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. Author manuscript; available in PMC: 2011 Apr 16.
Published in final edited form as: Cell. 2010 Apr 16;141(2):304–314. doi: 10.1016/j.cell.2010.02.035

Fig. 4. The effect of NudE and LIS1 on dynein force production.

Fig. 4

(A) Representative records of single motor force production in an optical trap for beads with bound dynein (D); dynein + NudE (DN); dynein + LIS1 (DL); and dynein + NudE + LIS1 (DNL). Red line marks center of optical trap. Dynein beads (D) typically detach before the motor can stall, but short (~0.5 sec) stalls occasionally occur (see B). DN beads (1:9 D:N) show almost no motion. However, DL and DNL beads (1:2 D:L and 1:9:2 D:N:L) exhibited dramatically longer force production events, some of which continued beyond the period shown (and see panel B). Similar behavior under load was also observed at higher amounts of LIS1 (1:10 D:L) in DL and DNL assays (see Fig. S4). The prolonged dynein stalls induced by LIS1 were eliminated by washing the beads (see Methods) in the DL assay (DL washed) but not the DNL assay (DNL washed). These observations support our biochemical results indicating that NudE retains LIS1 in a complex with dynein (Fig. 1A, B). (B) Representative stalls for dynein adsorbed to beads nonspecifically (D stall) or through anti-dynein IC monoclonal antibody (D Ab) illustrate maximal force production for single dynein motors. (DL) An extremely long (~110 sec) event (entire tracing is shown) demonstrating dramatic prolongation of dynein force production by LIS1. (C) Distribution of forces attained during prominent bead-microtubule binding events sampled in a 4 second window, and summed from multiple bead assays (n= 9–23 as indicated). DN shows minimal bead displacement almost symmetrically distributed around the trap center, indicating that bead motion is predominantly due to thermal noise. D alone exhibits a shift to higher forces. The shift is dramatically greater for DL and DNL, indicating higher average force production. Notably, this effect was retained when DNL beads were washed to remove excess proteins from solution (DNLw) but abolished when DL beads were similarly washed (DLw). This suggests that DNL association is stable but the DL one is not. (D) The fraction of beads exhibiting long force production events (>2 sec) is dramatically increased in the presence of LIS1. The effect is again abolished in DL-washed (DLw) but not in DNL-washed (DNLw) assays. Exact CI error bars are reported (see Supplement). See also Figure S4.