Combining Structure-Function and Single-Molecule Studies on Cytoplasmic Dynein

Abstract

Cytoplasmic dynein is the largest and most intricate cytoskeletal motor protein. It is responsible for a vast array of biological functions, ranging from the transport of organelles and mRNAs to the movement of nuclei during neuronal migration and the formation and positioning of the mitotic spindle during cell division. Despite its megadalton size and its complex design, recent success with the recombinant expression of the dynein heavy chain has advanced our understanding of dynein's molecular mechanism through the combination of structure-function and single-molecule studies. Single-molecule fluorescence assays have provided detailed insights into how dynein advances along its microtubule track in the absence of load, while optical tweezers have yielded insights into the force generation and stalling behavior of dynein. Here, using the S. cerevisiae expression system, we provide improved protocols for the generation of dynein mutants and for the expression and purification of the mutated and/or tagged proteins. To facilitate single-molecule fluorescence and optical trapping assays, we further describe updated, easy-to-use protocols for attaching microtubules to coverslip surfaces. The presented protocols together with the recently solved crystal structures of the dynein motor domain will further simplify and accelerate hypothesis-driven mutagenesis and structure-function studies on dynein.

Keywords: Cytoplasmic dynein; Fluorescence labeling; Microtubule immobilization; Microtubule motor proteins; Microtubules; Optical trapping; Optical tweezers; Recombinant proteins; Single-molecule assays; Yeast gene manipulation.

Figure 1

(a) Scheme of PCR-mediated two-step yeast transformation. During the first transformation, the targeted gene (yellow) is replaced by linear URA3 gene (pink) with flanking DNA (striped). The flanking DNA overlaps with yeast’s endogenous sequences so it can be integrated into yeast genome through homologous recombination. In the second step, the PCR product containing the target gene with intended modifications (green) replaces the URA3 in the genome via 5-FOA selection. (b) Scheme of primer design for generating URA3-containing PCR product. To generate a sequence with flanking DNA that overlaps with yeast genome, primer pairs that are upstream and downstream of the targeted sequence are obtained. The target sequence (yellow) together with ~500 bp both up- and downstream is selected, and the sequence is run through a primer design tool to obtain forward and reverse primers that are within ~150–300 bp from the targeted sequence (“F” and “R”). Next, pairs of primers are designed to stitch the flanking yeast sequence with URA3. To generate the stitching primer for upstream, 20–30 bp upstream of the 5′ end of the targeted site is combined with the first 20–30 bp of the 5′ end of the URA3 (pink), and then converted into its complementary sequence (“R-stitch” primer). To make the downstream stitching primer, the last 20–30 bp of the 3′ end of URA3 is combined with 20–30 bp downstream of the targeted site (“stitch-F” primer). Using yeast genome as template, the 5′-flanking sequence (along with the first 20–30 bp of the URA3 gene) can now be amplified with the “F” and “R-stitch” primers by PCR, while the 3′-flanking region can be amplified with the “stitch-F” and “R” primers. “UF” (5′-GTGATTCTGGGTAGAAGATCGG) and “UR” (5′-CGATGATGTAGTTTCTGGTTTTTAA) primers are used to amplify URA3 with its promoter and terminator. The final PCR product can then be generated using “F” and “R” as primers and the mixture of 5′-flanking DNA, 3′-flanking DNA, and URA3 as template. The same scheme applies for the second step of the yeast transformation, wherein the modified gene of interest (green in a) replaces the URA3 gene via homologous recombination.

Figure 2

(a) A representative SDS-PAGE 4–12% gradient gel for dynein purification. The dynein is a full-length dynein expressed behind its native promoter (Dyn1_471kDa) [65]. Due to the low concentration of native dynein, its associated subunits generally are not visible by InstantBlue staining. The lower bands in lane “B” are due to the IgG antibodies. L: molecular ladder, LY: lysate, FT: flow-through, W1: wash 1, W2: wash 2, S: sample, B: beads. (b) A representative SDS-PAGE 4–12% gradient gel for dynein microtubule binding release purification. In this example, the dynein is a truncated construct with an N-terminal GST tag (Dyn1_331kDa) [65]. L: ladder, S: sample, S1: supernatant 1, P1: pellet 1, S2: supernatant 2, P2: pellet 2.

Figure 3

(a) Scheme of microtubule immobilization via BSA-biotin-streptavidin attachment. BSA-biotin (green) adsorbs to a coverslip surface, followed by streptavidin (orange) that is bound to biotin. MTs with biotinlyated tubulin are then immobilized via streptavidin. (b) Scheme of microtubule immobilization via PLL. PLL (yellow) adsorbs to a coverslip surface, followed by Tween-20 (purple), which blocks the surface. MTs are immobilized via electrostatic interactions with the amine groups of PLL. (C) For optical trapping assays, the amine groups of PLL can be sparsely labeled with NHS-biotin, and biotinylated MTs can be further tightly attached via streptavidin.

Figure 4

(a) Micrograph of MTs attached to a coverslip surface via BSA-biotin-streptavidin attachment. (b) Micrograph of MTs attached to a coverslip using PLL.

Figure 5

(a) A representative kymograph of full-length dynein. The acquisition rate was 500 ms/frame. (b) A representative stall trace of full-length dynein. The spring constant k was 0.045 pN/nm.