(703f) Effect of Microtubule Motors On Microtubule Mechanics in Living Cells

Wu, J., University of Florida
Shekhar, N., University of Florida

Effect of microtubule motors on
microtubule mechanics in living cells

Introduction: The mechanical
properties of microtubules are important in enabling its various cellular functions
during division, migration and intracellular transport. Microtubules under
thermal forces have a persistence length on the order of millimeters yet
in vivo
microtubules exhibit bends on micron length scales. This suggests
that within a living cell, microtubules are exposed to large non-thermal
forces. We show with femtosecond laser ablation that microtubules are under
tension due to pulling forces generated by dynein motors1. These
pulling forces focus bends in microtubules to the periphery and also center the
centrosome. We also answer the question: can microtubule motors cause
fluctuations in the trajectories of the tips of growing microtubules?

Materials and Methods: Laser ablation
experiments were performed with a femtosecond laser on an inverted laser
scanning confocal microscope using a 63X, 1.4-NA Plan-Approchromatic oil
immersion lens.
growing tips of microtubules in EGFP-EB1 expressing NIH-3T3 cells were imaged
on a Leica SP5 DM6000 confocal microscope using a 63X objective. DsRed-CC1
expression was used to inhibit dynein. The growing tips were detected and
tracked over time using the software plusTipTracker2. Further
analysis was done using programs developed in MATLAB.

Results and Discussions: On severing a
single microtubule in a living cell using femtosecond laser ablation, we found
that the minus-ended microtubule increased in bending in a dynein-dependent
manner suggesting that the microtubule is under tension (not compression) along
its length due to dynein motor activity(Figure 1 A). Experimental results on
single microtubule severing and centrosome centering by dynein pulling forces
could be explained with a computational model. We also found that microtubule
growth trajectories in control cells spread out considerably at early times
compared to trajectories in dynein-inhibited cells (Figure 1 B). Dynein
activity thus contributes significantly to the bending of growing microtubules.
We have also performed preliminary experiments on understanding the role of
kinesin and myosin II in bending growing microtubule tips.

1. (A) Images highlighting changes in shape after laser-based severing of a
single microtubule in a living cell. The newly generated minus end microtubule
increases in bending after laser severing. The position of the cut is
indicated by
the flash and the severed microtubule is highlighted in red. New
plus and minus ends created upon severing are indicated by the yellow symbols;
the white symbols indicate the ends of the original microtubule
3. (B)
Trajectories of growing microtubules were reconstructed using plusTipTracker.
The plots show that fluctuations in growing microtubules are smaller in
dynein-inhibited cells (right) compared to control cells (left).


Conclusions: Dynein pulls
along the length of microtubules in living cells and also frictionally resists
any lateral motion. Compared to these forces, compressive stresses in the
microtubule are negligible (except very close to the periphery). In the absence
of dynein, microtubules grow straight while in the presence of dynein, there is
significant variance in the growth trajectories. Collectively, our results shed
new light on microtubule mechanics in living cells.

References: 1. Wu J. et al. MOL BIOL CELL, 2011, 22, 4834?4841
                    2. Applegate K. T. et al. J STRUCT BIOL, 2011, 176, 168?184
                    3. Wu J. et al. INTEGR BIOL, 2012, DOI: 10.1039/c2ib20015e