolysis functions to regulate spindle dynamics in cells. Finally, the identification of homologues in C. elegans (Inoue et al. 2000) will allow a comparison of phenotypes by using RNA-mediated interference to knockout the C. elegans protein. The identification of the human homologue (Inoue et al. 2000) will enable the use of cell lines to study the regulation of microtubule dynamics in cells using high-resolution imaging.
Submitted: 7 March 2000
Accepted: 14 March 2000
Abbreviations used in this paper: asp, abnormal spindle; CMFs, circular mitotic figures; CNS, central nervous system; MAPs, microtubule-associated proteins.
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