Laser Ablation Of Microtubules
During Cell Division Progression
The organization of a cell is critical for its function and understanding how organization
affects function is a major goal of cell biology. Researchers led by Dr. John A. Cooper at
the University of Washington in St. Louis and Dr Alexey Khodjakov at the Wadsworth
Center, Albany, New York used microtubule ablation to learn more about how cells use the
cytoskeleton to integrate spatial information into cell cycle regulation.
Budding yeast cells are one model organism used to study cell division.
During division in the budding yeast, Saccharomyces cerevisiae, one
end of the mitotic spindle is drawn through the bud neck to deliver
a genome to the daughter cell. Cytoplasmic microtubules projecting
from the spindle pole bodies carry out this process by interacting with
the cell cortex to orient the spindle along the mother-bud axis and then
move one spindle pole body through the neck and into the bud.
The cell has quality control mechanisms in place in case this
process goes wrong. For example, mutations can cause a delay in
the movement of the spindle. In this case mitosis proceeds in the
mother cell, but if things have not corrected themselves by anaphase
a cell-cycle checkpoint mechanism known as the spindle position
checkpoint will stop mitosis. Scientists have some understanding of
how this checkpoint halts mitosis, but it isn't fully known how the
checkpoint mechanisms detect that the spindles are not in the correct
position to proceed.
Previous work had suggested that dividing yeast prohibit cell cycle
progression when the mitotic spindle is not adequately positioned
between the nascent mother and daughter cells. This implies that the
cell must monitor the position of the spindle, and interpret it relative
to some other site, such as a landmark. To test the hypothesis that
cytoplasmic microtubules extending from the spindle to the bud neck
are important for this process Cooper’s team interrupted microtubule
interactions with the bud neck using laser ablation.
The researchers used a MicroPoint pulsed laser system with its
emission tuned to 539 nm to perform laser microsurgery of GFPlabeled
microtubules in dynein mutant budding yeast cells. Dynein
mutants cannot pull the spindle through the bud neck, and thus mitosis
is stopped by the spindle position checkpoint.
They had originally purchased the MicroPoint system to add a
photobleaching / photoactivation module to their microscope, but
the tunability of the system’s laser gave them the flexibility to also
use the system to ablate microtubules in this experiment. MicroPoint
allows the user to image the specimen during laser ablation and this
allowed the researchers to visually target dynamic microtubules for
ablation. Microtubules move quickly through the cell, so being able to
observe the cell during ablation was critical for nimble targeting and
subsequent verification of microtubule severing.
After using pulses of 539 nm light to sever individual cytoplasmic
microtubules between the bud neck and spindle pole bodies, the
researchers observed displacement of a distal fragment from the
remaining microtubule. The fragment and remaining microtubule
depolymerized and then grew back after several minutes. They
monitored the spindle of these cells for up to 90 minutes to see if
the cells remained arrested in anaphase. For imaging, they took timelapse
Z series of the cells using an inverted fluorescence microscope
with a 100 x, 1.35 N.A. oil objective lens, CSU spinning disk
confocal scanner with 488 nm laser excitation and EMCCD camera
for detection. The Z series covered 3 um in depth and were captured
at 30 or 60 s intervals.
When cytoplasmic microtubules in the bud neck were ablated most
of the cells exited mitosis, showing that the absence of cytoplasmic
microtubules in the bud neck activates the mitotic exit network. The
researchers next performed experiments to find out if disrupting
cytoplasmic microtubules not in the neck would disrupt the checkpoint.
In checkpoint-activated cells with microtubules extending from
one spindle pole body through the neck they ablated microtubules
at the other spindle pole body (microtubules not extending through
the neck). Most of these cells remained in mitosis. Finally, they
did experiments in which they damaged the spindle pole bodies or
severed microtubules near the spindle pole bodies. Neither of these
actions prompted mitotic exit.
Collectively these ablation experiments show that disrupting the
microtubules that extend from the spindle to the bud neck cause the
cells to fail to prohibit cell cycle progression and thus to exit mitosis.
This suggests that the cytoplasmic microtubules are important for
reading the position of the spindle upstream of cell cycle regulation.
These findings have implications across species. They may be
particularly important for the faithful distribution of genomes to
proper regions of the cytoplasm during the asymmetric cell divisions
that underlie metazoan development and the homeostasis of adult
- CSU laser dual spinning disk confocal
scanner, equipped with EMCCD detector
- Time-lapse imaging software
- MicroPoint with 539 nm dye resonator
Pulsed ablation targeting software