Fig 1.
Behaviors of DDB complexes with different DCTN1 isoforms.
(a) N-terminal regions of DCTN1 isoforms. Amino acid numbering is based on p150glued. (b) Kymographs of dynein movement on microtubules in the presence of 1 nM Alexa 647-labeled dynein, 2 nM 1A or 1B isoforms, and 20 nM BICD2. (c) Quantification of the behavior of dynein molecules on microtubules. The quantification data are presented as segmented vertical bars: the fraction of diffusive (red), stationary (blue), or unidirectional (green) dynein molecules on microtubules. Quantification of dynein molecules on microtubules in the presence of each dynactin isoform or mutant. The total residence time of dynein on microtubules under the condition of 1 nM Alexa 647-labeled dynein and 2 nM 1A or 1B isoforms. Mean ± S.D., n = 9 windows. (d) Histograms of the velocity and run length of unidirectional movement of the DDB complex (1A). Mean ± S.D., n = 168 particles (velocity) and n = 80 particles (run length).
Fig 2.
Behaviors of DD complexes with different DCTN1 isoforms.
(a) Kymographs of dynein movement on microtubules in the presence of each dynactin isoform. (b) The total residence time of dynein on microtubules was the same as that in Fig 1c under the condition of 1 nM Alexa 647-labeled dynein and 2 nM of each dynactin isoform. Mean ± S.D., n = 18 windows (dynein alone), n = 15 windows in the presence of 1A or 1B isoforms, n = 30 windows in the presence of vanadate, n = 9 windows in the presence of the p135 isoform. The total residence time of dynein under each condition was altered significantly compared with dynein alone. Quantification of the behavior of dynein molecules on MTs is presented as segmented vertical bars: the fraction of diffusive (red), stationary (blue), or unidirectional (green) dynein molecules on microtubules. The total residence time of dynein on microtubules was the same as that in Fig 1b. Mean ± S.D. (c) Histograms of the velocity and run length of unidirectional movement of dynein molecules in the presence of the 1A isoform. n = 92 particles. (d) Duration of dynein interacting with MTs in the presence of 1B. n = 23 particles (1B isoform).
Fig 3.
The CC1 fragment binds to dynein and inhibits microtubule binding of dynein.
(a) Mutant of the 1B isoform and the CC1 fragment. Amino acid numbering is based on p150glued. (b) Interactions between dynein and dynactin determined by TALON Dynabeads pull-down assays of purified proteins. The protein bands of dynein heavy chain (DHC) in SDS-PAGE gels were quantified by densitometry. (c) Binding ratio of dynein with the 1B isoform (○), CC1 fragment (●), and 1BΔCC1 mutant (▼). (d) Kymographs of dynein motility on microtubules in the presence of the 1BΔCC1 mutant or CC1 fragment. (e) Quantification of dynein molecules on microtubules in the presence of the 1BΔCC1 mutant or CC1 fragment. The total residence time of dynein on microtubules was the same as that in Fig 1b under the condition of 1 nM Alexa 647-labeled dynein and 2 nM 1BΔCC1 mutant or CC1 fragment. Mean ± S.D., n = 15 windows.
Fig 4.
Single molecule behavior of dynactin on microtubules.
(a) Mutant of the 1B isoform and the fragments of DCTN1. Amino acid numbering is based on p150glued. (b) Kymographs of dynactin including each isoform and mutant behavior on microtubules. (c) Quantification of each isoform and mutant on microtubules. The total residence time of dynactin on microtubules was the same as that in Fig 1b. Mean ± S.D., n = 9 windows (1A isoform, 1BΔCC1 mutant, 1BN-GCN4 fragment, CC1 fragment and p135 isoform) and n = 12 windows (1B isoform). (d) Time course of displacement of 1A (left panel) and 1B (right panel) isoforms on microtubules. The diffusion coefficients of 1A and 1B isoforms were 55.4 × 102 and 1.9 × 102 nm2/s, respectively. (e) Duration of 1B isoforms on microtubules. n = 454 particles.
Fig 5.
Relationships between regulatory domains.
Left: The CC1 domain inhibits the microtubule-binding ability of the Cap-Gly domain within the same molecule, and this inhibition is repressed by the K-rich domain. Right: The effect on dynactin itself still remains. The CC1 domain, which is bound to dynein, inhibits the microtubule-binding ability of the DD complex, and this inhibition is repressed by the K-rich domain.