Microtubule Defects Influence Kinesin-Based Transport In Vitro

Abstract

Microtubules are protein polymers that form "molecular highways" for long-range transport within living cells. Molecular motors actively step along microtubules to shuttle cellular materials between the nucleus and the cell periphery; this transport is critical for the survival and health of all eukaryotic cells. Structural defects in microtubules exist, but whether these defects impact molecular motor-based transport remains unknown. Here, we report a new, to our knowledge, approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo.

Figure 1

Single-microtubule (MT) measurements of cargo travel for few-kinesin transport. (A) Schematic of our single-MT assay (not to scale). An optical trap directs kinesin-coated beads to a unique position on a MT. (B) Mean travel distances of beads measured for individual MTs. MT1–3, 4–6, and 7–9 represent sets of three MTs; each set was measured in the same flow cell. Error bars, standard error. Asterisks, significant differences in cargo travel distance between MT pairs (p < 0.02, rank-sum test). Corresponding single-MT travel distributions are shown in Fig. S3. (C) Example of single-MT trajectories sharing the same initial travel position (red dashed line) on a single MT. Each trajectory represents a different bead trapped from the same bead population in the flow cell; the trajectories are offset with regard to their relative timing (x axis) to facilitate comparison. n, total number of trajectories measured for this MT. Magenta dashed line, MT position at which several beads disengage from transport. (D) Single-MT travel distribution corresponding to trajectories in (C). Blue line, best fit to a single exponential decay. Mean travel distance (d ± standard error), goodness of fit (R_adj²), and sample size (n trajectories) are indicated. Arrows, deviations from best fit (magenta, more counts; orange, fewer counts; see Materials and Methods). To see this figure in color, go online.

Figure 2

Pairwise comparisons of travel distance along two types of microtubule that differed in their respective likelihood of displaying supertwist (95% and 40%). (A and B) Experimental schematic (not to scale). We used the same kinesin/bead mixture to contrast transport between the two types of microtubules (A). We used the standard multiple-microtubule assay to minimize the influence of microtubule defects (B). (C) Distribution of few-kinesin travel along each microtubule type. Hatched bars at ∼9 μm indicate travel distances that exceeded our field of view. Solid line, best fit to a single exponential decay. Mean travel distance (d ± standard error) and sample size (n trajectories) are indicated. These distributions do not differ significantly from each other (p = 0.36, rank-sum test). To see this figure in color, go online.

Figure 3

Measurements of cargo travel along antibody-immobilized microtubules (MTs). (A) Experimental schematic (not to scale). Antitubulin antibody was used to elevate the MT above the coverslip surface and Pluronic F-127 was used to reduce nonspecific interactions between motor/bead complexes and the coverslip. (B) Single-MT travel distributions measured for three MTs in the same flow cell. Asterisks, significant differences in cargo travel distance between MT pairs (p < 0.02, rank-sum test). Blue line, best fit to a single exponential decay. Mean travel distance (d ± standard error) and sample size (n trajectories) are indicated. Hatched bars at 12 μm indicate cumulative counts of travel distance that exceeded our field of view. Arrows, substantial deviations between measurements and best-fitted values. To see this figure in color, go online.

Figure 4

Single-microtubule (MT) measurements of cargo pausing during many-kinesin transport. (A and B) Example trajectories (left) and the corresponding distribution of pauses along the MT axis (right) measured for two MTs. Each trajectory represents a different bead trapped from the same bead population in a single flow cell; the trajectories are offset with regard to their relative timing (x axis) to facilitate comparison. Red asterisks indicate common pause locations (>4 standard deviations above the mean number of pauses for that MT). (C–E) Example trajectories exhibiting static (C) and dynamic (D and E) pausing. Blue arrows indicate the direction of cargo travel. Mean cargo velocity (± standard error) during dynamic pausing (D and E) was 2.0 ± 0.4 μm/s (n = 11) during backward movement and 0.66 ± 0.16 μm/s (n = 11) during forward movement. To see this figure in color, go online.

Figure 5

Distributions of off-axis positions of beads during pausing and during motion. (A) Example of off-axis (top) and on-axis (bottom) positions for one bead trajectory. Vertical dash-dot lines indicate pausing. (B) Example distributions for six trajectories along the same microtubule. The distribution during cargo motion represents averages of all six trajectories; error bars, standard error. Distributions during cargo pausing were not averaged and represent individual trajectories. Pauses 1–3 shared the same on-axis location on the microtubule. Pause 6 corresponds to the off-axis trajectory shown in (A). (C) Normalized distributions averaged over 34 microtubules (MTs). Error bars, standard error. To see this figure in color, go online.

Figure 6

Probability of cargo pausing on microtubules with different defect frequencies. (A) Schematic of experimental setup (not to scale). A single population of kinesin-coated beads was introduced into two flow cells containing taxol-stabilized microtubules (blue) or taxol-polymerized microtubules (orange). Asterisks illustrate the relative defect frequencies as previously reported (2). (B) Probability of a trajectory pausing on each microtubule. Error bars, standard error. (C) Distributions of pausing probability measured for each microtubule type. Mean pausing probability (± standard error) and sample size (n microtubules) are indicated. To see this figure in color, go online.