Fig 2: Protrudin and KIF5 are required for the protrusions that develop in cells lacking spastin. a–c HeLa cells were mock-transfected, or transfected with the indicated spastin siRNAs, spastin and protrudin siRNAs, or spastin and KIF5B siRNAs, then labelled for acetylated tubulin. Selected representative images are shown in a, with the lower panels being higher magnification views of the boxed areas shown in the upper panels. The percentage of cells with protrusions was quantified in b, n = at least 4 biological repeats for each experimental condition. Protein depletion was confirmed by immunoblotting (c). In these experiments, as an antibody to endogenous protrudin was not then available, protrudin depletion was verified by carrying out siRNA transfection experiments in parallel, in cells transiently expressing GFP-protrudin. Anti-GFP immunoblotting of these cells is shown. Subsequent validation that the siRNAs used could deplete cells of endogenous protrudin is shown in Supplementary Figure 4. P values generated by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Scale bar = 20 µm

Fig 3: Knockdown of Kif5b does not compromise myosin-Va-driven centrifugal melanosome transport. melan-d1 cells were transfected with the indicated siRNA, then 72 h later infected with GFP–myosin-Va-expressing adenovirus, and fixed and stained with anti-GFP antibodies to reveal the expression of myosin-Va (as described the Materials and Methods). (A) Representative low-power bright-field and corresponding fluorescence images showing the subcellular distribution of melanosomes and expression of GFP–myosin-Va in fields of siRNA-transfected and/or GFP–myosin-Va-expressing melan-d1 cells. R27a, Rab27a siRNA. Scale bar: 100 µm. (B) A scatter plot showing the percentage of melanocytes manifesting perinuclear clustered melanosomes for each siRNA. Data are from 9 independent transfections, each performed in triplicate on different pools of cells. Plotted points represent the mean percentage of cells with perinuclear clustered melanosomes from each experiment (as described in the Materials and Methods). Bracketed numbers indicate the total number of cells analysed. Horizontal bars show the mean and 25th and 75th percentile of each population. ****P=0.0001 relative to NT cells (one-way ANOVA); no other significant differences were observed.

Fig 4: Forced targeting of Kif5b to melanosomes can drive centrifugal melanosome transport in melanocytes. melan-d1 cells were infected with adenovirus allowing expression of the indicated GFP fusion proteins then fixed 24 h later, processed for immunofluorescence and imaged using a confocal microscope (as described in the Materials and Methods). (A) Schematic representation of the domain organisation and expected oligomeric state of Kif5b (red and grey shapes), myosin-Va (green and grey shapes) and fusion proteins. RBD (orange shape) indicates the Rab27a-binding domain of murine Sytl2 [as described previously; Evans et al. (2014)]. (B) Pairs of confocal fluorescence and phase-contrast (transmitted light) images showing the distribution of GFP fusion proteins and melanosomes, respectively, in representative fields of infected cells. Scale bar: 50 µm. (C) High-magnification images of cells within the borders of the white boxes in lower magnification images phase-contrast images as shown in B, indicating the areas shown that highlight association between fusion proteins (green) and F-actin (for MVa–K5b) or melanosomes (other fusion proteins), both shown in magenta. (D) A scatter plot showing the pigment distribution as reported by percentage pigment coverage (pigment-filled area/total cell area), calculated as described previously (Hume et al., 2006). Horizontal bars show the median and 25th and 75th percentile of each population. The significance of differences in pigment coverage for each population compared with the GFP and wild-type myosin-Va are displayed below and above each scatter point. ****P=0.0001 (one-way ANOVA); no other significant differences were observed. Data are from one of three independent experiments and are representative of the results of all experiments. Number of cells analysed; GFP=14, Kif5b=12, myosin-Va=14, mini-K5b=10, mini-Va=9, k5b-mva=12 and mVa-k5b=11.

Fig 5: The intracellular distribution of Kif5b and Rab1a in mouse melanocytes. melan-a cells were transiently transfected with plasmid vectors allowing the expression of the indicated GFP or YFP fusion proteins. Cells were fixed after 48 h, stained with the indicated organelle marker-specific antibodies, and the intracellular distribution of GFP or YFP and organelle markers was observed using a confocal microscope (as described in the Materials and Methods). (A–E) Single confocal z-sections showing the distribution of GFP–Rab27a (A), Kif5b–YFP (B), GFP (C) and GFP–Rab1a (D,E) relative to organelle markers. Tyrosinase marks melanosomes (A-D) and giantin marks the Golgi apparatus (E), respectively. Phase-contrast and transmitted-light images show the distribution of pigmented melanosomes. In merge images, GFP is coloured green and organelle markers are coloured magenta; thus, white pixels indicate colocalisation. The white squares in the phase images indicate the part of the cell that is presented in the high-magnification panels below. Arrows in A and E highlight areas of colocalisation, while the arrows in B indicate the accumulation of Kif5b–YFP at the peripheral plus-tips of MTs. Scale bars: 20 µm. (F,H) Scatter plots showing the extent of colocalisation (measured using the PCC) between tyrosinase and Rab27a (n=6), Kif5b (n=8) and GFP (n=7) (F), and between GFP-Rab1a and tyrosinase (n=8) or giantin (n=7) in melan-a cells (H). Horizontal bars show the median and 25th and 75th percentile of each population. P-values were determined by one-way ANOVA for differences in PCC values (ns, not significant). (G) Western blot showing the expression of Kif5b and tyrosinase in subcellular fractions of melan-a cells (as described in Materials and Methods).