Lis1 activates dynein motility by modulating its pairing with dynactin

Abstract

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minus-ends of microtubules. It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 promotes the formation of an active complex with dynactin. Lis1 also favours the recruitment of two dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. We propose that Lis1 binding releases dynein from its autoinhibited state, which provides a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargos in cells.

Extended Data Fig. 1. Lis1 increases the velocity of complexes assembled with wtDyn

(a) Assembly of wtDDB and wtDDR. (b) Velocity distribution of wtDDB and wtDDR complexes assembled in the presence and absence of 600 nM Lis1. The line and whiskers represent the mean and SD, respectively. From left to right, n = 106, 72, 75, and 81, and mean values are 538, 718, 924, 1113 nm s^-1 (three independent experiments). p-values are calculated from a two-tailed t-test. (c) Velocity distribution of complexes assembled with wtDyn and mtDyn in the absence of Lis1. The line and whiskers represent the mean and SD, respectively. From left to right, n = 106, 132, 75, and 307, and mean values are 538, 652, 924, 1155 nm s^-1 (three independent experiments). p-values are calculated from a two-tailed t-test. (d) The percentage of processive wtDDB complexes that are dual-labeled when an equimolar mixture of TMR- and LD650-dynein motors were assembled with dynactin and BicD2N in the absence of Lis1 (mean ± SEM, n = 246 and 178 from left to right). Error bars represent SE calculated from multinomial distribution and the p-value is calculated from the two-tailed z-test.

Extended Data Fig. 2. Step analysis of mtDDB in the presence and absence of Lis1

(a) Additional examples of mtDDB stepping in the presence and absence of 600 nM Lis1. (b) The average size of steps taken in forward (μ_f), backward, (μ_b), and both (μ_cum) directions along the longitudinal axis of the MT. Error bars are SEM. In a and b, six independent experiments were performed per condition. (c) Stepping rates estimated from the exponential fit in Figure 1f. Error bars are SE of the fit. In b and c, p values are calculated from a two-tailed t-test; sample size (n) distribution of data are provided in Fig. 1f.

Extended Data Fig. 3. Lis1 does not increase the stall duration of dynein bound to dynactin and a cargo adaptor

(a) Inverse cumulative distribution of stall durations in the absence and presence of 600 nM Lis1. Solid curves represent fitting to a two-exponential decay (decay time ± SE). (b) Mean stall times of mtDDB and mtDDR in absence and presence of 600 nM Lis1 (± SEM). p values are calculated from a two-tailed t-test. In a and b, n = 53, 27, 50, and 39 from left to right, four independent experiments per condition.

Extended Data Fig. 4. Lis1 does not affect stall time and stepping rate of single dynein bound to dynactin

(a) Distribution of dwell times between consecutive steps along the longitudinal axis of the MT. A fit to an exponential decay reveals the decay rate (rate ± SE, n = 734 for mtDTR-Lis1 and 724 for mtDTR+Lis1). (b) Inverse cumulative distribution of stall durations of mtDTR in the presence and absence of 600 nM Lis1. Solid curves represent fitting to a two-exponential decay (decay time ± SE, n = 118 for mtDTR-Lis1 and 100 for mtDTR+Lis1, three independent experiments).

Extended Data Fig. 5. Lis1 does not stimulate the recruitment of dynein tail to dynactin

Representative kymographs show the motility of LD650-Dyn and TMR-Dyn_LT assembled with BicD2N or BicDR1 in the presence and absence of 600 nM Lis1. White arrows point to complexes that contain both LD650-mtDyn and TMR-Dyn_LT (three independent experiments were performed per condition).

Extended Data Fig. 6. Additional examples of binding events of Lis1 to mtDDB and mtDTR during processive movement

(a) Schematic depiction of mtDDB complex assembled in the presence of TMR-Lis1. (b) Representative kymographs show binding of Lis1 to motile mtDDB complexes assembled by mixing 1 nM LD650-mtDDB and 75 nM TMR-Lis1 and immediately recording motility with free proteins in solution (see methods). White arrows represent colocalization of LD650-Dyn (red) and Lis1-TMR (cyan). (c) Velocity distribution of mtDDB complexes not bound to Lis1 moves faster than complexes that are bound to Lis1 during single-molecule motility. The line and whiskers represent the mean and SD, respectively. From left to right, n = 270 and 117 and mean values are 921 and 813 nm s^-1. In b and c, three independent experiments were performed per condition. The p-value is calculated from a two-tailed t-test. (d) Rare events of dynamic binding of Lis1 to dynein as mtDDB walks along an MT assembled in the presence of 50 nM Lis1. White arrows represent the colocalization of LD650-Dyn (red) and TMR-Lis1 (cyan). In the top kymograph, Lis1 initially diffuses on an MT and then binds to mtDDB during processive movement. Lis1 binding reduces the velocity of the complex. In the middle kymograph, dissociation of Lis1 during mtDDB motility increases the velocity. In the bottom kymograph, a diffusing Lis1 initially binds and later dissociates from mtDDB, without significantly affecting the velocity of the complex (four independent experiments). (e) Additional kymographs show single- and dual Lis1 binding to motile mtDTR complexes assembled in the presence of 50 nM Lis1. Red arrows represent the colocalization of Atto488-Dyn_LT (green) and Cy5-Lis1 (red). White arrows represent the colocalization of Atto488-Dyn_LT (green) with both Cy5-Lis1 (red), and TMR-Lis1 (cyan). Three independent experiments were performed per condition.

Extended Data Fig. 7. At limiting dynein concentration, Lis1 recruits single dynein to dynactin and BicD2N

(a) Schematic depiction of wtDDB assembly using 5 nM LD650-wtDyn and TMR-wtDyn in the absence and presence of 600 nM Lis1. (b) Fraction of processive and static/diffusive wtDDB complexes on MTs (mean ± SEM, n = 59, 788, 303 and 984 from left to right, three independent experiments).

Figure 1. Lis1 increases the stepping rate of dynein-dynactin.

(a) Schematic depiction of the mammalian dynein-dynactin-cargo adaptor complexes. BicD2N primarily recruits single dynein to dynactin (DDB), whereas BicDR1 recruits two dyneins (DDR). Lis1 binds to the dynein motor domain. (b) Kymographs show the motility of mtDDB and mtDDR on MTs. (c) Velocity distribution of mtDDB and mtDDR with and without 600 nM Lis1. The centerline and whiskers represent the mean and SD, respectively. From left to right, n = 132, 217, 307, and 241, and mean values are 652, 854, 1155, and 1259 nm s^-1. In a-c, four independent experiments were performed per condition. (d) The velocity of mtDDB under different Lis1 concentrations (mean ± SEM). mtDDB complex was assembled in the presence of Lis1, followed by removing excess protein and introducing Lis1 into the flow chamber. The red curve represents a fit to a Hill equation with n = 1. From left to right, n = 132, 216, 177, 204, 156, 179, and 217 (three independent experiments). (e) Velocity distribution of mtDDB assembled in the absence and presence of 600 nM Lis1 under different assembly conditions. The line and whiskers represent the mean and SD, respectively. From left to right, n = 152, 161, 170, 183, and 387 and mean values are 622, 638, 838, 842, and 888 nm s^-1 (three independent experiments). (f) Step analysis of QD-labeled mtDDB (top insert) at 2 μM ATP in the presence and absence of 600 nM Lis1. Red staircases represent a fit to a step finding algorithm. (Bottom, left) Inverse cumulative distribution of dwell times between consecutive steps along the longitudinal axis. Solid curves represent fitting to an exponential decay (decay rate ± SE, n = 2138 for -Lis1 and 1441 for +Lis1). (Bottom, right) Normalized histograms of step sizes (n = 2,076 steps for -Lis1 and 1,374 for +Lis1, six independent experiments). Average forward and backward step sizes and the probability of backward stepping (p_b) are shown (± SEM). In c and e, p values are calculated from a two-tailed t-test.

Figure 2. Lis1 increases the force production of dynein-dynactin.

(a) Schematic of a fixed optical trapping assay for measuring the dynein stall force. (b) Typical stalls of beads driven by a single mtDDB or mtDDR. Red arrowheads denote the detachment of the motor from the MT after the stall event. Scale bars are 1 s. (c) Distribution of motor stall forces in the absence and presence of 600 nM Lis1. The centerline and whiskers represent the mean and SD, respectively. From left to right, n = 80 from 19 beads, 61 from 15 beads, 212 from 38 beads, and 152 from 32 beads, and mean values, are 4.1, 5.4, 5.4, and 6.1 pN. p-values are calculated from a two-tailed t-test. In b and c, four independent experiments were performed per condition. (d) Inverse cumulative distribution of stall durations of mtDDB and mtDDR in the presence and absence of Lis1. Solid curves represent fitting to a two-exponential decay (decay time ± SE, n = 53, 27, 50, and 39 from left to right). (e) Schematic depiction of the in vitro tug-of-war assay. DDB and kinesin were labeled with different-colored fluorescent dyes and tethered using a DNA scaffold. (f) Representative kymographs show the motility of LD650-dynein (red) and TMR-kinesin (cyan) in the absence and presence of 600 nM Lis1. White arrows show DDB-kinesin colocalizers. (g) Velocity distribution of mtDDB, kinesin, and mtDDB-kinesin assemblies in the absence and presence of Lis1. The centerline and whiskers represent the median and 65% CI, respectively. From top to bottom, n = 33, 45, 217, 132, and 210, and median values are 233, 185, −836, −604, and 670 nm s^-1. In f and g, three independent experiments were performed per condition. Negative velocities represent movement towards the MT minus-end.

Figure 3. Lis1 does not affect the force generation and velocity of single dynein complexed to dynactin and a cargo adaptor.

(a) Schematic depiction of the mtDTR complex. Full-length dynein and DynLT are labeled with LD650 and TMR dyes, respectively. (b) Representative kymographs show the motility of mtDyn and Dyn_LT. Arrows represent the colocalization of TMR and LD650. (c) Velocity distribution of mtDDB, mtDTB, mtDDR and mtDTR in the presence and absence of 600 nM Lis1. The centerline and whiskers represent the mean and SD, respectively. From left to right, n = 144, 117, 65, 88, 209, 134, 213, and 126 and mean values are 584, 778, 783, 809, 1108, 1248, 1111, and 1154 nm s^-1. In b and c, three independent experiments were performed per condition. (d) Example traces of beads driven by mtDTR in the presence and absence of 600 nM Lis1 against 1 pN hindering force. The raw stepping data are shown in black and the steps fitting are in red. (e) Normalized histograms of mtDTR steps taken in the longitudinal direction. In d and e, n = 734 for -Lis1 and 724 for +Lis1 (three independent experiments per condition). Average sizes of steps taken in forward and backward directions (± SEM) and the probability of backward stepping in the presence and absence of Lis1 are indistinguishable (p = 0.6, two-tailed t-test). (f) (Top insert) Streptavidin (SA)-coated beads are sparsely decorated with biotin-Dyn_LT in the presence of mtDyn, dynactin, and BicDR1, and trapped with a focused laser beam. Traces represent typical stalls of beads driven by mtDTR in the absence and presence of 600 nM Lis1. Red arrowheads denote the detachment of the motor from the MT after the stall event. Scale bar is 1 s. (g) Distribution of mtDTR stall force. The centerline and whiskers represent the mean and SD, respectively. From left to right, n = 111 stalls from 23 beads and 101 stalls from 21 beads, and mean values are 3.7 and 3.8 pN. In f and g, three independent experiments were performed per condition. In c and g, p-values are calculated from a two-tailed t-test.

Figure 4. Lis1 favors the recruitment of two dyneins to dynactin.

(a) Representative kymographs show the motility of LD650- and TMR-labeled dynein in the presence of dynactin, BicD2N, and 600 nM Lis1. Arrows represent TMR and LD650 colocalization. (b) The percentage of processive complexes that contain both TMR and LD650 signals (mean ± SEM, n = 178, 190, 289, 290 from left to right). Error bars represent SE calculated from multinomial distribution and p-values are calculated from a two-tailed z-test. (c) Velocity distribution of single-colored and dual-colored complexes of DDB and DDR in the presence and absence of Lis1. The line and whiskers represent the mean and SD, respectively. From left to right, n = 153, 25, 145, 45, 204, 85, 193, and 97 and mean values are 544, 840, 766, 899, 1082, 1248, 1263, and 1390 nm s^-1. p-values are calculated from a two-tailed t-test. In a-c, four independent experiments were performed per condition. (d) Schematic shows the assembly of mtDDB and mtDTB complexes using TMR-Dyn_LT, LD650-mtDyn, dynactin, and BicD2N. (e) The ratio of processive runs by TMR-Dyn_LT to LD650-mtDyn on individual MTs in the presence and absence of Lis1. The line and whiskers represent the mean and SD, respectively (n = 10, 9, 11, and 10 MTs from left to right, three independent experiments). p values are calculated from a two-tailed t-test.

Figure 5. Lis1 binding decreases the velocity of dynein/dynactin.

(a) Representative kymographs show the motility of mtDDB and Lis1 on MTs. White arrows represent the colocalization of LD650-Dyn (red) and TMR-Lis1 (cyan). (b) Velocity distribution of mtDDB and mtDDB-Lis1 assemblies. The centerline and whiskers represent the mean and SD, respectively. From left to right, n = 512, and 49 from left to right and mean values are 726 and 447 nm s^-1. The p-value is calculated from a two-tailed t-test. In a and b, four independent experiments were performed per condition. (c) The percentage of processive complexes that contain both LD650-mtDyn and Lis1-TMR signals using different assembly conditions (see Methods; mean ± SEM, n = 561 and 387 from left to right). Error bars represent SE calculated from multinomial distribution and p-values are calculated from a two-tailed z-test. (d) Schematic depiction of mtDTR complex assembled in the presence of 50 nM TMR- and Cy5-Lis1. (e) Representative kymographs show the motility of mtDTR and Lis1 on MTs. Yellow arrows represent the colocalization of Dyn_LT (green) and one color of Lis1. White arrows represent the colocalization of Dyn_LT with Cy5-Lis1 (red), and TMR-Lis1 (cyan). (f) Velocity distribution of mtDTR that colocalizes with zero, one and two colors of Lis1. The centerline and whiskers represent the mean and SD, respectively. From left to right, n = 357, 172, and 40 and mean values are 985, 701, and 582 nm s^-1. In e and f, three independent experiments were performed per condition. p-values are calculated from a two-tailed t-test.

Figure 6. Lis1 promotes assembly of the dynein transport machinery.

(a) Representative kymographs show the motility of DDB at 5 nM concentration of dynein in the absence and presence of 600 nM Lis1. (b) Ratio comparison of the number of processive runs by DDB to the total number of landed motors on MT (mean ± SEM). From left to right, n = 508, 355, 491, 262, 1244, and 392 for wtDyn, and 234, 459, 426, 1352, 457, and 859 for mtDyn. In a and b, three independent experiments were performed per condition. Error bars represent SE calculated from multinomial distribution and p values are calculated from a two-tailed z-test. (c) Representative kymographs show the motility of LD650- (red) and TMR- (cyan) wtDDB at 2 nM concentration of dynein in the absence and presence of 600 nM Lis1. Left kymographs show single-colored runs and right kymographs show rare events of TMR-LD650 colocalization (white arrows). (d) Velocity distribution of wtDDB motility assembled at 5 nM dynein concentration in the absence and presence of 600 nM Lis1. The centerline and whiskers represent the mean and SD, respectively. From left to right, n = 51 and 257, and mean values are 572 and 604 nm s^-1. In c and d, three independent experiments were performed per condition. (e) Fraction of processive complexes that contain TMR, LD650, and TMR-LD650 colocalizers (mean ± SEM, n = 59 for -Lis1 and 303 for + Lis1). The p values are calculated from a two-tailed t-test in d and a two-tailed z test in e. (f) A model for Lis1-mediated assembly of the dynein-dynactin complex. (1) Lis1 binds to the open-conformation of dynein with one Lis1 dimer for each dynein motor domain. (2) Lis1 binding prevents transitions of the open conformation to the phi conformation, which increases the affinity of dynein to dynactin. This mechanism also favors the recruitment of second dynein to the complex resulting in higher force production and faster movement. Lis1 dissociates from active dynein-dynactin-cargo adaptor motors, either after pairing of two dyneins with dynactin (3-5) or during the assembly of the complex (6-8).