Journal: The Journal of Cell Biology

Article Title: Kinesin-5 inhibitor resistance is driven by kinesin-12

doi: 10.1083/jcb.201507036

Figure Lengend Snippet: KIRC-2 and -3 express a spontaneous Eg5 rigor mutant, Eg5-G268V. (A) Each KIRC line is clonally distinct. Unsupervised cluster and heatmap analysis of indicated cell types. The dendrogram (bottom) shows that KIRC-1 and KIRC-2 are most similar based on gene expression. HeLa is more similar to KIRC-1 and KIRC-2 than KIRC-3. KIRC-3 is the most distinct cell line. (B) Eg5 binds spindle MTs in KIRC-2 and -3 cells despite the presence of STLC. Single optical sections of indicated cell types stained with antibodies targeting Eg5 (grayscale and red) and tubulin (green). DNA, blue. Lookup tables (LUTs) for grayscale and red channel are scaled identically. Bar, 10 µm. See also Fig. S1 . (C) Purification of recombinant Eg5 proteins. (left) Protein gel of Eg5-WT and Eg5-G268V preparations stained with Coomassie-blue. (right) Western blot of Eg5-WT and Eg5-G268V preparations probed with antibodies targeting Eg5 (αEg5). (D) Eg5-G268V is immotile. Kymographs of single XR-MTs in gliding assays propelled by Eg5-WT or Eg5-G268V. Bars: (x axis) 1 µm; (y axis) 1 min. See also Videos 1 and 2 . (E) Histogram showing gliding velocities from D. Values represent the number of MTs observed to move at indicated velocities in gliding assays powered by Eg5-WT (green) or Eg5-G268V (red). Velocities are reported in micrometers per minute and binned every 0.1 µm/min. n = 300 from three independent experiments. Distributions of Eg5-WT velocities are fit to a single Gaussian (R 2 = 0.91). (F) Eg5-G268V does not release from MTs in the presence of ATP. Blot of pellet (P) and supernatant (S) fractions from reactions containing Eg5-WT or Eg5-G268V with tubulin or microtubules in the presence of DMSO or FCPT as indicated, probed with antibodies targeting Eg5 (αEg5) and tubulin (DM1α).

Article Snippet: For display ( and S3 B), Gaussian fits to the binned data were calculated with SigmaPlot (Systat Software).

Techniques: Mutagenesis, Gene Expression, Staining, Purification, Recombinant, Western Blot

Journal: The Journal of Cell Biology

Article Title: Kinesin-5 inhibitor resistance is driven by kinesin-12

doi: 10.1083/jcb.201507036

Figure Lengend Snippet: Enhanced spindle MT bundling in KIRC-2 and -3 cells affects Kif15 partitioning. (A) Kif15 mislocalizes to non–K-MTs in KIRC-2 and -3 cells. Maximum intensity z-projections of indicated cell types 2 d after transfection with nontargeting (Control) or Nuf2-targeting (-Nuf2) siRNAs and stained with antibodies targeting Kif15 (grayscale and red) and tubulin (green). DNA, blue. LUTs for grayscale and red channels are scaled identically. Bar, 10 µm. (B) Kif15 protein levels are not elevated in KIRC-2 or -3. Western blot of WCLs prepared from indicated cell types probed with antibodies targeting Kif15 (αKif15) and tubulin (DM1α). (C) Quantitation of B. Values represent the levels of Kif15 protein in indicated cell types normalized to the parental HeLa line. n = 2. Error bars, ± SEM; *, P ≤ 0.1 relative to parental HeLa line. (D) Cellular Kif15 primarily organizes into dimers. Histogram showing initial single-molecule fluorescence intensities of EGFP-Kif15 (red) from HeLa cell extracts. XMAP215-EGFP (green) represents monomer control. EGFP-HSET (blue) represents dimer control. Intensities are indicated in au × 10 −3 . n ≥ 1945 particles from ≥ 3 fields of view. Single Gaussian fits of fluorescence intensity distributions are overlayed. XMAP215-EGFP (green, R 2 = 0.96), EGFP-HSET (blue, R 2 = 0.90), and EGFP-Kif15 (red, R 2 = 0.93). (E) Example traces showing photobleaching steps of single EGFP-Kif15 molecules from HeLa cell extracts. XMAP215-EGFP (green) represents monomer control. EGFP-HSET (blue) represents dimer control. Two example traces are shown for EGFP-Kif15 (red, cyan) highlighting the variability observed in photobleaching steps. Fluorescence intensities are indicated in au × 10 −3 , and the baseline is set to the background fluorescence. See also Fig. S3 .

Article Snippet: For display ( and S3 B), Gaussian fits to the binned data were calculated with SigmaPlot (Systat Software).

Techniques: Transfection, Control, Staining, Western Blot, Quantitation Assay, Fluorescence

Journal: Scientific Reports

Article Title: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

doi: 10.1038/s41598-019-42331-6

Figure Lengend Snippet: PAS-mutant hERGs are sorted for lysosomal delivery from the cell surface. ( a ) hERG is targeted to LAMP1-positive endo-lysosomal compartments. Endocytic WT, M124R and C64Y hERG pool labelled by Ab capture (15 min at 37 °C) and remaining cell-surface hERG blocked with unconjugated secondary F(ab′) 2 (1 h on ice). Cells then chased at 37 °C for 3 h prior to fixation. Lysosomal compartments labelled with LAMP1 pAb. hERG (green) and LAMP1 (magenta) staining visualized by LCFM. Whole-cell (scale bar: 10 µm, left) and high-magnification (scale bar: 5 µm, right) images shown. Magnified area indicated by white box. Analysis of additional mutants (F29L, R56Q, T65P) in Supplementary Fig. . ( b ) Representative distribution of vesicular pH for WT and T65P hERG containing endocytic vesicles following 3 h chase. Overlay of multi-Gaussian peak-fits shown and mean pH ± SD indicated. N indicates total number of vesicles analyzed in a representative experiment. ( c ) PAS-mutations accelerate hERG endo-lysosomal delivery kinetics. Mean luminal pH of vesicles containing WT or T65P hERG measured by FRIA. Anti-HA Ab and FITC-Fab were bound on ice and FRIA was performed after 1- to 6-h chase. ( d ) Mean luminal pH of vesicles containing WT and PAS-mutant hERG following 3 h chase. ( e , f ) Lysosomal activity contributes to degradation of mature hERG proteins. Metabolic stability of WT and PAS-mutants hERG evaluated by immunoblotting following translational inhibition with cycloheximide (CHX, 150 µg/ml). V-ATPase inhibition with Bafilomycin A1 (BafA1, 200 nM), or proteasome inhibition with Bortezomib (Bort, 3 µM) or Ixazomib (Ixa, 3 µM) attenuated the rapid degradation of PAS-mutants. Mature complex-glycosylated (~155 kDa) and ER-resident core-glycosylated (~135 kDa) hERG indicated by solid and empty arrows, respectively. Representative immunoblots shown (uncropped images in Supplementary Fig. ). Solid line: different parts of the same gel. White space: separate gels. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See methods and materials for explanation of statistical analysis).

Article Snippet: Multiple-Gaussian fits for hERG vesicular pH performed using Origin (OriginLab).

Techniques: Mutagenesis, Staining, Activity Assay, Western Blot, Inhibition

Journal: Scientific Reports

Article Title: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

doi: 10.1038/s41598-019-42331-6

Figure Lengend Snippet: Peripheral quality control engagement is dependent on conformational destabilization. ( a ) Mature hERG is destabilized at elevated temperature. Metabolic stability of mature WT, PAS-mutant (F29L and T65P) or temperature-rescued G601S (48 h at 26 °C, rG601S) hERG evaluated at 37 °C or 41 °C by immunoblotting following translational inhibition with cycloheximide (CHX, 150 µg/ml). Representative immunoblots shown (uncropped images in Supplementary Fig. ). Solid line: different parts of the same gel. White space: separate gels. ( b ) Turnover kinetics of mature WT and F29L hERG fit using single-exponential decay functions. Similar results obtained for T65P and rG601S hERG (Supplementary Fig. ). ( c ) Turnover rate-constants determined by curve fitting as in ( b ) and expressed as fold increase relative to 37 °C. ( d ) Pharmacological correction of hERG folding restores cell-surface stability. PM-turnover of WT and select PAS-mutants hERG measured by cell-surface ELISA following overnight (16 h) E4031 treatment (10 µM). ( e ) Pharmacochaperone treatment improves folding of nascent hERG at the ER but does not promote refolding of mature channels at the PM. Internalization of WT and select PAS-mutant hERG measured by PM-ELISA following acute (1 h) or overnight (16 h) E4031 pre-treatment (10 µM). ( f ) Delivery of PM-labelled T65P hERG to LAMP1-positive compartments evaluated by LCFM following 3 h chase. Lysosomal delivery is prevented by overnight pre-treatment with E4031 (10 µM). Whole-cell (scale bar: 10 µm, left) and high-magnification (scale bar: 2 µm, right) images shown. Magnified area indicated by white box. Analysis of WT and additional PAS-mutants in Supplementary Fig. . ( g ) Pharmacochaperone pre-treatment prevents endo-lysosomal trafficking of T65P hERG. Representative histogram of T65P hERG vesicular pH following 3 h chase. Overlay of multi-Gaussian peak-fits (mean ± SD) shown. N indicates total number of vesicles evaluated. ( h ) Mean luminal pH of hERG-containing endocytic vesicles measured by FRIA following overnight treatment with E4031 (10 µM) and 3 h chase at 37 °C. ( i ) Subset of temperature-rescued PAS-mutants are resistant to unfolding at physiological temperature. Internalization of WT and PAS-mutant hERG measured by PM-ELISA following low-temperature rescue (30 °C for 24 h) and unfolding (37 °C for 2 h). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference (See Methods for explanation of statistical analysis).

Article Snippet: Multiple-Gaussian fits for hERG vesicular pH performed using Origin (OriginLab).

Techniques: Control, Mutagenesis, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay