Journal: Cellular signalling

Article Title: Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y(1) receptors.

doi: 10.1016/j.cellsig.2010.09.007

Figure Lengend Snippet: Fig. 4. Co-expressed Rab5aS34N or down-regulation of all three Rab5 isoforms by siRNAs decrease constitutive internalization of Y1Δ32 receptors. A) Western blots probed with an anti-Rab5a antibody revealed endogenous Rab5a (A, control HEK293 cells; B, Y1 cells; C, Y1Δ32 cells; D, Y1Δ32 cells co-transfected with mRFP1-Rab5aS34N) and exogenous Rab5aS34N (D). B) Confocal images showing EGFP-tagged Y1Δ32 receptors and mRFP1-Rab5aS34N expression. Note the apparent retention of Y1Δ32 at the cell membrane. Bar, 10 μm. The scatter plot represents the colour and intensity distributions of pixels from the pair of images. High intensities pixels (ROI outlined in yellow) were extracted and displayed in the overlay as white dots. C) Co-transfection of cells with Rab5aS34N (■) inhibits internalization upon transfer from 4 °C to 37 °C compared to Y1Δ32-expressing cells (□), as assessed by FACS. Data represent the mean±sem of 4–5 independent experiments. **Pb0.01 vs control. D) siRNA-inhibited expression of the three isoforms of Rab5 (▲) inhibits internalization upon transfer from 4 °C to 37 °C compared to control Y1Δ32-expressing cells (□), as assessed by FACS. Data represent the mean±sem of 6–8 independent experiments. *Pb0.05 vs control; **Pb0.01 vs control. E) Whole-cell lysates were prepared and subjected to immunoblot (IB) analysis using primary antibodies recognizing Rab5a, Rab5b and Rab5c. Whole-cell lysates were also analyzed utilizing antibody recognizing β-actin for control. The immunoblot was repeated for each siRNA transfection experiment as a control of siRNA-inhibited expression of the three isoforms of Rab5. Shown is a representative immunoblot of such an experiment with essentially identical results for the others.

Article Snippet: After blocking at (1 h at room temperature, I-Block Reagent; Tropix), membranes were incubated overnight at 4 °C with either a mouse monoclonal antiGFP antibody (Roche; 1/1000 dilution) or a rabbit polyclonal antiRab5a antibody (Santa Cruz; 1/1000 dilution) or a rabbit polyclonal anti-Rab5b antibody (Santa Cruz; 1/1000 dilution) or a goat polyclonal anti-Rab5c antibody (Santa Cruz; 1/300 dilution).

Techniques: Western Blot, Control, Transfection, Expressing, Membrane, Cotransfection

Journal: eLife

Article Title: Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate

doi: 10.7554/eLife.82111

Figure Lengend Snippet: ( A ) The endocytic recycling pathway during sheath initiation and loss. ( B ) Lateral images of oligodendrocytes in the early stages of the ensheathment process in the spinal cord of living larvae at 2.5 dpf labeled with sox10:mScarlet-CAAX (magenta) and expressing either myrf:eGFP-RAB5C , myrf:eGFP-RAB7A , or myrf:eGFP-RAB11A (green). White boxes outline immature sheaths with Rab+ endosomal puncta for each fusion protein. (Scale bar = 5 μm). ( C ) Top panels are grey inset images from the outlined regions in B. The bottom panels are 3D reconstructions of these insets (Membrane in magenta, endosomes in green). ( D ) Quantification of Rab+ endosomal puncta in immature sheaths. Number of puncta in each sheath was normalized by the length of the sheath. Rab5 n = 36 sheaths (9 ventral cells/2 dorsal cells/11 larvae), Rab11 n = 34 sheaths (8 ventral cells/2 dorsal cells/10 larvae), Rab7 n = 33 sheaths (9 ventral cells/2 dorsal cells/11 larvae). Dashed lines represent average values and error bars are SD. (See associated source data and supplementary video files). Figure 7—source data 1. Excel spreadsheet with the Rab5, -7, -11 localization data.

Article Snippet: Plasmids encoding the RAB5C , RAB7A , and RAB11A zebrafish coding sequences (Addgene, RAB5C =80518, RAB7A =80522, and RAB11A =80529) were sequenced, and the RAB7A and RAB11A sequences each had a point mutation ( D63G and Q166R, respectively) relative to the NCBI protein sequences ( RAB7A =NM_200928.1, RAB11A =NM_001007359.1) and relative to other previous work in zebrafish ( ).

Techniques: Labeling, Expressing, Membrane

Journal: eLife

Article Title: Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate

doi: 10.7554/eLife.82111

Figure Lengend Snippet: ( A ) Representative lateral images of ventral oligodendrocytes in the spinal cord of living larvae at 4 days post fertilization (dpf) labeled by sox10:eGFP-CAAX (green) and one of the following: myrf:tagRFP , myrf:tagRFP-RAB5C , myrf:tagRFP-rab5C S36N , myrf:tagRFP-RAB7A , myrf:tagRFP-rab7A T22N , and myrf:tagRFP-RAB11A , myrf:tagRFP-rab11A S25N (all in magenta). The image and data for the control is the same as for the ventral group in (scale bar = 5 μm). ( B ) Sheath number per cell. ( C ) Average sheath length per cell. ( D ) Total sheath length per cell ( myrf:tagRFP n=26 cells/26 larvae, myrf:tagRFP-RAB5C n=28 cells/28 larvae, myrf:tagRFP-rab5C S36N n=27 cells/27 larvae , myrf:tagRFP-RAB7A n=29 cells/29 larvae, myrf:tagRFP-rab7A T22N n=32 cells/32 larvae, and myrf:tagRFP-RAB11A n=30 cells/30 larvae, myrf:tagRFP-rab11A S25N n=27 cells/27 larvae). The dashed lines in each plot represent average values with all data points shown. Error bars are standard deviation. Global significance was determined using a Kruskal-Wallis test for B–D. This global p-value is shown for C since it was not significant. Post hoc multiple comparison tests were not performed for this analysis. Post hoc Dunn’s multiple comparison tests were performed to compare groups in B and D. We compared everything with the control group and compared each wild-type and associated mutant with each other. The different Rab groups were not compared with each other (see associated source data). Figure 8—source data 1. Excel spreadsheet with the sheath analysis data for the Rab5, -7, -11 dominant-negative mutants.

Article Snippet: Plasmids encoding the RAB5C , RAB7A , and RAB11A zebrafish coding sequences (Addgene, RAB5C =80518, RAB7A =80522, and RAB11A =80529) were sequenced, and the RAB7A and RAB11A sequences each had a point mutation ( D63G and Q166R, respectively) relative to the NCBI protein sequences ( RAB7A =NM_200928.1, RAB11A =NM_001007359.1) and relative to other previous work in zebrafish ( ).

Techniques: Labeling, Control, Standard Deviation, Comparison, Mutagenesis, Dominant Negative Mutation

Journal: eLife

Article Title: Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate

doi: 10.7554/eLife.82111

Figure Lengend Snippet: ( A ) Lateral images from the ventral spinal cord of living larvae labeled with sox10 : eGFP-CAAX (in green) and one of the following: myrf:tagRFP , myrf:tagRFP - RAB5C , myrf:tagRFP-rab5C S36N (all in magenta); and time-lapsed for 15 hours from 2.5-3dpf. The first panel is an image taken immediately before starting the time-lapse experiment. The subsequent panels are the same cells at the peak of sheath accumulation, at 3dpf, and at 4dpf. The images and data for the control are the same as for the ventral group in . (Scale bar = 5 μm). ( B ) Total ensheathment attempts per cell. ( C ) Peak sheath number per cell. ( D ) Sheath number at 3dpf per cell. ( E ) Final sheath number per cell at 4dpf. ( F ) Net sheaths lost from the peak to 4dpf. ( G ) Percent of sheaths stabilized during the accumulation phase (peak sheath number/total ensheathment attempts). (H) Percent of sheaths stabilized during the stabilization phase (final sheath number/peak sheath number). ( I ) Percent of total sheaths stabilized across both the accumulation and stabilization phases (final sheath number/total ensheathment attempts). ( J ) Simple linear regression comparing the total number of ensheathment attempts to the final sheath number at 4dpf for each cell. (control n=18 cells/18 larvae, wild-type Rab5 n=18 cells/18 larvae, Rab5DN n=18 cells/17 larvae). The dashed lines in each plot represent average values with all data points shown. The error bars are standard deviation. Significance was determined using global Kruskal-Wallis tests. These p-values are shown for B-D and G since they were not significant. Post hoc multiple comparisons tests were not performed for these analyses. Post hoc Dunn’s multiple comparisons tests were done for E, F, H, and I and the individual p-values are shown. ( J’ ) The slopes of the Rab5WT and Rab5DN regression lines from J were compared to the control in Graphpad by (two-tailed) testing the null hypothesis that the slopes are identical (the lines are parallel). P-values are shown in the table. (See associated source data and supplementary video files). Figure 9—source data 1. Excel spreadsheet with the summary data for the Rab5 dominant-negative mutant oligodendrocyte ensheathment dynamics experiment.

Article Snippet: Plasmids encoding the RAB5C , RAB7A , and RAB11A zebrafish coding sequences (Addgene, RAB5C =80518, RAB7A =80522, and RAB11A =80529) were sequenced, and the RAB7A and RAB11A sequences each had a point mutation ( D63G and Q166R, respectively) relative to the NCBI protein sequences ( RAB7A =NM_200928.1, RAB11A =NM_001007359.1) and relative to other previous work in zebrafish ( ).

Techniques: Labeling, Control, Standard Deviation, Two Tailed Test, Dominant Negative Mutation

Journal: eLife

Article Title: Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate

doi: 10.7554/eLife.82111

Figure Lengend Snippet:

Article Snippet: Plasmids encoding the RAB5C , RAB7A , and RAB11A zebrafish coding sequences (Addgene, RAB5C =80518, RAB7A =80522, and RAB11A =80529) were sequenced, and the RAB7A and RAB11A sequences each had a point mutation ( D63G and Q166R, respectively) relative to the NCBI protein sequences ( RAB7A =NM_200928.1, RAB11A =NM_001007359.1) and relative to other previous work in zebrafish ( ).

Techniques: Plasmid Preparation, Membrane, Mutagenesis, Transgenic Assay, Expressing