Journal: Nature Communications

Article Title: A conserved interaction of the dynein light intermediate chain with dynein-dynactin effectors necessary for processivity

doi: 10.1038/s41467-018-03412-8

Figure Lengend Snippet: The conserved Helix-1 within the LIC1-effector-binding domain binds the Hook domain. a Alignment of LIC sequences from different species and isoforms around the predicted Helix-1 within the C-terminal effector-binding domain (top) and domain diagram of human LIC1 showing the constructs used in this study (bottom). The name of each sequence includes the organism of origin and UniProt accession code. Yellow and orange backgrounds indicate 70% and 100% sequence conservation, respectively. Red stars highlight residues F447 and F448 that were mutated to alanine. The predicted Helix-1 and Helix-2, coinciding with regions of higher sequence conservation (see Supplementary Fig. ), are highlighted in the domain diagram, and Helix-1 is also depicted above the sequence alignment. The region corresponding to the Helix-1 (LIC1 433–458 ) peptide is contoured red. b – g ITC titrations of Hook1 11–166 and Hook3 1–160 into LIC1 constructs (as indicated). Listed with each titration are the concentrations of the protein in the syringe and in the cell, as well as the temperature of the experiment and parameters of the fit (stoichiometry N , dissociation constant K D ). Errors correspond to the s.d. of the fits. Open symbols correspond to control titrations into buffer

Article Snippet: Constructs Hook3 1–143 and Hook3 1–160 were cloned between NotI and SalI sites of a modified pMAL-c2x (NEB) vector in which the Sac1 site after MBP residue N367 was replaced with a NotI site.

Techniques: Binding Assay, Construct, Sequencing, Titration

Journal: Nature Communications

Article Title: A conserved interaction of the dynein light intermediate chain with dynein-dynactin effectors necessary for processivity

doi: 10.1038/s41467-018-03412-8

Figure Lengend Snippet: Crystal structure of the Hook domain in complex with LIC1 Helix-1. a Ribbon and surface representation of the structure of Hook3 1–160 (magenta) in complex with Helix-1 (LIC1 433–458 , blue). The side chains of Helix-1 are shown using a sticks representation, colored by atom type. b Close-up view of the Helix-1 binding site, showing the 2 F o- F c electron density map (blue mesh) at 1.5 Å resolution, contoured at 1 σ around an all-atom representation of Helix-1. c Close-up view of the Helix-1 binding site, showing the residues at the hydrophobic contact interface. d Superimposition of the structure of the Hook domain from the Helix-1-bound complex (magenta) and unbound structure (gray) . A conformational change in the C-terminal helix α8, which distinguishes this domain from the CH domain, leads to the formation of two helices (α8a and α8b) that constitute the binding site for Helix-1. e Sequence conservation of the Hook domain (see also Supplementary Fig. ) mapped onto the surface of the structure and colored from low to high conservation using a red to green gradient. In the crystal lattice, the Hook domain contacts a second Helix-1 from a neighboring complex (light blue). f Surface representation of the Hook domain (magenta), showing in yellow the two amino acids mutated (A138D and M140D) to test the functional relevance of the two Helix-1 interactions. g – i ITC titrations of the indicated Hook3 1–160 mutants into MBP-LIC1 FL . Experimental conditions and fitting parameters are listed. Errors correspond to the s.d. of the fits. Open symbols correspond to titrations into buffer

Article Snippet: Constructs Hook3 1–143 and Hook3 1–160 were cloned between NotI and SalI sites of a modified pMAL-c2x (NEB) vector in which the Sac1 site after MBP residue N367 was replaced with a NotI site.

Techniques: Binding Assay, Sequencing, Functional Assay

Journal: Nature Communications

Article Title: A conserved interaction of the dynein light intermediate chain with dynein-dynactin effectors necessary for processivity

doi: 10.1038/s41467-018-03412-8

Figure Lengend Snippet: The Helix-1-effector interaction is important for processive motility in vitro and in cells. a , b Time series and kymographs (1 min) of Halo-Hook3 1–552 and Halo-BICD2 1–572 runs on microtubules (magenta) in the absence (control) or the presence of Helix-1 or Helix-1 F447A,F448A peptides (as indicated) analyzed by TIRF microscopy. Arrows indicate a motile particle and arrowheads indicate the beginning and end of the trajectory in a maximum projection (max). Scale bar, 5 μm. Quantifications (right) show that the number of motile events declines with increasing Helix-1 concentrations, but not Helix-1 F447A,F448A . The statistical significance of the measurements was determined using a One-way Anova test, analyzing N = 6–21 videos and a minimum of 3 individual cell lysates per condition (n.s., non-significant; * p ≤ 0.05; ** p ≤ 0.01; *** p < 0.001). Error bars correspond to the s.e.m. c Representative images of LAMP1 staining of fixed HeLa cells expressing GFP, LIC1 WT -GFP or LIC1 F447A,448A -GFP. Note that the LAMP1 puncta become more dispersed with the expression of LIC1 F447A,448A -GFP, but not LIC1 WT -GFP. Cell perimeters are outlined in white. Scale bar, 10 μm. d Percentage of cells with abnormal LAMP1 staining from fixed HeLa cells expressing GFP, LIC1 WT -GFP and LIC1 F447A,448A -GFP. The statistical significance of the measurements was determined using a One-way Anova test, analyzing N = 148 (GFP), N = 77 (LIC1 WT -GFP), and N = 48 (LIC1 F447A,448A -GFP) cells from three independent repeats (n.s., non-significant; * p ≤ 0.05; ** p ≤ 0.01). Error bars correspond to the s.e.m

Article Snippet: Constructs Hook3 1–143 and Hook3 1–160 were cloned between NotI and SalI sites of a modified pMAL-c2x (NEB) vector in which the Sac1 site after MBP residue N367 was replaced with a NotI site.

Techniques: In Vitro, Microscopy, Staining, Expressing