Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors

Abstract

Many cellular cargoes move bidirectionally along microtubules, driven by teams of plus- and minus-end-directed motor proteins. To probe the forces exerted on cargoes during intracellular transport, we examined latex beads phagocytosed into living mammalian macrophages. These latex bead compartments (LBCs) are encased in membrane and transported along the cytoskeleton by a complement of endogenous kinesin-1, kinesin-2, and dynein motors. The size and refractive index of LBCs makes them well-suited for manipulation with an optical trap. We developed methods that provide in situ calibration of the optical trap in the complex cellular environment, taking into account any variations among cargoes and local viscoelastic properties of the cytoplasm. We found that centrally and peripherally directed forces exerted on LBCs are of similar magnitude, with maximum forces of ~20 pN. During force events greater than 10 pN, we often observe 8-nm steps in both directions, indicating that the stepping of multiple motors is correlated. These observations suggest bidirectional transport of LBCs is driven by opposing teams of stably bound motors that operate near force balance.

Fig. 1.

Latex bead-containing phagosomes exhibit bidirectional motility in the cell. (A) In the cell, motor proteins function collectively in a crowded, viscoelastic environment to transport vesicular cargoes. (B) Polystyrene beads 1 μm in diameter (example indicated by arrow) are phagocytosed by J774A.1 mouse macrophage cells. (C) These LBCs are transported bidirectionally in the cell, as shown by typical trajectories. The trajectories were projected onto the black line (drawn parallel to trajectories based on a maximum projection image) to quantify displacements in the retrograde and anterograde directions. (D) Anterograde displacements are plotted in the upward direction; retrograde displacements are directed downward. Black dots indicate reversals. (E) LBC motility is typical of bidirectional transport, with short directed runs interspersed with apparent diffusion and pausing. At the stage of maturation used in these experiments (1–2 h after internalization), the LBCs exhibit approximately equal fractions of plus- and minus-end–directed motility, with similar average velocities in the anterograde and retrograde directions. Error bars indicate SEM (n = 52 processive runs, n = 1,261 total runs between reversals, n = 153 trajectories from six cells).

Fig. 2.

The optical trap was calibrated in the viscoelastic cellular environment. (A) The diagram depicts the forces on the LBC that result from the optical trap and the viscoelastic cytoplasm. (B) The calibration uses a global fit to the response of the LBC to sinusoidal oscillations of the stage or optical trap and the portion of the power spectrum of spontaneous fluctuations of the LBC greater than 300 Hz assumed to be thermal motions (black line). At frequencies of less than 300 Hz, the power spectrum shows disturbances as a result of nonequilibrium, biological processes in the cell, and vibrations of the stage caused by the coupling of the stage and the LBC in the viscoelastic cytoplasm. Motions of the beads in cells are subdiffusive, as the slope of the power spectrum is less than 2 (red line indicates a slope of 2). Insets: Spectra from a bead in water. Note that for a purely viscous fluid like water (k_cyt = 0), the magnitude of the forced response continues to decrease at low frequencies, and the slope of the high-frequency fluctuations is near 2. (C) The calibration gives the optical trap stiffness (k_trap) and sensitivity (β), shown here for five LBCs in separate cells. (D and E) The storage and elastic moduli of the cytoplasm for several cells (10⁶ pN/nm² = 1 Pa). Results for water, 1% methylcellulose, and 2% methylcellulose solutions are shown for comparison.

Fig. 3.

Collective dynamics of kinesin and dynein motors drive LBC motility. (A) Forces are exerted bidirectionally on LBCs in living cells. The optical trap was calibrated separately for each cargo in the same position within the cell where forces were recorded. Signals were acquired at 2 kHz (gray), then median-filtered to 4 Hz (black). (B) Boxed portions in A are shown in detail, and black lines are filtered to 20 Hz. (C) Similar forces are exerted by plus- and minus-end–directed motors. Force events are defined as excursions from the trap center greater than ±0.5 pN. The maximum force is recorded for each event. [Event criteria: abs(force) >0.5 pN, event duration > 250 ms; n = 2,165 events from 14 recordings.] (D) When only force events longer than 1 s are included, the retrograde forces show components at 1.6- to 2.3-pN intervals, whereas anterograde forces show a broader distribution consistent with ∼6-pN stalls by single kinesin motors. Low-force events are likely the result of detachments before reaching kinesin’s stall force (n = 855 events). The number of fitted Gaussian components was chosen by using Bayes information criterion (SI Appendix, Fig. S10). (E and F) Force events greater than ±10 pN were analyzed to select for transport driven by multiple motors. For net anterograde (E) and net retrograde (F) runs, step size distributions are centered around ∼8 nm, with occasional back-steps also centered around ∼8 nm (SI Appendix, Figs. S6 and S7). Note that the position data (Inset) are taken from the traces in B.

Fig. 4.

Bidirectional forces are exerted by motors stably bound to isolated LBCs along paclitaxel-stabilized microtubules in vitro. (A and B) Force traces were acquired at 2 kHz (gray) and median-filtered to 20 Hz (black). (C) As in live cells, most events are short and low-force (n = 1,137 events from 44 recordings). (D) Retrograde force events >1 s exhibit components at ∼1.5-pN intervals, at peaks strikingly similar to those observed in living cells. Anterograde force events >1 s exhibit a component at ∼5 pN, corresponding approximately to the stall force of single kinesin motors and low-force components likely caused by early detachments (n = 401 events).