1. Molecular Adaptations Allow Dynein to Generate Large Collective Forces inside Cells 
Arpan K. Rai, Ashim Rai, Avin J. Ramaiya, Rupam Jha, Roop Mallik 
Cell 
Volume 152, Issue 1, Pages (January 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Microtubule-Dependent Transport of LBPs in J774.2 Cells
(A) DIC image of elongated adherent cells on a coverslip (outline of one cell marked in black; nucleus with dashed line). Microtubules are oriented with plus ends at the periphery of elongated regions (marked “+”). LBPs (refractile spheres) are dispersed inside the cell. One minus-moving LBP (circled with black line) is shown to pass through the optical trap (schematic gray spot).
(B) X-Y trajectories obtained from video tracking of plus-moving LBPs.
(C) X-Y trajectories obtained from video tracking of minus-moving LBPs. The linear motion is representative of linear microtubule arrangement. Lower inset (Correct) magnifies motion and stalls of an LBP that was selected for further analysis. Direction of motion before, during and after trapping is exactly same. This shows that the trap (shown as gray circle) was centered precisely on the LBP and the MT (gray line). This is required for correct force measurement (see text). Trap on/off positions are indicated. Tracked positions are closer to each other in the trap because the LBP slowed down. Upper inset (Incorrect) magnifies motion and stalls of an LBP that was rejected. The optical trap is centered ∼30 nm away from the microtubule. The excursions of the LBP from trap occur in arbitrary directions (not necessarily along microtubule; compare with “Correct”).
See also Figures S1, S2, S3, S4, S5, and S7 and Movies S1 and S2.
Cell  , DOI: ( /j.cell )
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4. Figure 2
Force Measurement of Kinesin on Plus-Moving LBPs in Cells and Beads In Vitro
(A) Representative stalls for one and two kinesin-driven LBPs in cells. Tenacity of kinesins against load (measured as TSTALL) is unchanged. Black arrow points to a shoulder in the two kinesin stall, presumably corresponding to single-kinesin force.
(B) Histogram of kinesin stall force (from 51 LBPs) in cells with fit (black line) to sum of two Gaussians. Peaks represent force from one (5.8 ± 1.0 pN) and two kinesins (10.5 ± 0.8 pN) inside cells (mean ±SD).
(C) Stalls for kinesin-1-coated beads in vitro. TSTALL is almost same for one, two, and three kinesins. Note similar quality of data inside cells and in vitro.
(D) Histogram of stall force on kinesin-1-coated beads (42 beads) in vitro. Peaks in Gaussian fit represent force from one (5.7 ± 1.0 pN) and two (10.6 ± 1.3 pN) kinesins (mean ±SD). Mean value and width of stall-force histograms match closely inside cells and in vitro, establishing the precision of measurements.
See also Figures S5, S6, and S7 and Movie S3.
Cell  , DOI: ( /j.cell )
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5. Figure 3
Force and Tenacity of Dynein Teams on Beads In Vitro and on LBPs inside Cells
(A) Representative stalls for dynein-driven beads in vitro for varying dynein concentration. Tenacity of dyneins against load (measured as TSTALL; double-headed arrows) increases with stall force. The displacement for each stall is between 80 and 120 nm, depending on the trap stiffness.
(B) Stalls for dynein-driven LBPs in cells. Noise in experimental data is shown by the gray band at zero force and was obtained by recording fluctuations of an LBP trapped inside a cell with MTs depolymerized by nocadozole treatment. Tenacity of dyneins against load is measured as TSTALL (see text). Putative dynein number is also indicated, assuming ∼1 pN force/dynein. Note how TSTALL increases with dynein number. Black arrows indicate steps at ∼2 pN intervals that are presumably attachments of dynein pairs to MT (see text).
(C) Histogram of stall force from in vitro dynein experiments over a range of dynein concentration. A periodicity of ∼1 pN (likely unit-dynein force) is demonstrated by a fit to sum of three Gaussian peaks. The peak values are 1.1 ± 0.3, 2.0 ± 0.4, 3.1 ± 0.4 pN (mean ±SD).
(D) Histogram of stall force on dynein-driven LBPs inside cells (80 LBPs used). The force from dyneins is usually 6–10 pN in cells. Thick black line is fit to sum of five unconstrained Gaussians. Arrows point to peaks at ∼2 pN intervals, possibly due to pairing of dyneins on LBP (see text). Peak positions (mean ±SD) are 4.3 ± 0.5, 6.0 ± 0.4, 7.7 ± 0.5, 9.7 ± 0.7, and 12.1 ± 0.7 pN. The counts are lower at <4 pN because such LBPs moved over short distance before/after stalls and could not be aligned reliably (see main text).
See also Figures S5, S6, and S7 and Movie S3.
Cell  , DOI: ( /j.cell )
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6. Figure 4
Collective Function and Force-Velocity Curves for Dynein and Kinesin-1
(A) Tenacity of motor teams (measured as TSTALL) as a function of stall force. The stall force is representative of active motor number. In vitro and intracellular data are shown together. Tenacity increases linearly with putative dynein number (1 dynein ≈1 pN). In contrast, there is no improvement in tenacity on increasing kinesin number (1 kinesin ≈5.8 pN). Lines are shown through each set of data points. Error bars are SEMs. The number of stalls used for each point varies and can be estimated from counts in stall-force histograms.
(B) Force-velocity curves of dynein and kinesin-1. Stalls were recorded for single kinesin or dynein-driven beads at 1 mM ATP in an in vitro assay. Stalls (13 for each motor) were fitted by a third-order polynomial to determine the F-V curves. An average F-V curve was then determined. Error bars are SD.
Cell  , DOI: ( /j.cell )
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7. Figure 5 Dynein Reduces Step Size under Load
(A) Stepping of an LBP inside a cell against load during dynein-driven (minus) and kinesin-driven (plus) motion. This LBP paused before reversing along the same MT (break in time axis). For dynein-driven stall, a 24 nm step followed by 16 and 8 nm steps at higher load are seen. In contrast, only 8 nm steps are seen for kinesin-driven motion (one step is missed; M). Inset shows the variation in step size as a function of load (normalized to stall load) obtained from stalls of dynein and kinesin-driven LBPs. Dynein step sizes are clustered around 24, 16, and 8 nm with a clear reduction in step size with load (black line). However, kinesin largely takes steps of 8 nm irrespective of load.
(B) Video track of stepping of a multiple-dynein-driven bead at ∼50 μm ATP in the absence of load (no trap). Velocity is ∼60 nm/s. Well-resolved successive steps can be seen with a mean size of ∼24 nm. Upper inset shows a pairwise distance analysis (see text) of part of the video track to reveal the 24 nm periodicity. Lower inset shows a multiple-kinesin-driven bead at ∼50 μm ATP under no load. Velocity is ∼100 nm/s. Only steps of 8 nm can be seen in video and QPD.
(C) Experiment as above with multiple-dynein-driven beads under load from an optical trap at 1 mM ATP. Note the dominance of successive large steps (∼24 nm; values indicated) at low load, shortening to ∼16 and 8 nm steps at higher load. Inset magnifies the motion for another multiple-dynein-driven bead under load approaching stall. Steps of 8 nm can be seen clearly.
Cell  , DOI: ( /j.cell )
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8. Figure 6 Tenacity of a Dynein Team under Superstall Load
(A) A piezo stage (shown as schematic) is moved to displace a multiple-dynein-driven phagosome after it stalled at ∼7 pN (curved arrow). Only a single dynein is shown for the sake of clarity. This brings the phagosome under superstall load (∼11 pN), whereupon it stays attached for ∼1 s ( = TSUPERSTALL) before detachment (see upper schematic; note the lower value of θ, which would lead to a catch bond in superstalled state).
(B) The detachment time (TSUPERSTALL) of dyneins at superstall load (i.e., load > stall force) is plotted against the stall force. Stall force is proportional to the putative number of dyneins driving motion (stall force of 1 dynein ≈1 pN). There is a 10-fold increase in TSUPERSTALL with stall force. Error bars are SDs. Each data point is a mean of at least seven stalls.
Cell  , DOI: ( /j.cell )
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9. Figure 7 Model for Collective Force Generation by Dynein Teams
Cartoons of an LBP (730 nm dia; shown partially) attached to four dyneins (black lines; 100 nm length) is shown as it moves under increasing load. LBP and dynein are drawn to relative scale. Dyneins are assumed attached equidistant to each other at fixed points on the LBP membrane. L0 (no load): LBP is at trap center (vertical dashed line). Dyneins take large (32 and 24 nm) steps (curved arrows on MT). The spread of dyneins along MT (double-headed arrow) is large. The average angle (θ) between dyneins and MT is large. L1 (low load): Leading dyneins have just reduced step size to slow down, while the lagging ones still take large steps. Bunching starts. L2 (intermediate load): Dyneins are bunched close together due to differential stepping, and share load equitably. L3 (high load): All dyneins have shortened step to 8 nm and are straining against load (θ small). Note how the dynein-MT-binding positions are closer (length of double-headed arrow shortens from L0 → L3). Clean 8 nm steps are now visible in the stall record. L4 (stalled): Two dyneins have detached from MT, bringing the remaining two in superstall catch-bonded state (θ small; see text).
Cell  , DOI: ( /j.cell )
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10. Figure S1
Internalized Latex Beads Mature into a Homogeneous Population of Late Phagosomes after a 1 hr Chase and Acquire Kinesin-1 and Dynein Motors, Related to Figure 1
(A) Cells loaded with beads and chased for 1 hr were fluorescently labeled using established endo-lysosome markers. The chased LBPs did not label for EEA1 (early endosome marker) but were uniformly and intensely labeled for Rab7 (late endosome/phagosome marker) and LAMP-2 (lysosomal marker). In the top right panel, note how the phagocytosed beads are not fluorescently labeled for EEA1 (compare with DIC image). However, many other early endosomes in the cell are labeled. Note how all the LBPs cluster around the nucleus for EEA1 and Rab7 labeled cells. The LAMP-2 labeled cell had phagocytosed too many beads to allow motility and perinuclear clustering.
(B) Phagocytosed beads were chased for 1 hr inside J774.2 cells and isolated on a sucrose gradient after lysing the cells. Purified LBPs were subjected to immunoblotting. Equal amount of total protein was loaded for EEA1 and Rab7. Absence of EEA1 and presence of Rab7 on LBPs shows that LBPs have matured into a homogeneous population of late phagosomes. Kinesin-1 was detected with two different antibodies in cytosol and on LBPs. Dynein and dynactin were also immunodetected abundantly on LBPs. We could not immunodetect kinesin-2 with two different antibodies on LBPs even after concentrating phagosomes several-fold. Kinesin-2 could be detected in cytosol. Similarly, we could not detect kinesin-3 (KIF 1C) on concentrated LBPs. The results of these immunoblots were reproducible for LBPs isolated in three different preparations.
Cell  , DOI: ( /j.cell )
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11. Figure S2
LBP Motion Occurs Predominantly along Microtubules in J774.2 Macrophages, Related to Figure 1
(A) Fluorescent labeling of MTs (green) and actin (red) in fixed J774.2 cells. In > 90% of the cells, a unique MT-organizing center close to the nucleus was seen (marked with ∗). MTs were organized almost parallel to each other and extended linearly to the periphery spanning the elongated “arms” of the cell (blue dashed arrows). Motion of LBPs was measured in these arm-like regions, and away from the cell periphery to minimize contribution from the actin cytoskeleton. The uniform polarity (plus ends at cell periphery marked with “+,” minus end near nucleus) and parallel organization of MTs made it easy to analyze transport of cargoes on MTs and ascribe motion to specific motors (kinesins or dynein). Unlike MTs, F-actin was concentrated in the cortical region. This is consistent with the hypothesis that the acto-myosin system is required for phagocytic uptake, following which the ingested phagosome is briefly trapped by cortical actin and then moves onto MTs for long-distance motion and maturation. Other cells such as fibroblasts have a complicated MT arrangement with multiple MTOCs, which could make it difficult to understand motility and infer motor identity from direction of cargo motion.
(B) Magnified view of the microtubule cytoskeleton. Note linear and parallel organization of microtubules, which makes J774.2 cells an ideal system to study microtubule dependent motility.
(C) The fraction of LBPs that moved over distance > 500nm in minus and plus directions within an observation period of 30sec is plotted for control, actin-depolymerized (–actin) and microtubule-depolymerized (–MT) cells. Cells observed = 8; total LBPs observed = 67. Error bars represent SD.
(D) Actin was depolymerized in J774.2 cells by cytochalasin treatment (40 μM, 20min) after phagocytosis. Motion of LBPs (after 1 hr chase) was observed in presence of cytochalasin. Actin depolymerization had insignificant effect on LBP velocity. Error bars represent SD.
Cell  , DOI: ( /j.cell )
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12. Figure S3
Analysis of LBP Motility inside J774.2 Cells, Related to Figure 1
(A) Video tracks of LBP motion were parsed into linear segments of constant velocity to analyze the motion. Histogram of LBP velocities in minus and plus directions. Vigorous, but episodic motion of LBPs was observed. A stationary LBP could move rapidly, and then pause for a while before moving again in the same or different direction. Individual tracks were fitted to a straight line (the assumed MT) to obtain the position along MT as a function of time, keeping note of the direction of motion (plus or minus). The position-time data were then parsed into segments of constant velocity to obtain a histogram of velocities. The velocity was 1.6 ± 0.7 μm/sec in plus and 1.7 ± 0.8 μm/sec (mean ± s.d.) in minus direction.
(B) A plot between segment velocity and segment length shows a clear positive correlation (red line with positive slope) between these two quantities, both for minus and plus motion. Thus, long uninterrupted motion is likely to occur with higher velocity. Such motion is expected if the LBP is driven by multiple motors. Since long segments are more common for minus motion, this contributes to the larger LBP flux in minus direction. Segment length should not be confused with run length of LBPs (see main text). A run (period of uninterrupted motion between pauses) may contain multiple segments of varying velocities. Inset: The fraction of LBPs moving in plus and minus directions within a given time across many cells. This shows that there is net minus bias in motion. Error Bars represent SD.
Cell  , DOI: ( /j.cell )
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13. Figure S4
Role in Motility and Relative Abundance of Dynein and Kinesin-1 on Latex Bead Phagosomes, Related to Figure 1
(A) LBP motion was assayed in the absence and presence of CC1, a peptide fragment of dynactin that inhibits dynein function in cells. CC1 was introduced into cells by glass-bead loading. The fraction of cells with > 60% LBPs at cell periphery (away from nucleus) is plotted. In control cells, most beads were clustered at the nucleus after a chase of 3 hr. In CC1 loaded cells most of the phagocytosed beads were still at the periphery of the cell because dynein-driven inward motion was inhibited. Error bars represent SD.
(B) Phagocytosed beads were chased for 3 hr, at the end of which all beads were perinuclear. Acetate ringers (pH 6.9) was added to slightly acidify the cytosol. The fraction of cells with > 60% LBPs at cell center (near nucleus) is plotted. In control cells, almost all LBPs moved to the cell periphery (motion in plus direction) as a result of this acidification. However, in cells loaded with a kinesin-1 inhibitory tail domain (KTD), outward LBP motion was significantly inhibited. Error bars represent SD.
(C) Quantitative western blotting was done to measure relative abundance of dynein and kinesin-1 on LBPs. The molar ratio (MD/K) of dynein to kinesin measured was 19 ± 3. Antibodies were used in western blots with known amount (standards) of kinesin-1 purified from goat brain and recombinant DIC. A serial dilution of these standards was used. The experiment was done for three independent phagosome preparations. The intensity of bands for standards varied linearly as a function of dilution in the investigated range. This linear variation was used as a calibration curve to determine the amounts of kinesin-1 and dynein in the phagosome sample. The numbers mentioned are known micromolar concentrations of standards and calculated micromolar concentration of kinesin-1 and dynein on LBPs.
Cell  , DOI: ( /j.cell )
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14. Figure S5
Observed LBP Motion inside J774.2 Cells Occurs along Single Microtubules, Related to Figures 1, 2, and 3
(A) Representative video tracks of motile LBPs were first fitted to a straight line (assumed MT). The perpendicular displacement of LBP (“flop”; see schematic on right) from the assumed linear MT (gray horizontal band of 25nm diameter) was determined from the video track. Similarly, the flop of a bead (same size as LBP) driven by dynein along a single MT in an in vitro assay was determined. The dynamics of the bead in vitro is faster due to lower viscosity of buffer compared to cytoplasm.
(B) A histogram of perpendicular displacements (flop) from MT is plotted for minus-moving LBPs inside cells and dynein-driven beads in vitro. Note the approximately similar flop in both cases – the slightly larger flop for LBPs is because the MTs in cells are not exactly as straight as MTs stuck on a coverslip in vitro. This provides strong evidence that the LBPs are attached by motors to a single MT during the long linear segments of motion. This also indicates that there is no intermittent attachment/force-generation by myosin motors along randomly oriented actin during our period of observation (since that would increase the flop).
Cell  , DOI: ( /j.cell )
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15. Figure S6
Goat Brain Dynein Exerts a Low Force, Related to Figures 2 and 3
Lower panel shows representative video tracks of the motion of 500nm beads coated with dynein purified from goat brain. Note that the velocity of bead translocation by dynein is high (∼500nm/sec). Both runs ended in detachments from the MT. Stall force records taken during both these runs are shown in the upper panel. The trap stiffness for single-dynein experiment was 0.02pN/nm and for multiple dynein experiment it was 0.05pN/nm. Single dynein moved over a short distance and exerted ∼1.2pN force. The long run is likely too long to be driven by a single dynein, and is characteristic of motion driven by 4-5 dyneins. The stall force during this motion was 4pN. This shows that single dynein extracted from goat brain exerts a low force (likely ∼1pN).
Cell  , DOI: ( /j.cell )
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16. Figure S7 Dynein Is a Low-Force Motor, Related to Figures 1, 2, and 3
(A) Representative stall force records for dynein-driven beads at increasing dynein concentrations. Stall force record is also shown for a dynein-driven endosome from dictyostelium. The displacements (mentioned on each stall) are not to scale because each experiment was done with different trap stiffness.
(B) Video tracks of dynein-driven beads and endosome on which stall forces were measured during motion (upper panel). Black: short motion at single-molecule limit; Green: longer ∼2 dynein driven motion; Magenta: ∼4 dynein driven motion; Red: endosome driven by ∼6 dyneins. The average velocity for all runs is 1.5 micrometers/sec or more. The run length of beads and force increased as dynein concentration was increased. Beads that stalled at ∼4 pN routinely moved for > 5 μm, and were likely driven by multiple dynein because single dynein has limited processivity (∼1 μm). Similarly, dynein-driven endosomes translocated over long distances, sometimes ∼15 μm.
(C) Long (∼15 μm) run of an LBP inside a J774 cell, and two stall force measurements yielding 6–8 pN force at the beginning and end of motion. Following the above arguments for beads and endosomes, this is consistent with the idea that many dyneins (each presumably generating low ∼1 pN force) drive intracellular motion of LBPs.
Cell  , DOI: ( /j.cell )
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