The offloading model for dynein function: differential function of motor subunits

Abstract

During mitosis in budding yeast, dynein moves the mitotic spindle into the mother-bud neck. We have proposed an offloading model to explain how dynein works. Dynein is targeted to the dynamic plus end of a cytoplasmic microtubule, offloads to the cortex, becomes anchored and activated, and then pulls on the microtubule. Here, we perform functional studies of dynein intermediate chain (IC) and light intermediate chain (LIC). IC/Pac11 and LIC/Dyn3 are both essential for dynein function, similar to the heavy chain (HC/Dyn1). IC and LIC are targeted to the distal plus ends of dynamic cytoplasmic microtubules, as is HC, and their targeting depends on HC. Targeting of HC to the plus end depends on IC, but not LIC. IC also localizes as stationary dots at the cell cortex, the presumed result of offloading in our model, as does HC, but not LIC. Localization of HC to cortical dots depends on both IC and LIC. Thus, the IC and LIC accessory chains have different but essential roles in dynein function, providing new insight into the offloading model.

Figure 1.

Dyn3 is required for dynein function and coimmunoprecipitates with Dyn1. (A) The percentage of cells with two nuclei in the mother in a nuclear segregation cold assay is plotted for the indicated strains. Error bars represent standard error of proportion (n = 300–500 cells for each strain). P > 0.4 for dyn1Δ to dyn3Δ; P > 0.1 for dyn1Δ dyn3Δ to dyn1Δ; P > 0.02 for dyn1Δ dyn3Δ to dyn3Δ; P > 0.5 for dyn1Δ to pac11Δ; P > 0.5 for dyn1Δ pac11Δ to dyn1Δ; P > 0.2 for dyn1Δ pac11Δ to pac11Δ. (B) An extract from cells expressing Myc-tagged Dyn1 and TAP-tagged Dyn3 was incubated with IgG/protein A beads, and the bound proteins were immunoblotted with anti-Myc. Dyn1-Myc coprecipitated with IgG/protein A beads, but not with control beads carrying protein A without IgG. (C) A similar cell extract as in B was incubated with anti-Myc beads. Bound proteins were immunoblotted with rabbit IgG to detect the TAP tag. Dyn3-TAP coprecipitated from a Dyn1-Myc extract, but not from the control extract in which Dyn1 was not tagged.

Figure 2.

Localization of Dyn3-3YFP and Pac11-3YFP. (Top row) Dyn3-3YFP and CFP-Tub1 wide-field fluorescence images of wild-type cells. Dyn3-3YFP is observed in the cytoplasm as dots moving toward and away from the bud (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200407036/DC1). The merged image shows cytoplasmic Dyn3-3YFP dots (red) at the distal ends of microtubules (blue). (Middle and bottom rows) Pac11-3YFP and CFP-Tub1 wide-field fluorescence images of wild-type cells. Pac11-3YFP is observed in the cytoplasm as motile dots and at the mother cortex as stationary dots (see Video 2), similar to what was observed for Dyn1-3GFP (Lee et al., 2003). The merged image shows Pac11-3YFP dots (red) at the distal ends of microtubules (blue) and the mother cortex.

Figure 3.

2D projections from 3D image reconstructions of Dyn3-3YFP, Pac11-3YFP, and Dyn1-3GFP in living wild-type cells. Rotating 3D images are shown as videos in the online supplemental materials (available at http://www.jcb.org/cgi/content/full/jcb.200407036/DC1). (A) Dyn3-3YFP is absent from the cortex (see Video 3 for rotating 3D image). The Dyn3-3YFP dot at the distal end of a microtubule moved during Z-collection, causing it to appear spread out in the bud cytoplasm arrow. (B and C) Pac11-3YFP and Dyn1-3GFP are observed as dots at the cortex, predominantly in the mother (dots in the bud are at microtubule ends arrows, and occasionally at the cortex in large buds arrowhead; also see Fig. 4). See Videos 4–6.

Figure 4.

Dyn1-3GFP at the cortex of large buds. Dyn1-3GFP (red) and CFP-Tub1 (blue) wide-field fluorescence images of wild-type cells, showing Dyn1-3GFP dots at the bud cortex (arrows) of large budded cells in which the mitotic spindle had moved into the neck. These dots are not at the distal ends of cytoplasmic microtubules.

Figure 5.

Localization dependence of Dyn1-3GFP, Pac11-3YFP, and Dyn3-3YFP. (A) 2D projection of a Z-stack of confocal images of Dyn1-3GFP in living wild-type, pac11Δ, and dyn3Δ cells. Wild-type and dyn3Δ cells showed Dyn1-3GFP motile dots in the bud. pac11Δ cells showed the absence of Dyn1-3GFP motile dots. See Videos 7–8 (available at http://www.jcb.org/cgi/content/full/jcb.200407036/DC1). (B) 2D projection of a Z-stack of confocal images of Pac11-3YFP (top row) and wide-field fluorescence images of Dyn3-3YFP (bottom row) in living wild-type and dyn1Δ cells. dyn1Δ cells showed no focal accumulations of Pac11-3YFP or Dyn3-3YFP.