Analysis of the dynein-dynactin interaction in vitro and in vivo

Abstract

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.

Figure 1.

Schematic representations of expression constructs used in this study. (A) p150^Glued contains a CAP-Gly microtubule-binding motif near the N terminus and two predicted coiled-coil domains, CC1 (AA 217-548) and CC2 (AA 926-1049). (B) The IC 2C isoform contains a predicted N-terminal coiled-coil domain (AA 1-66), light chain binding regions (RP3, Tctex-1, and LC8: AA 107-138; roadblock: AA 223-261) and WD repeats (AA 262-612). Arrows indicate two sites of alternative splicing (after AA 76 and 106), and the S indicates a region (AA 81-99) that contains eight serine residues. The regions encoded by different expression constructs are shown beneath.

Figure 2.

Direct interaction between dynein IC fragments and dynactin p150^Glued CC1. Recombinant proteins (CC1, IC2-N237, IC2-S84A, IC2-S84D, IC2-N106, and dynamitin) were used alone or mixed pairwise and chromatographed on a Superose 12 gel filtration column. The two S84 mutants (S84A and S84D) were made in the IC2-N237 fragment. Individual column fractions were analyzed by SDS-PAGE and Coomassie Blue staining. The identity of polypeptides present in each column run is provided to the right. The A₂₈₀ chromatographic positions of molecular standards of known Stokes' radii are indicated at the top.

Figure 3.

Velocity sedimentation of detergent lysates from HeLa cells overexpressing dynein IC fragments. Lysates were sedimented into 5-20% sucrose gradients. Fractions were analyzed by Western blotting with antibodies to GFP, dynein IC, the dynein LC, rp3, or the dynactin subunit p150^Glued. The construct being expressed and the size of each immunoreactive species are indicated; sedimentation standards are at the bottom. Similar sedimentation patterns were obtained when IC polypeptides were overexpressed in COS-7 cells (our unpublished data).

Figure 4.

Effects of dynein IC overexpression on the integrity of the microtubule array and pericentriolar material in COS-7 cells. (A) Representative images of the centrosome component, γ-tubulin (top), in cells transfected with IC2-FL, -N237 or -N106. GFP staining (to identify overexpressing cells) is shown below. N indicates the nuclei of untransfected control cells in the population. (B) Bar graphs (percentage of cells) illustrating how each expression construct affected the integrity of the microtubule array (MT) or pericentriolar material (pericentrin, γ-tubulin, Arp1, p150). The construct being expressed is indicated below each set of bars. Overexpressing cells were scored as normal if they had radially focused microtubules, one or two perinuclear foci of γ-tubulin, or a perinuclear focus of Arp1, p150^Glued, or pericentrin; anything else was considered abnormal. The numbers given are the percentage of overexpressing cells with normal phenotypes ± SD.

Figure 5.

Effects of dynein IC overexpression on endomembrane localization. HeLa cells were used in this experiment because they exhibit more homogeneous endomembrane localization patterns than COS-7 cells. (A) Representative images of cell populations overexpressing IC2-N237 stained for giantin (Golgi complex; Golgi), ERGIC-53 (endoplasmic reticulum-Golgi intermediate compartment; ERGIC), EEA1 (early endosomes; EE), transferrin receptor (recycling compartment; RE), and LAMP-1 (late endosomes and lysosomes; LE) are shown. GFP staining (to identify overexpressing cells) is shown below. N indicates the nuclei of untransfected control cells in the population. (B) Bar graphs (percentage of cells) illustrating how each expression construct affected endomembrane compartment morphology. The construct being expressed is indicated below each set of bars. Only cells expressing moderate-to-high levels of GFP-tagged protein were counted and of these, only cells showing complete fragmentation or dispersion were scored as abnormal. The numbers given are the percentage of overexpressing cells with normal membrane distribution ± SD.