Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

Abstract

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.

FIGURE 1:

Dyn2 is the yeast LC8 homologue. (A) Dyn2 is highly similar to the LC8 family of mammalian dynein light chains, based on sequence alignment and secondary structure prediction. Dyn2 is 46.7% identical and 71.7% similar to H. sapiens LC8 (Accession number Q96FJ2), 47% identical and 72% similar to D. melanogaster LC8 (Accession number NP_726942.1), and 46% identical and 71% similar to Mus musculus LC8 (Accession number NP_080832). Amino acids highlighted in red are involved in light chain dimerization, and amino acids highlighted in purple are involved in LC8 interaction with the dynein IC. (B) The requirement of Dyn2 for dynein function was tested in vivo using a single–time point nuclear segregation assay. yJC5919, yJC5603, and yJC7259 strains were used. Error shown is SD. (C) Epistasis experiments place Dyn2 in the dynein pathway, not the Kar9 pathway. yJC2588, yJC3601, yJC3607, yJC3754, yJC3756, and yJC3832 strains were used. (D, E) Analysis of preanaphase spindle movement in representative HU-arrested wild-type cells (panel D, yJC5919) and dyn2Δ cells (panel E, yJC7259). Images represent single time points at 4-min intervals. Bar is 1 μm. Graphs represent the distance that the daughter-bound SPB is from the bud neck (0) at each time point.

FIGURE 2:

Dyn2 localization in cells. (A) Dyn2 colocalizes with dynein heavy chain, Dyn1, at SPBs and cytoplasmic foci and (B) dynein IC, Pac11, at cytoplasmic foci. (C) Dyn2 colocalizes with tubulin at SPBs, along cytoplasmic microtubules, at the plus-ends of cytoplasmic microtubules, and at the nuclear envelope. Strains were yJC4883 (A), yJC4966 (B), and yJC4371 (C). White arrows denote sites of colocalization. Bar is 1 μm.

FIGURE 3:

Dynein IC domain structure and light-chain binding sites. (A) Domain structure of Pac11 and human IC (IC2C isoform, GenBank Accession number NP_001369). Pac11 contains a short predicted coiled-coil domain followed by two putative LC8 binding sites and a C-terminal WD repeat domain. Domains determined using NCBI BLAST. Sequences of the predicted Dyn2/yLC8 binding sites on Pac11 are shown above schematic. (B) The two putative Dyn2/yLC8 binding sites identified in Pac11, site 1 and site 2, are aligned with human LC8 binding sites in human IC (isoforms IC1 and IC2) (C) and the Dyn2 binding sites identified in Nup159 (Stelter et al., 2007). Alignments created using ClustalW2 at EMBL-EBI (Larkin et al., 2007).

FIGURE 4:

Biochemical analysis of putative LC8-binding sites. (A) Native gel analysis of Drosophila LC8 and its interaction with wild-type Pac11 1–86aa fragment, Pac11 site 1 mutant 1–86aa, Pac11 site 2 mutant 1–86aa, and Pac11 double mutant 1–86aa. (B) Analysis of Drosophila LC8 and interaction with Pac11 fragments by size exclusion chromatography. The combination of the Pac11 1–86aa fragment and LC8 elutes at 9.3 ml. Both the single site mutants (sites 1 and 2) elute at 10.2 and 10.1 ml, respectively. No association is seen between the Pac11 double mutant fragment and LC8.

FIGURE 5:

Sedimentation equilibrium using analytical ultracentrifugation. (A) Radial absorbance (280 nm) of LC8 and wild-type Pac11 at 20, 25, 30, and 40K rpm. The data were fitted as a single species with the molecular weight as the only variable parameter. The resulting molecular weight is 54 kDa, consistent with 2 LC8 dimers (20.6 kDa) and two wt Pac11 fragments (10 kDa each) with some association/dissociation; (B) LC8 and the Pac11 double mutant fragment were mixed at 2:1 stoichiometry and equilibrated overnight before the analysis. Fitting the data as a single species did not produce an adequate fit. Fitting the data as two noninteracting species at the appropriate concentrations and molar extinction coefficients afforded a fit to the dimeric weight of LC8 and monomeric weight of Pac11, indicating no interaction. (C) Same as (A) but using the Pac11 site 1 mutant fragment. (D) Same as (A) but using the Pac11 site 2 mutant fragment. (E) Radial absorbance (280 nm) of Pac11 indicates that the 1–86 fragment of Pac11 is monomeric with a calculated molecular weight of 11,557 ± 527 kDa (χ² = 3.2).

FIGURE 6:

Functional analysis of putative Dyn2 binding sites in vivo. (A) TAP-tagged Pac11 constructs were used in a pull-down assay to test the association of Dyn2–13Xmyc with wild-type and mutants forms of Pac11. Strains were yJC7271 through yJC7278. (B) Single–time point nuclear segregation assay for dynein function in cells. Data is averaged from six or nine independent experiments: ∼300 cells were counted per experiment per strain. Error shown is SD. Strains were yJC5919, yJC5603, yJC6354, yJC7259, yJC6376, yJC6375, yJC6377, yJC7261, yJC7262, and yJC7263.

FIGURE 7:

Preanaphase spindle dynamics in HU arrested pac11 mutant cells. Cells expressing GFP-tubulin (Tub1) were arrested in S phase using HU, and preanaphase spindle dynamics were analyzed over time. Images (above) represent single time points at 4-min intervals. Bar is 1 μm. Graphs (below) represent the distance (μm) that the daughter-bound SPB is from the bud neck (0) at each time point. (A) pac11Δ (yJC6354), (B) Pac11 site 1 mutant (yJC6376), (C) Pac11 site 2 mutant (yJC6375), (D) Pac11 double mutant (yJC6377).

FIGURE 8:

Fluorescence intensity measurements of (A) Dynamitin/Jnm1 and (B) Dynein HC/Dyn1 at the plus-ends of microtubules in pac11 mutant cells. Histograms represent the percentage of cells containing Jnm1 or Dyn1 foci displaying fluorescence intensity at bud-proximal microtubule plus-ends in G₂/M cells, identified by spindle length and grown in log-phase cultures. Microtubule ends were identified in the CFP-Tub1 image, and intensity measurements were taken from the corresponding plane of the GFP or tdimer2 stack. Wild-type and mutant cells are expressing CFP-tubulin (Tub1), Jnm1-tdimer2, and Dyn1–3XGFP. Control cells are expressing CFP-tubulin alone. Modes calculated for each data set are listed in each upper right corner (* represents data sets that are significantly different from respective wild-type data sets (p ≤ 0.005), and ** represents data sets that are significantly different from all other data sets collected (p ≤ 0.0004).) Strains were yJC5668, yJC7354, yJC7355, yJC7356, and yJC7358.