During every cell generation, chromosomes are replicated
and sister chromosomes are segregated to each of the daughter cells.
The segregation of chromosomes involves a complex cellular machine
called the mitotic spindle. The major components of the mitotic spindle
are molecular fibers that connect chromosomes to the spindle poles
and the poles to each other. These fibers are called microtubules
and are composed of two proteins, alpha and beta-tubulin. In addition,
a large number of other proteins are involved in spindle function.
Although many of these proteins are as yet uncharacterized, they are
thought to include microtubule motor proteins that mediate movements
of chromosomes, regulatory proteins that influence microtubule assembly,
and structural proteins that are required for spindle assembly and
function. These proteins work together to form an extremely efficient
machine; in yeast, a chromosome is missegregated only once in every
100,000 cell divisions.
The aim of our lab is to identify proteins involved in mitotic spindle
function in the yeast Saccharomyces cerevisiae and to study
the function of these proteins at the molecular level. With its sophisticated
molecular genetics, increasingly powerful cell biology tools, and
relatively simple mitotic spindle, yeast is a particularly tractable
organism for these studies.
Our research is focused on several microtubule-binding proteins that
are necessary for proper chromosome segregation. These proteins, which
were identified in a variety of genetic screens, bind to microtubules in
vitro and colocalize with microtubules in vivo. We have
made mutations in the genes encoding these proteins and are examining
the effects of these mutations in vivo. Our phenotypic analysis
of mutants relies on fluorescence microscopy which we use to look
for defects in chromosome segregation, spindle orientation, spindle
elongation, and microtubule dynamics. By using a variety of GFP constructs,
we are able to examine these processes in real time in living cells.
For example, recent work has shown that loss of the microtubule-binding
protein Stu2p dampens microtubule dynamics, interferes with spindle
orientation, and blocks spindle elongation and chromosome segregation.
This work demonstrates that Stu2p plays a role in the regulation of
microtubule dynamics and that this regulation is essential for spindle
In a collaboration with Tony Bretscher's lab, we
are examining the connection between microtubules and actin filaments
in yeast. Both cytoplasmic microtubules and actin cables are known
to play roles in orienting the spindle in yeast. We have shown that
this process requires a type V myosin of yeast, Myo2p, and established
a molecular linkage between actin cables and cytoplasmic microtubules.
Our results suggest that Myo2p, in a complex with one or more microtubule-binding
proteins, moves along actin cables and pulls the cytoplasmic microtubules
in the bud.