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 α,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.
The 165-kD product of the orbit gene was isolated and contains a highly basic central
region, characteristic for microtubule-binding proteins (Fig. 2). This region contains
two consensus sites for phosphorylation by p34cdc2 that may be important for regulating
the association of Orbit with microtubules. Sequence analysis of the putative microtubule-binding
domain revealed that it is similar to that of MAP4, a nonneuronal mammalian MAP important
in stabilizing interphase microtubules, and to Stu1, a MAP found in budding yeast.
Consistent with these findings, Orbit was found to cosediment with microtubules in
embryo extracts. In addition, immunolocalization of Orbit showed that it colocalized
with spindle microtubules suggesting a role in stabilizing microtubules during mitosis.
Perhaps the most interesting finding reported by Inoue and colleagues is that Orbit
appears to bind microtubules in a GTP-dependent manner. Sequence analysis indicates
the presence of two putative GTP-binding motifs within the highly basic central region
of Orbit. These motifs closely resemble those found in β-tubulin and other members
of the GTPase superfamily (Downing and Nogales 1998; Nogales et al. 1998). Orbit was
shown to bind to microtubules in a GTP-dependent manner in blot overlay assays. Consistent
with this observation, microtubule pelleting assays indicate that Orbit must bind
GTP to co-sediment with microtubules. MAPs are known to bind and release microtubules;
however, this is usually dependent on salt concentration and is often regulated by
phosphorylation. If Orbit can bind and release from microtubules in a nucleotide-sensitive
manner, this would be a unique MAP function, suggesting the possibility of a nucleotide-regulated
association with microtubules. Other microtubule-binding proteins that also bind nucleotide
include a vast array of motor proteins important in spindle organization (Goldstein
and Philp 1999). The recent identification of kinesin-related proteins that destabilize
microtubules or that act as signaling molecules suggest that motor proteins can have
more diverse functions other than microtubule translocation (reviewed in Goldstein
and Philp 1999). Perhaps Orbit is the first example of a more dynamic MAP that functions
in the spindle. If Orbit uses its nucleotide-binding domain to regulate its association
with microtubules, then it will be interesting to compare its activity to conventional
motor proteins.
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.