Tug-of-war between dissimilar teams of microtubule motors regulates transport and fission of endosomes

Abstract

Intracellular transport is interspersed with frequent reversals in direction due to the presence of opposing kinesin and dynein motors on organelles that are carried as cargo. The cause and the mechanism of reversals are unknown, but are a key to understanding how cargos are delivered in a regulated manner to specific cellular locations. Unlike established single-motor biophysical assays, this problem requires understanding of the cooperative behavior of multiple interacting motors. Here we present measurements inside live Dictyostelium cells, in a cell extract and with purified motors to quantify such an ensemble function of motors. We show through precise motion analysis that reversals during endosome motion are caused by a tug-of-war between kinesin and dynein. Further, we use a combination of optical trap-based force measurements and Monte Carlo simulations to make the surprising discovery that endosome transport uses many (approximately four to eight) weak and detachment-prone dyneins in a tug-of-war against a single strong and tenacious kinesin. We elucidate how this clever choice of dissimilar motors and motor teams achieves net transport together with endosome fission, both of which are important in controlling the balance of endocytic sorting. To the best of our knowledge, this is a unique demonstration that dynein and kinesin function differently at the molecular level inside cells and of how this difference is used in a specific cellular process, namely endosome biogenesis. Our work may provide a platform to understand intracellular transport of a variety of organelles in terms of measurable quantities.

Fig. 1.

Tug-of-war between kinesin and dynein on endosomes inside Dictyostelium cells. (A) Trajectory of a reversal of an endosome inside a Dictyostelium cell in an x–y plane obtained from video tracking. Prereversal (grey circles) and postreversal motion (black circles) shows a close overlap over long distances, indicating that the reversal is along a single microtubule. (Inset) Part of an agar-flattened Dictyostelium cell is shown. The cell boundary is outlined (black dotted line). Two endosomes (circled) and their trajectories (white lines) are schematized. Note the reversals in direction. (Scale bar, 5 μm.) (B) Motion of another endosome close to a reversal is projected along a microtubule by assuming the microtubule to be a straight line. A slow tug-of-war (TOW) segment is sandwiched between fast motion. The direction of motion (plus or minus) is not determined (see main text). (Upper Inset) A time series of images (150 msec apart) of an endosome during reversal. The microtubule orientation is approximately vertical. Note the slowing down and elongation of the endosome, interpreted as a TOW between motors (also see Movie S1 and Movie S2). (Lower Inset) Elongation of this endosome is quantified manually using images from successive frames of Movie S1. Endosome length (distance between front and rear ends along direction of motion) is plotted as a function of time. There is a 33% increase in the length during TOW. TOW lasts for ≈1.3 sec.

Fig. 2.

Motion of Dictyostelium endosomes on polarity-labeled microtubules. (A) In vitro reversals of endosomes along polarity-labeled microtubules. Video tracks of a plus → minus (Left) and minus → plus (Right) reversal are shown. Sharp changes in velocity occur at the beginning and end of the tug-of-war (TOW). The TOW segment has negative velocity for both reversals, indicating elongation toward the minus end with the plus end of the endosome static (see main text; also see Table S1). The microtubule orientation is schematized. The Inset shows a time series of images (150 msec apart) of the endosome undergoing minus → plus reversal. Note slowing down and elongation along the microtubule during TOW. Also note how the plus end of the endosome is static during TOW. (B) Motion in the optical trap of a plus-moving endosome likely driven by one kinesin. Note the long plateaus before motor detachment, where kinesin is “stalled.” The stall force for this endosome is 5.6 pN, corresponding to a mean displacement of ≈180 nm from the center of the optical trap. (C) Motion in the optical trap for a minus-moving endosome likely driven by multiple dyneins. Note frequent detachments against load applied by the trap (compare with kinesin). This endosome walked out of the trap at an ≈4.5-sec time point

Fig. 3.

Force and response to applied load of kinesin and dynein using motor-coated beads in an optical trap. (A) Stall in an optical trap for dyneins and DdUnc104 kinesin. T_STALL (thick double-headed arrows) is time spent above half-maximal load before detachment of motor(s). T_STALL increases with increasing dynein number and approaches the large value for a single tenacious kinesin (also see Table S1). (B) Histogram of stall force for dynein. The fit to the sum of three Gaussians (thick line) shows that the motor forces are additive. The obtained values of stall force for one, two, and three dyneins are 1.1 ± 0.3 pN, 2.0 ± 0.4 pN, and 3.1 ± 0.4 pN, respectively (Table S1). (C) Monte Carlo simulated trajectory of an endosome using experimentally determined input parameters. Four to eight weak dyneins are in a tug-of-war against one to two kinesins (see main text). Efficient minus transport with occasional reversals is seen. (Inset) Magnified view of a plus → minus reversal shows the zero-velocity TOW segment.

Fig. 4.

Asymmetric motor competition model (AMCM). A sequence of events for tug-of-war-mediated plus → minus and minus → plus reversals is schematized. A single kinesin is shown in a tug-of-war against six dyneins. Note endosome stretching in the minus direction in both cases, with the kinesin-attached plus end being almost stationary. One dynein exerts ≈1.1 pN force, whereas kinesin exerts ≈5.5 pN. Dynein is also more prone to detachment than kinesin under applied backward force (load). These asymmetries in number and single-motor properties combine to ensure net minus transport of endosomes with occasional reversals and fission (see main text).