Regulation of the processivity and intracellular localization of Saccharomyces cerevisiae dynein by dynactin

Abstract

Dynactin, a large multisubunit complex, is required for intracellular transport by dynein; however, its cellular functions and mechanism of action are not clear. Prior studies suggested that dynactin increases dynein processivity by tethering the motor to the microtubule through its own microtubule binding domains. However, this hypothesis could not be tested without a recombinant source of dynactin. Here, we have produced recombinant dynactin and dynein in Saccharomyces cerevisiae, and examined the effect of dynactin on dynein in single-molecule motility assays. We show that dynactin increases the run length of single dynein motors, but does not alter the directionality of dynein movement. Enhancement of dynein processivity by dynactin does not require the microtubule (MT) binding domains of Nip100 (the yeast p150(Glued) homolog). Dynactin lacking these MT binding domains also supports the proper localization and function of dynein during nuclear segregation in vivo. Instead, a segment of the coiled-coil of Nip100 is required for these activities. Our results directly demonstrate that dynactin increases the processivity of dynein through a mechanism independent of microtubule tethering.

Fig. 1.

Purification of dynactin from S. cerevisiae. (A) Diagram of the dynactin complex. Intact dynactin complexes were affinity purified by using a ZZ-TEV tag on the N terminus of Arp10, and the Nip100 subunit was labeled with a TMR-conjugated C-terminal HaloTag (white asterix). (B) Reconstituted dynein–dynactin complexes move processively and unidirectionally along axonemes. Kymographs of dynein-TMR (Left) or unlabeled dynein and dynactin-TMR (Right) moving along axonemes are shown.

Fig. 2.

Single dynein–dynactin complexes exhibit robust minus-end-directed motility with enhanced processivity. (A) Histograms of dynein and dynein–dynactin velocities. The velocity of dynein is 87 ± 36 nm/s (mean ± SD; n = 1499), and the velocity of dynein–dynactin is 77 ± 37 nm/s (n = 560). Curved lines represent Gaussian fits of the data. A few outlying points are truncated from histograms of run lengths and velocity for display purposes. (B) Histograms of dynein and dynein–dynactin run lengths. The run length of dynein is 1.15 ± 0.04 μm (mean ± SE), and the run length of dynein–dynactin is 2.54 ± 0.17 μm (determined from cumulative probability functions; see Fig. S2). Dashed lines indicate the mean. (C) Histogram of bleaching events. All bleaching events observed occurred in 1 or 2 steps. GFP-dynein, n = 18; GFP-dynein–dynactin, n = 19; dynein–dynactin-TMR, n = 26.

Fig. 3.

High spatial precision measurements of dynactin movement along MTs. Gaussian fits of the position of moving dynein–dynactin-TMR spots at each point in time are displayed as black circles connected by lines, and steps extracted from these fits are displayed as cyan lines. Horizontal lines are separated by 8 nm. (Inset) Histogram of the length of unidirectional segments (distance traveled before a ≥8-nm reversal) of dynein–dynactin movment.

Fig. 4.

The N-terminal coiled-coil (CC1) of Nip100, but not its N-terminal MT binding domains, is required for dynein processivity enhancement. (A) Diagram of truncations of the Nip100 subunit of dynactin. The N-terminally truncated Nip100 subunits lack the following residues: ΔCAP-Gly, 1–68; Δbasic, 1–102; ΔCC1a, 1–182; ΔCC1, 1–377. (B) Mean velocities of dynein–dynactin containing truncations in Nip100 or lacking Arp1, determined from a Gaussian fit. Error bars show SD. Dynein, n = 1,499; dynein plus dynactin, n = 560; dynein plus ΔCAP-Gly-dynactin, n = 899; dynein plus Δbasic-dynactin, n = 487; dynein plus ΔCC1A-dynactin, n = 496; dynein plus ΔCC1-dynactin, n = 939; dynein plus ΔArp1-dynactin, n = 599. (C) Mean run lengths of dynein–dynactin variants, determined from cumulative probability distributions (Fig. S2). Error bars show SE.

Fig. 5.

Effect of Nip100 truncations on dynactin function in yeast cells. (A) The fidelity of nuclear segregation in cells containing truncated Nip100, as indicated by the percentage of anaphase cells with binucleate mothers. Error bars show SEM; n > 200 for each strain. (B) Localization of dynein in cells containing truncated Nip100, displayed as Z-projections of confocal stacks. Images on the right are duplicates of those to their left, scaled such that wild-type localization is easily visible. Dyn1–3xGFP is shown in green, and mCherry–Tubl is shown in red. Examples of astral MT plus-end and cortical localization are indicated with blue and white arrows, respectively. (C) Quantification of Dyn1–3xGFP intensity at astral MT plus-ends. Intensities are normalized to intensity in WT cells. Error bars show SEM. WT, n = 22; ΔCAP-Gly-NIP100, n = 8; Δbasic-NIP100, n = 6; ΔCC1A-NIP100, n = 12; ΔCC1-NIP100, n = 11; ΔNIP100, n = 11.