Journal: Journal of Cell Science

Article Title: The adaptor proteins HAP1a and GRIP1 collaborate to activate the kinesin-1 isoform KIF5C

doi: 10.1242/jcs.215822

Figure Lengend Snippet: GRIP1 and HAP1 form a complex in cells and in brain. (A) Schematic of GRIP1 and HAP1 domains. PDZ, PDZ domain; CC, coiled-coil; A, acidic domain; tail, variable C-terminal tail. (B) C-terminal sequences of rat HAP1a and HAP1b. (C–F) COS cells co-transfected with GFP–GRIP1a and HA–HAP1a show recruitment of GRIP1 to HAP1a puncta (C,E). Yellow line, cell periphery. Scale bars: 10 μm. (D,F) Line scans through the merged images at the section highlighted with the white line; peaks correspond to punctate structures. (G) Western blot (WB) of immunoprecipitation from COS cells co-transfected with GFP–GRIP1a and either HAP1a or HAP1b, immunoprecipitated with anti-GFP antibody. The interaction is specific to HAP1a. (H) Western blot of GRIP1 co-immunoprecipitated with HAP1 from rat brain homogenate.

Article Snippet: Mouse anti-HAP1 (clone 1/HAP1, cat. no. 611302; 1:250) and mouse anti-GRIP1 (clone 32/GRIP, cat. no. 611319; 1:200) were both from BD Biosciences.

Techniques: Transfection, Western Blot, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: The adaptor proteins HAP1a and GRIP1 collaborate to activate the kinesin-1 isoform KIF5C

doi: 10.1242/jcs.215822

Figure Lengend Snippet: HAP1a but not GRIP1 redistributes to the periphery of HeLa cells with KIF5C. (A) Singly transfected HeLa cells showing the distribution of HAP1a, GRIP1 and KIF5C, respectively. Scale bar: 10 µm. (B) KIF5C recruits HAP1a to the periphery of co-transfected HeLa cells, highlighted by white arrowhead. An enlarged area shows superposition of HAP1a and KIF5C puncta. Scale bars: 10 μm (main image) and 2 μm (enlargement). (C) KIF5C is unable to recruit GRIP1 to the periphery of co-transfected HeLa cells. The black arrowhead highlights KIF5C-positive GRIP1-negative peripheral puncta. Scale bars: 10 μm (main image) and 2 μm (enlargement).

Article Snippet: Mouse anti-HAP1 (clone 1/HAP1, cat. no. 611302; 1:250) and mouse anti-GRIP1 (clone 32/GRIP, cat. no. 611319; 1:200) were both from BD Biosciences.

Techniques: Transfection

Journal: Journal of Cell Science

Article Title: The adaptor proteins HAP1a and GRIP1 collaborate to activate the kinesin-1 isoform KIF5C

doi: 10.1242/jcs.215822

Figure Lengend Snippet: GRIP1 can co-complex with KIF5C in the presence of HAP1a through the KIF5 stalk. (A) KIF5C is able to recruit GRIP1 to the periphery of co-transfected HeLa cells when HAP1a is also present. The boxed area is enlarged on bottom row. Scale bars: 10 μm (main image) and 2 μm (enlargement). (B) Plot of cumulative distribution of HAP1a signal according to distance from the centre of a cell (HAP1a probability map). Displacement to the right compared to that in HAP1a only (denoted H) indicates that the HAP1a signal is accumulated further from the centre of the cell. H+K, HAP1a plus KIF5C; H+K+G, HAP1a plus KIF5C and GRIP1a. Analysis was performed from three independent experiments ( n =number of cells; in H, 53; H+K, 43; H+K+G, 39). (C) The distance from the cell centre at which 95% of the HAP1a signal is found. Analysis was performed from three independent experiments ( n =number of cells; in H, 53; H+K, 43; H+K+G, 39). ** P <0.01 (one-way ANOVA test). (D) Plot of the cumulative distribution of GRIP1a signal according to distance from the centre of a cell (GRIP1a probability map). Displacement to the right compared to GRIP1a only (denoted G) indicates that GRIP1a signal is accumulated further from the centre of the cell. G+K, GRIP1a plus KIF5C; G+K+H, GRIP1a plus KIF5C and HAP1a. Analysis was performed from three independent experiments ( n =number of cells; in G, 31; GK, 29; GKH, 35). (E) The distance from the cell centre at which 95% of the GRIP1a signal is found. Analysis was performed from three independent experiments ( n =number of cells in G, 31; G+K, 29; G+K+H, 35). ** P <0.01; *** P <0.001 (one-way ANOVA test). (F) Schematic representation of KIF5 polypeptide chain showing functional regions and constructs used. (G) Western blot of co-IP from COS cells showing that Myc–GRIP1 preferentially binds to the stalk region of KIF5.

Article Snippet: Mouse anti-HAP1 (clone 1/HAP1, cat. no. 611302; 1:250) and mouse anti-GRIP1 (clone 32/GRIP, cat. no. 611319; 1:200) were both from BD Biosciences.

Techniques: Transfection, Functional Assay, Construct, Western Blot, Co-Immunoprecipitation Assay

Journal: Journal of Cell Science

Article Title: The adaptor proteins HAP1a and GRIP1 collaborate to activate the kinesin-1 isoform KIF5C

doi: 10.1242/jcs.215822

Figure Lengend Snippet: Activation of KIF5 motility in vitro requires both HAP1a and GRIP1. (A) Representative motility of KIF5C–Halo in the presence of adaptor proteins as shown by kymographs and corresponding tracking data. (B) Histograms of the track speed of motile particles for each condition; n =39, 63, 66 and 302 for Control, GRIP1, HAP1a and HAP1a and GRIP1, respectively, from four independent experiments. Histograms are also overlaid with a scaled Gaussian kernel density plot (solid line). (C) Landing events per micrometre of microtubule; n =14–16 microtubules from four independent experiments. (D) Motile percentage from the total number of landing events; n =14–16 microtubules from four independent experiments. Box plots are presented as described in Materials and Methods. (E) Example trace of AMP-PNP-immobilised GFP puncta fluorescence intensity bleaching over time. (F) Photobleaching events per molecule. Parameters of fitted Gaussians are listed in Tables S1 and S2 . (G) Model of KIF5 activation by GRIP1 and HAP1. HAP1a binding is insufficient to stabilise the open confirmation on its own, but upon co-addition of GRIP1, KIF5 is robustly activated.

Article Snippet: Mouse anti-HAP1 (clone 1/HAP1, cat. no. 611302; 1:250) and mouse anti-GRIP1 (clone 32/GRIP, cat. no. 611319; 1:200) were both from BD Biosciences.

Techniques: Activation Assay, In Vitro, Fluorescence, Binding Assay