Shaping the Mitotic Spindle: Investigating
the Interactions between Dynein and
Augmin
KC Gandy
with Jay Gatlin
Department of
Molecular Biology
PROPOSED RESEARCH PROJECT
Introduction
The normal mitotic spindle is a bipolar structure consisting
of microtubules and two centrosomes. This spindle shape
leads to accurate segregation of chromosomes:
The motor protein dynein is involved in sliding and
crosslinking microtubules1 and is thus central to bipolar
spindle formation. The role of other proteins in the process,
including a complex known as augmin, has not yet been
elucidated. Augmin appears to be involved in the
nucleation of spindle microtubules2.
Methods
1
2
Hypothesis
Augmin interacts with dynein to facilitate the formation of a
bipolar mitotic spindle.
In this procedure, magnetic beads are
suspended in a solution containing primary
antibody (ligand). In our case this antibody
recognizes the intermediate chain of dynein.
The beads are coated with a specific protein
to which the antibody tail binds.
Next the beads are added to Xenopus egg
extract, which contains the target protein.
The protein binds to the antibody on the
beads.
 Collect the eggs
3
 Centrifuge the eggs to
crush them
4
 Remove extract layer
5
The inhibition of centrosome clustering in cancer cells will
result in multipolar divisions and death of resultant progeny.
As such, identifying the responsible molecular mechanisms
should provide novel therapeutic targets for anticancer
drugs.
 Inject Xenopus laevis
(African Clawed frog) with
hormones to induce egg
production
 Rinse eggs with buffers,
including a cytostatic
factor solution (to “freeze”
cell division)
Unresolved, this typically leads to cell death or inviable
offspring, as the cell divides into three or more cells with
unequally distributed chromosomes.
Statement of the Problem
Co-Immunoprecipitation
Frog Egg Extracts
In some instances, a multipolar spindle with more than two
centrosomes is formed3. Experiments have shown that
perturbation of augmin also results in the formation of
abnormal spindles4.
However, some cancer cells resolve this issue by clustering
the centrosomes into two poles3, allowing the cell to divide
into just two cells with a more equal distribution of genetic
material. These cells may then be able to survive and
proliferate:
Conclusions
The beads, along with the attached
antibodies and antigens, are pulled to the
side of the tube. The rest of the solution is
pipetted off. The isolated protein can then be
eluted for use in experiments.
For this experiment, the goal is to isolate the
entire complex of proteins associated with
dynein during mitosis. If augmin is among
these proteins, it suggests an interaction
between augmin and dynein.
• The antibody recognized a protein band of the correct
molecular weight.
• It appears that our approach does not allow us to pull
down intact complexes.
Future Directions
1. Optimize immunoprecipitation experiments: use a
different antibody to rule out epitope masking
2. Repeat the experiment using an antibody against
augmin.
3. Based on these findings, determine if dynein and augmin
co-localize
on
the
mitotic
spindle
via
immunofluorescence microscopy.
NuMa, one of the proteins that
associates with dynein, co-localizes at
the spindle poles during mitosis. In
this image, fluorophore-conjugated
anti-NuMa antibodies are shown in
green.
(Blue:
dapi-stained
chromosomes. Red: fluorescentlylabeled tubulin.)
References
 Extract is used as source of
protein complexes for IP
experiments
Preliminary Results
Coomassie Stained Gel
Western Blot
Characterizing a new antidynein antibody
1. Goshima, G., F. Nédélec, and R.D. Vale. 2005.
Mechanisms for focusing mitotic spindle poles by minus
end–directed motor proteins. J. Cell Biol. 171:229-240.
2. Goshima, G., M. Mayer, N. Zhang, N. Stuurman, and
R.D. Vale. 2008. Augmin: a protein complex required for
centrosome-independent microtubule generation within
the spindle. J. Cell Biol. 181:421-429.
3. Ganem N.J., S.A. Godinho, and D. Pellman. 2009. A
mechanism linking extra centrosomes to chromosomal
instability. Nature. 460:278-283.
4. Lawo, S., M. Bashkurov, M. Mullin, M.G. Ferreria, R.
Kittler, et al. 2009. HAUS, the 8-subunit human Augmin
complex, regulates centrosome and spindle integrity.
Current Biology. 19:816-826.
Acknowledgements
1 – molecular weight marker
2 – extract proteins prior to immunoprecipitation
3 – extract proteins following immunoprecipitation
4 – immunoprecipitated proteins
Jay Gatlin
Miroslav Tomschik
James Hazel
Paul Mooney
University of Wyoming McNair Scholars Program
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