Single-molecule observation of anomalous electrohydrodynamic orientation of microtubules.

We use fluorescence microscopy to measure the orientation and shape of microtubules-which serve as a model system for semiflexible rods-that are electrophoretically driven. Surprisingly, a bimodal orientation distribution is observed, with microtubules in either parallel or perpendicular orientations to the electric field. The occupancy of these states varies nonmonotonically with the microtubule length L and the electric field E. We also observe a surprising bending deformation of microtubules. Interestingly, all data collapse onto a universal scaling curve when the average alignment is plotted as a function of B proportional, variantEL3, which reflects the ratio between the driving force and a restoring elastic force. Our results have important implications for the interpretation of electrical birefringence experiments and, more generally, for a better understanding of the electrokinetics of rods.

Microtubule curvatures under perpendicular electric forces reveal a low persistence length.

The mechanics of microtubules, cylindrical protein filaments that constitute the cytoskeleton, have been well characterized on long length scales. Here, we investigate the persistence length of short (approximately 0.1 microm) ends of microtubules by measuring the trajectories of kinesin-propelled microtubules under perpendicular electric forces. We relate the measured trajectory curvatures to the biased thermal fluctuations of the leading microtubule end, and upon including all electrohydrodynamic forces, we find that the persistence length of the microtubule ends is only 0.08 +/- 0.02 mm. This is significantly shorter than the well established value of approximately 4-8 mm that is measured for long microtubules. Our data are in good agreement with recent theoretical predictions that microtubules mechanically behave as a loose assembly of independent protofilaments on these short length scales.

Persistence length measurements from stochastic single-microtubule trajectories.

We present a simple method to determine the persistence length of short submicrometer microtubule ends from their stochastic trajectories on kinesin-coated surfaces. The tangent angle of a microtubule trajectory is similar to a random walk, which is solely determined by the stiffness of the leading tip and the velocity of the microtubule. We demonstrate that even a single-microtubule trajectory suffices to obtain a reliable value of the persistence length. We do this by calculating the variance in the tangent trajectory angle of an individual microtubule. By averaging over many individual microtubule trajectories, we find that the persistence length of microtubule tips is 0.24 +/- 0.03 mm.

Electrophoresis of individual microtubules in microchannels.

We use micrometer-sized fluidic channels to confine and measure electrophoresis of freely suspended individual microtubules. We measure orientation-dependent velocities of microtubules and the electro-osmotic flow mobility in our channels to infer the anisotropic electrophoretic mobility of microtubules under physiological conditions. We discuss the difference between electrophoresis and purely hydrodynamic motion and its implications for interpreting mobility measurements. We show that the mobility anisotropy is a factor of 0.83, clearly different from the well known anisotropy factor of 0.5 in Stokes drag coefficients for cylindrical objects. We also show that the velocity is independent of microtubule length, which would be different for hydrodynamic motion. We demonstrate that the electric force on the counterions has important consequences for the interpretation of electrophoresis experiments and that ignoring this can lead to an underestimation of the effective charge by orders of magnitude. From the electrophoresis measurements, we calculate an effective surface-charge density of -36.7 +/- 0.4 mC/m2 for microtubules. Electrophoretic measurements of subtilisin-digested microtubules, which have the negatively charged C termini on the outer surface removed, show a 24% decrease in mobility and, correspondingly, in surface charge, but no change in anisotropy.