A fluid membrane enhances the velocity of cargo transport by small teams of kinesin-1

Abstract

Kinesin-1 (hereafter referred to as kinesin) is a major microtubule-based motor protein for plus-end-directed intracellular transport in live cells. While the single-molecule functions of kinesin are well characterized, the physiologically relevant transport of membranous cargos by small teams of kinesins remains poorly understood. A key experimental challenge remains in the quantitative control of the number of motors driving transport. Here we utilized "motile fraction" to overcome this challenge and experimentally accessed transport by a single kinesin through the physiologically relevant transport by a small team of kinesins. We used a fluid lipid bilayer to model the cellular membrane in vitro and employed optical trapping to quantify the transport of membrane-enclosed cargos versus traditional membrane-free cargos under identical conditions. We found that coupling motors via a fluid membrane significantly enhances the velocity of cargo transport by small teams of kinesins. Importantly, enclosing a cargo in a fluid lipid membrane did not impact single-kinesin transport, indicating that membrane-dependent velocity enhancement for team-based transport arises from altered interactions between kinesins. Our study demonstrates that membrane-based coupling between motors is a key determinant of kinesin-based transport. Enhanced velocity may be critical for fast delivery of cargos in live cells.

FIG. 1.

Experimental schematic (not to scale). (a) Optical trapping-based experiments were carried out in flow chambers, identically for membrane-enclosed cargos [panels (b) and (c)] and standard membrane-free cargos (not shown). The flow chamber was constructed by adhering a cover glass and a microscope slide together using two thin strips of double-sided Scotch tape. Microtubules were immobilized on the cover-glass surface; kinesin/cargo complexes were freely dispersed in solution in the flow chamber. (b) A single-beam optical trap was used to confine individual cargos to the vicinity of a microtubule to reduce the initial interaction distance between the microtubule and kinesins on the cargo. The trap was turned off upon observation of directed cargo motion along the microtubule, eliminating external load. For the membrane-enclosed cargos illustrated here, a silica bead supports a DOPC-based lipid bilayer, ensuring compatibility with the optical trap and matching the size of membrane-enclosed and membrane-free cargos for direct comparison. (c) Polyhistidine-tagged kinesins were specifically recruited to membrane-enclosed cargos via a nickel-functionalized lipid incorporated in the bilayer membrane.

FIG. 2.

Run length does not differ between membrane-enclosed cargos (red) and membrane-free cargos (black). (a) Cumulative probability distributions of cargo run length at three motile fractions. Mean run length (±standard error) and p-value (rank-sum test) are indicated (n = 117-197). The two distributions measured at each motile fraction do not differ significantly from each other (p ≥ 0.27, rank-sum test). (b) Mean run length (±standard error) at six motile fractions (n = 117-197). The two mean run lengths determined at each motile fraction do not differ significantly from each other (p ≥ 0.21, rank-sum test).

FIG. 3.

Pausing does not differ between membrane-enclosed (red) and membrane-free (black) cargos. Arithmetic mean (a) frequency (n = 4-5 experiments) and (b) duration (n = 11-48 pauses) of pauses at six motile fractions. (c) Cumulative probability distribution of pause duration, pooled from measurements at all six motile fractions (n ≥ 175 pauses). Mean pause duration (±standard error) is indicated. These two distributions do not differ significantly from each other (p = 0.23, rank-sum test).

FIG. 4.

Changes in velocity distributions at distinct motile fractions differ between cargo types. (a) Dot plot of velocity measurements for each experimental condition (n = 97-208). Horizontal bars—30th percentile, median, and 70th percentile. **p < 0.005 versus a motile fraction of 0.3 (Welch’s t-test). [(b) and (c)] Fraction and median velocity of cargos moving at <0.6 μm/s. (d) Median velocity of cargos moving at ≥0.6 μm/s. Error bars in [(b)–(d)]—standard error. Blue lines are guides to the eye. At higher motile fractions, we detected a substantially smaller fraction of slow runs (b) and a somewhat faster median velocity of these slow runs (c) for membrane-enclosed cargos than membrane-free cargos.

FIG. 5.

Membrane-enclosed cargos (red) move faster than membrane-free cargos (black) at higher motile fractions. (a) Cumulative probability distributions of velocity at three motile fractions. Median velocity (±standard error) and p-value (Welch’s t-test) are indicated (n = 97-139). (b) The difference in the median velocity of two cargo types at each motile fraction. A positive value indicates that the membrane-enclosed cargos move faster than membrane-free cargos. The pink line is a guide to the eye.