Molecular mechanisms of mitotic spindle assembly and
microtubule nucleation; cell-cycle dependent regulation of spindle
assembly proteins; centrosome structure and function
Many fundamental cellular processes take advantage of the intrinsic
polarity of microtubules. Processes that use microtubules as molecular
tracks include vesicular trafficking, cell motility, organelle
translocation, and, most prominently, cell division. In animal cells,
microtubules are organized by the centrosome, which aids in microtubule
initiation (termed 'nucleation'), anchoring, and organization.
Centrosome defects are thought to be involved in a variety of human
diseases, including male infertility and cancer. Centrosomes nucleate
radial arrays of microtubules during interphase and mark the poles of
the elaborate macromolecular structure required for the proper
segregation of chromosomes, the mitotic spindle, during mitosis.
Because defects in cell division are central to the progression of
cancer, a molecular understanding of spindle structure and function
could lead to a better understanding of cancer and its potential
therapies.
Our long-term
goal is to elucidate the principles that underlie the assembly and
function of the mitotic spindle and to identify and study the roles of
individual proteins that are involved in this process. We focus our
current efforts on proteins required for microtubule nucleation,
stabilization, and organization. The small Ras-like GTPase, RanGTP,
which is required for nuclear transport during interphase, has recently
been identified as a regulator of spindle assembly in mitotic Xenopus
laevis egg extracts. We discovered that Ran exerts its effects through
importin β, a protein which functions as a nuclear import receptor
during interphase. We showed that during mitosis, importin β functions
as a potent inhibitor of spindle assembly. This inhibition by importin
β is relieved in the presence of RanGTP, which is known to interact
with importin-β and to cause it to release its `cargo'. We are
currently trying to understand the molecular mechanism of the effect of
importin β on spindle assembly factors. Some of our questions are: How
does importin β inhibit spindle assembly? Does its binding to spindle
assembly factors prevent these from performing their functions? Does
its binding prevent phosphorylation (or other secondary modifications)
of the spindle assembly factors? Does it prevent interaction with other
proteins?
In another line of investigation, we are interested in understanding the molecular mechanism of a protein complex involved in the nucleation of microtubules from centrosomes. This protein complex, known as the γ-tubulin ring complex (γTuRC), can nucleate microtubules in vitro. In addition, it also caps the so-called minus ends of microtubules, preventing their growth and shrinkage. We want to understand the biophysical properties of the interaction between the γTuRC and the end of the microtubule. In a more cell biological approach, we want to identify cellular factors that regulate the interaction of the γTuRC with the microtubule, as well as those that regulate the interaction of the γTuRC with the centrosome.
Two models for the interaction between the γTuRC (red, blue, green) and the end of the microtubule (yellow, light blue) have been proposed, differing mainly by the arrangement of γ-tubulin (green) and the other γTuRC subunits (red, blue) with respect to tubulin (yellow, light blue).