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Results: 4

Fig. 3

Fig. 3. Binding of vesicles to MTs is motor-driven and dependent on inhibitory antibodies to dynein, dynactin and kinesin. From: Motor Coordination Via Tug-Of-War Mechanism Drives Bidirectional Vesicle Transport.

(A) The binding of vesicles to MTs is ATP-dependent. Addition of AMP-PNP increases binding, likely due to rigor binding of kinesin. Similarly, depletion of ATP through hexokinase increases binding, likely due to the rigor binding of dynein. (B) Antibodies (Abs) to dynein partially inhibit vesicle binding to MTs (yellow, *: P<0.05 and dark green bars, **: P<0.005). (C) Vesicle binding was inhibited by addition of either mAb-p150 (light purple, ***: P>0.0001) or pAb-p150 (dark purple, *: P>0.05). (D) Vesicle binding was inhibited by an antibody to kinesin-2 (orange, *:P>0.05). However, an antibody known to inhibit motility of kinesin-1 in vitro (SUK4, gray) did not have a significant effect on binding. The control for (B), (C), and (D) is binding in the presence of an anti-Myc antibody.

Adam G. Hendricks, et al. Curr Biol. ;20(8):697-702.
Fig. 4

Fig. 4. A tug-of-war model predicts the observed parameters of bidirectional transport in vitro. From: Motor Coordination Via Tug-Of-War Mechanism Drives Bidirectional Vesicle Transport.

A tug-of-war model [21] was used to analyze the observed motility of neuronal transport vesicles moving bidirectionally along MTs in vitro. Model parameters were based on previous experimental observations [21, 29]. The two free parameters in the model are the number of actively engaged plus- and minus-end directed motors. (A) The relative fraction of time vesicles are moving toward either the plus or minus ends of the MT depends strongly on the ratio of the number of engaged plus- and minus-end directed motors. Predictions are shown over a range of dynein:kinesin-1 and dynein:kinesin-2 ratios. (B) Experimental data from vesicle motility along MTs is best described by a mole ratio of 7:1 dynein to kinesin-1 motors or 3:2 dynein to kinesin-2 motors. Data from vesicles treated with an Ab to DHC and DIC suggest a fraction of the dynein is inhibited under these conditions. (C) The Gillespie method [28] was used to generate simulated trajectories for control vesicles as well as vesicles incubated with pAb-DHC for motility driven by dynein and kinesin-1 (red), and dynein and kinesin-2 (black). Simulated trajectories are compared to kymographs of vesicle motility (excerpted from Fig. S4). (D) Regulation of bidirectional transport likely occurs at several levels. Recruitment, activation, or inhibition of motor proteins regulates the number of active motors associated with vesicular cargo. Regulation on a longer time scale (τlong) likely involves motor effectors. At shorter time scales (τshort), net directionality of movement results from a stochastic “tug-of-war” among opposing motor proteins bound to the same cargo and actively engaged with the MT.

Adam G. Hendricks, et al. Curr Biol. ;20(8):697-702.
Fig. 1

Fig. 1. MT motor proteins dynein and kinesin co-purify with axonal transport vesicles and drive active motility in vitro. From: Motor Coordination Via Tug-Of-War Mechanism Drives Bidirectional Vesicle Transport.

(A) Top, GFP-dynamitin is distributed in a punctate pattern throughout the cell soma and processes of motor neurons in vivo. Middle, GFP-dynamitin is localized to vesicles distributed along the axon of a motor neuron in vivo. Bottom, GFP-dynamitin is distributed to vesicles along the processes of DRG neurons cultured from TgGFP-dynamitin mice. A corresponding line scan of relative fluorescent intensity along the neurite emphasizes the punctate nature of the localization. (B) MT motor proteins cytoplasmic dynein (DHC and DIC), kinesin-1 (KHC), and kinesin-2, and dynactin (p50), axonal transport markers synaptotagmin and synaptophysin, and late endosome markers LAMP-1 and Rab-7 co-purify with isolated vesicles. GFP-labeled dynamitin is efficiently incorporated into the vesicle-associated dynactin complex. Fractions from the vesicle purification include initial cytosolic (S) and membrane (P) fractions from mouse brain homogenate and the 0.6 M (0.6), the 0.6/1.5 M (V), and 1.5 M and 2.5 M (1.5 and 2.5) steps from a discontinuous sucrose gradient. (C) Vesicles isolated from the 0.6/1.5 M interface were incubated with MTs and analyzed by negative stain EM (left panel). Vesicles had a mean diameter of 90.0 ± 2. 9 nm (SE, n=311). (D) Stepwise quantitative photobleaching of dispersed vesicles produced a bimodal distribution. For comparison, photobleaching data for soluble purified dynein/dynactin [2] is shown (red bars). (E) Quantitative western blotting was performed to measure vesicle-bound cytoplasmic dynein, dynactin, kinesin-1 and kinesin-2.

Adam G. Hendricks, et al. Curr Biol. ;20(8):697-702.
Fig. 2

Fig. 2. Bidirectional transport of purified vesicles along MTs in vitro closely models the motility of Lysotracker-positive vesicles in live cells. From: Motor Coordination Via Tug-Of-War Mechanism Drives Bidirectional Vesicle Transport.

(A) Automated tracking analysis of in vitro motility. Top: Fluorescent vesicles (in green) moving on a polarity-marked MT (in red, plus end is bright). Bottom: Custom Matlab programs were used to automatically track the vesicles. The MT coordinates (in gray) were tracked manually in ImageJ. (B) The top left panels show a rhodamine-labeled MT and a time series of the movement of a GFP-labeled vesicle along the MT. A kymograph of distance moved over time is shown in the top right panel. The bottom panel shows the distribution of absolute values of run lengths between reversals, |LRev|, from automated tracking of the vesicle population as a whole (blue curve). Intervals of motility were categorized as stationary when |LRev| < 33 nm (twice the standard deviation of the tracked position of an immobile vesicle attached to the coverslip). The distribution of |LRev| shows a peak <100 nm corresponding primarily to vesicle diffusion. To differentiate between diffusive and processive motility, the long tail of the distribution was fit to an exponential (black curve, characteristic decay length = 300 nm, R-value = 0.8), which provides a good fit for runs >500 nm (see inset). (C) A time series and kymograph of LysoTracker-labeled vesicles in live cells shows bidirectional motility (top). The distribution of |LRev| for Lysotracker-positive vesicles and purified vesicles in vitro indicates similar motility (bottom). (D) Bidirectional motility in vitro and in the cell show a similar distribution. The corresponding MSD s for all tracked vesicles (inset) are consistent. (E) ~40% of processive motility is plus-end directed while ~60% is towards the minus end for purified vesicles and Lysotracker-labeled vesicles. (F) Distribution of observed velocities for processive motility of vesicles in vitro (100 μM ATP) and in live cells. (G) The average velocity of processive runs in the plus- and minus-end directions is higher for Lysotracker-labeled vesicles in live cells than for purified vesicles in vitro.

Adam G. Hendricks, et al. Curr Biol. ;20(8):697-702.

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