Figure 5. Inhibition of dynein 1 activity by dynapyrazole-A (compound 8).

(A) Gel filtration trace (Superose 6) for GFP-dynein 1, with volume at elution peak indicated. V_o, void volume. (B) SDS-PAGE analysis (Coomassie blue stain) of GFP-dynein 1, ~0.5 µg protein loaded. (C) Montages of fluorescent microtubules moving on GFP-dynein 1-coated glass coverslips in the presence of 1 mM ATP and either DMSO, ciliobrevin D or 8 (10µM). The interval between successive images is 2 s and total time elapsed is 20s. Horizontal scale bar, 5 µm. (D) Inhibition of GFP-dynein 1-driven motility by 8 and ciliobrevin D (mean ± S.D., n = 3). IC₅₀ values for 8: 2.3 ± 1.4 µM (n = 3); ciliobrevin D: 15 ± 2.9 µM (n = 3). Number of microtubules quantified: 8: 20 µM-98, 10 µM-105, 5 µM-108, 2.5 µM-97, 1.3 µM-134, 0.6 µM-99, 0.3 µM-43, 0.2 µM-29; ciliobrevin D: 40 µM-64, 20 µM-74, 10 µM-82, 5 µM-79, 2.5 µM-81, 1.3 µM-87. IC₅₀ values reported reflect the mean (± S.D.) of separate IC₅₀ values obtained from independent dose-response analyses. Data were fit to a sigmoidal dose-response curve and the fit constrained such that the value at saturating compound >0. All motility assays were performed at 1 mM MgATP, 0.05 mg/mL casein, and 2% DMSO. Velocity distribution histograms for inhibition of dynein-1 driven microtubule motility are presented in Figure 5—figure supplement 1. Analysis of microtubule attachment to dynein-coated coverslips is presented in Figure 5—figure supplement 2. (E - J) Images of CAD cell neurites stained with Lysotracker Red. in the presence of DMSO control (0.1%), 3.5 µM and 5 µM (8). Scale bar, 10 µm. (E–G) Phase contrast microscopy images of CAD cells. (H–J) Overlay of successive images of lysosome motility in CAD cell neurites. Sixty images, spaced 1s apart, are stacked and successive images colored using FIJI according to the temporal color code shown. (K–M) Kymographs corresponding to images in H-J. The kymograph size is 60 s (vertical) by 37 µm (horizontal) and the anterograde and retrograde orientations are indicated. (N) Quantitation of lysosome velocity. (O) Quantitation of total lysosome displacement over the time course of imaging (1 min). Data are mean of n ≥ 2 experiments with ≥150 particles counted per experiment. Number of frame-to-frame velocities measured: DMSO-anterograde: 14167, DMSO-retrograde: 14973, 3.5 µM 8-anterograde: 11283, 3.5 µM 8-retrograde: 11340, 5 µM 8-anterograde: 9449, 5 µM 8-retrograde: 10458. For O, number of particles counted: DMSO-3770, 5 µM 8–2400, 3.5 µM 8–840. Error bars: S.D. (DMSO, 5 µM 8), or range of values (3.5 µM 8).

DOI: http://dx.doi.org/10.7554/eLife.25174.015

Figure 5—figure supplement 1. Microtubule velocity distribution histograms for dynein-1-driven microtubule gliding in the presence of different concentrations of compound dynapyrazole-A (compound 8).

At least 88 microtubules were analyzed per experimental condition.

DOI: http://dx.doi.org/10.7554/eLife.25174.016

Figure 5—figure supplement 2. Analysis of the number of microtubules associated with coverslips in gliding assays.

(A, B) Images from time-lapse movies of microtubule motility driven by GFP-dynein 2 (A) or GFP-dynein 1 (B) at 1 mM MgATP. Scale bar, 10 µm. (C) Quantification of microtubule number. Each data point represents the average count of microtubules in four fields of view in a each independent experiment. Mean ± S.D from n ≥ 3 experiments.

DOI: http://dx.doi.org/10.7554/eLife.25174.017

Figure 5—figure supplement 3. Reversibility of inhibition by dynapyrazole-A (compound 8).

The GFP-dynein 1 and GFP-dynein 2 motility assays were conducted at 1 mM ATP and 0.05 mg/mL casein. Chambers exposed to inhibitor (5 µM 8) were imaged and then buffer A (see motility methods) supplemented with 1 mM ATP was flowed into the chamber, followed by a 1-min incubation. This process was repeated twice and then reaction mix containing 2% DMSO and 1 mM ATP was flowed into the chamber using ~4 chamber volumes and the chambers were then sealed. Time-lapse movies of the washout condition were taken 10–15 min after last addition of reaction mix. Data shown reflect average of n = 3 washout experiments (+S.D.) and n = 2 for the DMSO controls (+ range).

DOI: http://dx.doi.org/10.7554/eLife.25174.018

Figure 5—figure supplement 4. Analysis of the effect of blocking agent on dynein inhibition by dynapyrazole-A (compound 8).

The GFP-dynein 1 motility assay was conducted at 1 mM ATP in a reaction mix containing 0.5 mg/mL casein. Mean data from three experiments are shown (± S.D). In the presence of 0.5 mg/ml casein, the IC₅₀ was 10.5 ± 4.6 µM (n = 3, mean of 3 separate IC₅₀ fits, ± S.D.). Curves shown represent fit to average value for a given compound concentration. The curve shown in grey reflects the same data as used in Figure 5D (IC₅₀ = 2.3 µM).

DOI: http://dx.doi.org/10.7554/eLife.25174.019

Figure 5—figure supplement 5. Analysis of the effect of dynapyrazole-A (compound 8) on intracellular ATP concentrations.

IMCD3 cells incubated in either low-serum (A-0.2% FBS) or serum-free (B) media were treated with dynapyrazole-A (5 µM, 15 µM) over the course of 3 hr. Cellular ATP content was measured using the CellTiter-glo assay system. Bars represent mean ± S.D. of n = 3 independent replicates.

DOI: http://dx.doi.org/10.7554/eLife.25174.020