Reconstitution of dynein transport to the microtubule plus end by kinesin
- Harvard Medical School, United States
- School of Molecular and Cellular Biology, University of Leeds, United Kingdom
Figures
Figure 1 with 1 supplement
Purification and analysis of the putative dynein plus-end transport machinery.
(A) SDS-PAGE and diagrams of purified proteins. Kip2 from S. cerevisiae migrates as a doublet of bands, corresponding to differently phosphorylated isoforms as verified by phosphatase treatment …
Figure 1—figure supplement 1
Analysis of interactions between Bik1, Bim1 and Kip2 by size-exclusion chromatography.
The void volume (V0) and elution volumes of the individual proteins are shown on the top axis. SDS-PAGE analysis of the corresponding fractions is shown below, pseudo-colored to match the traces. …
Figure 2 with 3 supplements
Reconstitution of dynein transport to the microtubule plus end.
(A) Diagram of dynamic microtubule assay, after Bieling et al. (2007). A bright, biotinylated, GMP-CPP-stabilized microtubule seed is attached to the coverslip via streptavidin and biotin-PEG. Dimly …
Figure 2—figure supplement 1
Examples of plus-end-directed motion of GST-Dynein331 kDa and full-length dynein.
Dynein at the growing microtubule plus end is marked with arrowheads. Plus (+) and minus (−) denote microtubule polarity.
Figure 2—figure supplement 2
Weakening dynein's microtubule affinity increases the velocity of its plus-end-directed transport.
(A) Four charged amino acids were mutated to weaken dynein's microtubule affinity (K3116A, K3117A, E3122A and R3124A). These residues are depicted in red sphere representation using the cryo-EM …
Figure 2—figure supplement 3
Kymographs showing colocalized, plus-end-directed runs of weak dynein and Kip2 in the presence of Bik1 and Lis1.
Plus (+) and minus (−) denote microtubule polarity.
Figure 3 with 2 supplements
Bik1 and Bim1 are Kip2 processivity factors.
(A) Top: diagram of Kip2, Bik1 and Bim1 domain structure. Arrows indicate reported and putative interactions (see main text for details). Bottom: Kip2 contains an SxIP motif (orange) within an …
Figure 3—figure supplement 1
While increasing Kip2 processivity, Bik1 and Bim1 confer a small reduction in Kip2 velocity and do not change Kip2 copy number.
(A) Impact of Bik1/Bim1 on dynein and Kip2 velocity on Taxol-stabilized microtubules. Graphs show mean velocity ± SEM (N = 175–331 events per condition). Bik1 and Bim1 together confer a small …
Figure 3—figure supplement 2
Bik1 and Bim1 increase Kip2’s microtubule on-rate and decrease its off-rate.
(A) Left: kymographs showing Kip2 at a fixed concentration in the absence or presence of Bik1 and Bim1. Right: quantification of Kip2 landing rate on the microtubule, expressed as events per micron …
Figure 4 with 1 supplement
Bik1 and Bim1 regulate the outcome of a tug-of-war between dynein and Kip2.
(A) Coupling dynein and Kip2 to a DNA origami ‘chassis’ allows them to be pitted directly against each other in a tug-of-war. Single-stranded DNA oligonucleotides were attached to dynein and Kip2 …
Figure 4—figure supplement 1
Dynein and Kip2 engage in a tug-of-war when coupled via the DNA origami chassis.
Graph shows the average velocity ± SD of chassis movements toward the microtubule minus end (gray bars) or plus end (purple bars). 1D denotes a chassis with one dynein-attachment site. 1K denotes a …
Figure 5
Model of the minimal machinery for transporting dynein to the microtubule plus end.
Based on results from this study and earlier work. Dynein is connected to the plus-end-directed motor Kip2 by Lis1 and Bik1. Specifically, Lis1's ß-propeller domain binds to the AAA4 subdomain of …
Tables
Table 1
Protein homolog names
| Generic | S. cerevisiae | S. pombe |
|---|---|---|
| Kinesin | Kip2 | Tea2 |
| Clip170 | Bik1 | Tip1 |
| EB1 | Bim1 | Mal3 |
| Lis1 | Pac1 | – |
Table 2
Yeast strains used in this study
| Strain | Genotype | Used to purify | Source |
|---|---|---|---|
| RPY753 | PGAL1-ZZ-Tev-GFP-3xHA-GST-DYN1331kDa-gs-DHA-KanR; pac1Δ::klURA3; ndl1Δ::cgLEU2 | GST-dynein331 kDa | (Huang et al., 2012) |
| RPY780 | ZZ-Tev-3xHA-DYN1-gs-DHA-KanR nip100Δ pac1Δ::HygroR ndl1Δ::cgLEU2 | Full-length dynein | (Huang et al., 2012) |
| RPY816 | PGAL1-8HIS-ZZ-Tev-PAC1; dyn1D::cgLEU2; ndl1D::HPH | Lis1 | Julie Huang |
| RPY1084 | PGAL1-ZZ-Tev-GFP-3xHA-SNAP-gs-GST-DYN1331kDa-gs-DHA-KanR | Dynein for DNA labeling | (Derr et al., 2012) |
| RPY1099 | PGAL1-8HIS-ZZ-Tev-KIP2-g-FLAG-ga-SNAP−KanR | Kip2 | This work |
| RPY1235 | PGAL1-ZZ-Tev-GFP-3xHA-GST-DYN1E3197K-gs-DHA-KanR | Polarity marker dynein for chassis experiments | (Redwine et al., 2012) |
| RPY1536 | PGAL1-ZZ-Tev-GFP-3xHA-GST-DYN1K3116A, K3117A, E3122A, R3124A-gs-DHA-KanR | Weak dynein | This work |
-
All strains were made in the W303a background (MATa his3-11,15 ura3-1 leu2-3,112 ade2-1 trp1-1) with genes encoding the proteases Pep4 and Prb1 deleted (prb1Δ; pep4Δ::HIS5). DHA and SNAP refer to the HaloTag (Promega) and SNAP-tag (NEB), respectively. Tev indicates a Tev protease cleavage site. PGAL1 denotes the galactose promoter, which was used to induce protein expression. Amino acid spacers are indicated by g (glycine), ga (glycine–alanine), and gs (glycine–serine).
Additional files
-
Supplementary file 1
ImageJ macro used to calculate motor velocities and run lengths from kymographs.
- https://doi.org/10.7554/eLife.02641.017
