Reconstitution of the human cytoplasmic dynein complex

Abstract

Cytoplasmic dynein is the major motor protein responsible for microtubule minus-end-directed movements in most eukaryotic cells. It transports a variety of cargoes and has numerous functions during spindle assembly and chromosome segregation. It is a large complex of about 1.4 MDa composed of six different subunits, interacting with a multitude of different partners. Most biochemical studies have been performed either with the native mammalian cytoplasmic dynein complex purified from tissue or, more recently, with recombinant dynein fragments from budding yeast and Dictyostelium. Hardly any information exists about the properties of human dynein. Moreover, experiments with an entire human dynein complex prepared from recombinant subunits with a well-defined composition are lacking. Here, we reconstitute a complete cytoplasmic dynein complex using recombinant human subunits and characterize its biochemical and motile properties. Using analytical gel filtration, sedimentation-velocity ultracentrifugation, and negative-stain electron microscopy, we demonstrate that the smaller subunits of the complex have an important structural function for complex integrity. Fluorescence microscopy experiments reveal that while engaged in collective microtubule transport, the recombinant human cytoplasmic dynein complex is an active, microtubule minus-end-directed motor, as expected. However, in contrast to recombinant dynein of nonmetazoans, individual reconstituted human dynein complexes did not show robust processive motility, suggesting a more intricate mechanism of processivity regulation for the human dynein complex. In the future, the comparison of reconstituted dynein complexes from different species promises to provide molecular insight into the mechanisms regulating the various functions of these large molecular machines.

Fig. 1.

Reconstitution of the human cytoplasmic dynein complex. (A) Schematic of the subunit composition of the dynein complex studied here. A His₆ tag, followed by mGFP (green), is fused to the N terminus of the CDHC (Fig. S1A, Top). (B) Purification scheme: lysates of cells expressing His₆-mGFP-CDHC (C, lane 1; 560 kDa; referred to here as CDHC) and cells expressing IC1 (C, lane 2; 71 kDa) were mixed, supplemented with purified LIC2 (D, lane 1; 54 kDa), and subjected to immobilized metal-ion–affinity chromatography (IMAC). The eluate was supplemented with purified light chains (D, lanes 2–4; 10–13 kDa) and gel-filtered (GF). (C and D) Coomassie-stained SDS gels showing lysates of cells expressing CDHC and IC1 (C) and purified LIC2 and light chains (D), as indicated. (E) Gel-filtration profile around the position where the dynein complex elutes showing normalized absorbance values at 280 nm (black line) and 488 nm (green line). The fraction between the red lines was collected and analyzed. (F) Coomassie-stained (Left) and SYPRO Ruby–stained (Right) SDS gel showing the dynein complex after gel filtration.

Fig. 2.

Oligomerization state and subunit stoichiometry of the reconstituted human dynein complex. (A, Upper) Sedimentation coefficient distribution [g(s*) plot] of the dynein complex. Gaussian functions (lines) were fitted to the data (blue circles), with the peak center at 22S for the major species (82%) of the dynein complex (red line). (A, Lower) Comparison of normalized fits to the g(s*) plots of the dynein complex (red; as above), the dynein motor domain (Dyn380kD, green; Fig. S2A, Upper), and the dynein motor domain fused to a dimerizing coiled coil (cc) sequence (cc-Dyn380kD, blue; Fig. S2A, Lower). (B, Upper) Analytical gel-filtration profile of the dynein complex. Gaussian fits (colored lines) to the data (black line) reveal a peak center at the K_av value of 0.04 (corresponding to Stokes radius, R_s, of 17 nm) for the main eluate (red line). The blue line shows the sum of the two Gaussian curves. (B, Lower) Comparison of normalized fits to the gel-filtration profiles of the dynein complex and truncated dynein constructs (color code as in A, Lower; see also Fig. S2B). (C) Summary of results from the sedimentation-velocity ultracentrifugation [s(20,w)] and analytical gel filtration (Stokes radius) and calculated partial specific volumes for the dynein complex, CDHC alone, and truncated control constructs. Mw, molecular mass. Errors are SEM. (D) Summary of the mean molar ratios of subunits in the reconstituted dynein complex. Errors are SEM. For experimental data, see Fig. S3.

Fig. 3.

EM images of negatively stained dynein constructs. Overview of samples (large images) and galleries of single dynein particles (small images): motor domain Dyn380kD (A); artificially dimerized motor domains cc-Dyn380kD (B); dynein complex (C); and CDHC (D). Scale is the same for all single particles images in A–D. (E) Histogram of the distribution of the Dyn380kD motor domain (MD) diameter [measured as indicated by red arrowheads in the EM image (30 × 30 nm)]. (F) Histograms of the distribution of the dynein complex MD diameter (Left) and the tail domain length (Right) [measured as indicated on the EM image (60 × 80 nm) by red and yellow arrowheads, respectively]. Indicated are mean values ± SD. d, diameter; l, length.

Fig. 4.

Single-molecule behavior of human dynein. (A–C, Upper) Schematic of the assay showing individual mGFP-labeled dyneins on fluorescent MTs that are imaged using TIRFM. (A–C, Lower) Exemplary kymographs (space–time plots) show individual monomeric Dyn380kD (A), dimeric cc-Dyn380kD (B), and dynein complex (C) molecules interacting with MTs in the presence of ATP or AMP-PNP, as indicated. (D) Total number of binding events per minute and per concentration of motor (nM) and MT length (µm). Error bars are SEM. Number N of independent experiments: dynein complex with ATP (n = 4) and AMP-PNP (n = 2), cc-Dyn380kD with ATP (n = 3) and AMP-PNP (n = 2), and Dyn380kD with ATP and AMP-PNP (both n = 2). The motor concentrations in this figure refer to monomers for Dyn380kD and dimers for cc-Dyn380kD and the dynein complex.

Fig. 5.

Dynein motility in MT-gliding assays. (A) Schematic of the assay on Tris-Ni-NTA-PEG–functionalized glass: oligo-His–tagged motors are immobilized on a Tris-Ni-NTA–functionalized surface in an oriented manner. Fluorescently labeled MTs are imaged by TIRFM. (B) Exemplary kymograph of a polarity marked MT transported by the dynein complex. (C–E) Histograms showing MT-gliding velocity distributions of the dynein complex (C), Dyn380kD (D), and cc-Dyn380kD (E). (F) Histogram showing the MT-gliding velocity distribution of the dynein complex immobilized on nonfunctionalized glass. Gaussian fits (red lines) with mean velocity (v) ± SD as indicated. (G) Landing-rate profile for the dynein complex (red stars) immobilized on nonfunctionalized glass and Dyn380kD (green circles) and cc-Dyn380kD (blue diamonds) both immobilized on Tris-Ni-NTA–functionalized glass. The density of immobilized motor is expressed as the measured mGFP intensity of the dynein constructs. The continuous curves are fits to the data (SI Materials and Methods) yielding the minimal number n of motors required to support MT gliding.