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First published online 14 August 2007
doi: 10.1242/jcs.010207

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Rab27a and MyoVa are the primary Mlph interactors regulating melanosome transport in melanocytes

¹ Molecular and Cellular Medicine, National Heart and Lung Institute, Imperial College London, London, SW7 2AZ, UK
² Biological Nanosciences, National Heart and Lung Institute, Imperial College London, London, SW7 2AZ, UK

View larger version (24K): [in this window] [in a new window]   Fig. 1. Mapping of EB1 and Mlph domains required for interaction. For A and B the left-hand part shows the relationship of truncated test proteins, indicated with solid lines, to the domain structures of EB1 (A) and Mlph (B). The right-hand part shows the results of the -galactosidase reporter gene assay for each test protein with either Mlph aa367-590 (A) or EB1 aa1-268 and MyoVa MSGTA (B). 1 and 2 denote the position of -helices in EB1; ABD, actin-binding domain; CC, coiled coil; CH, calponin homology domain; EFBD, MyoVa exon F binding domain; GTBD, MyoVa globular tail binding domain; MBD, MyoVa binding domain; SHD1, synaptotagmin homology domain; R27BD, Rab27a binding domain; ZnF, Zn2+ finger. Panel C is an alignment of part of Mlph and MyRIP sequences from human (Hs Mlph accession NP_077006.1 and Hs MyRIP accession BAC15555.1), mouse (Mm accession BAB41087.1), rat (Rn accession NP_001012135.1), chicken (Gg Mlph accession XP_421876.2) and zebrafish (Dr Mlpha accession NP_001073147.1 and Dr Mlphb accession XP_685065.2) showing the position of the Mlph IP motifs (red) and the coiled coil region (green). Alignment of sequences was conducted using Muscle protein multiple sequence alignment software available at http://phylogenomics.berkeley.edu/cgi-bin/muscle/input_muscle.py. Numbering indicates the amino acid position of each sequence.

View larger version (42K): [in this window] [in a new window]   Fig. 2. Measurement of the interaction of Mlph with EB1 and MyoVa-MSGTA in vitro. Panels A and B show interaction with EB1 and MSGTA involves different regions of Mlph (A) and that Mlph may interact simultaneously with EB1 and MSGTA (B). One hundred pmol of GST-Mlph protein (A) or MBP-EB1 (B) was incubated with equimolar amounts of MBP-EB1 or MBP-MSGTA (A) or his6-Mlph and/or his6-MSGTA (B) in the presence of glutathione-sepharose (A) or amylose agarose (B) as described in the Materials and Methods. Bound proteins were precipitated, eluted from the beads, and subjected to immunoblotting using indicated antibodies. In A, relative binding (see bar chart) was calculated by dividing band intensity of EB1 or MSGTA by the band intensity of Mlph (as described in Materials and Methods). Experiments were carried out in triplicate and the migration of molecular-weight standards is indicated on the left of each blot.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Intracellular distribution of endogenous Mlph and EB1 in wild-type melan-ink melanocytes. Melan-ink cells were fixed and stained using antibodies that specifically recognise EB1 (A) and Mlph (B) as described in Materials and Methods. Panel C is the corresponding transmitted light image showing the distribution of melanosomes, panel D is an inverted and filtered copy of the transmitted light images showing the position of melanosomes and panel E is a merged image showing EB1 (green), Mlph (red) and melanosomes (blue). Panels A`-E` are higher magnification images of the indicated regions of A-E. Bar, 20 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 4. EB1 comet movements in wild-type and melan-ln melanosomes. Wild-type and melan-ln melanocytes were transiently transfected with plasmid encoding EB1-EGFP and the movements of EB1 comets and melanosomes were recorded as described in Materials and Methods. Panel A shows the distribution of EB1-EGFP in a melan-ln melanocyte and a magnified section of this cell in which arrows indicate individual comet structures. Panel B shows the tracks of melanosomes (red) and EB1-EGFP comets (green) in individual transfected wild-type and melan-ln cells over the course of a 95 second time-lapse recording (these tracks correspond to Movies 1 and 2 in supplementary material). Bars, 10 µm (A); 5 µm (B). Panel C shows the velocity of EB1-EGFP comets in representive wild-type () and melan-ln () melanocytes. The velocities of individual comets (n=2541 comets from 20 cells for wild-type and n=1711 comets from 10 cells for melan-ln) were binned into velocity ranges of 0.025 µm/sec and then plotted showing the percentage of the total population that falls within each bin.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Disruption of Mlph interaction with Rab27a and MyoVa, but not EB1, affects rescue of melan-ln melanosome transport defects. Melan-ln cells were transfected with plasmids encoding the indicated Mlph point mutant molecules and control proteins. Cells were fixed 48 hours later and stained with antibodies to detect Myc-tagged Mlph and Slp1 protein. Panels A, E, I, M and Q show the distribution of overexpressed protein, panels B, F, J, N and R are transmission images showing the distribution of pigment; panels C, G, K, O and S are inverted and filtered copies of the transmission images showing the distribution of melanosomes and panels D, H, L, P and T are merged images in which the distribution of myc tagged Mlph and Slp1 protein (green) and melanosomes (red) are superimposed. Bars, 20 µm. Arrows in panels A-H and M-T indicate colocalisation of Mlph protein with the cytoplasmic surface of melanosomes. Arrows in panel I indicate association of Mlph with linear actin filaments. Panel U shows quantification of the extent of rescue of melanosome transport by the Mlph point mutants. Rescue efficiency for populations of transfected cells was determined as described in Hume et al. (Hume et al., 2006). Shaded boxes indicate 25th/75th percentile; bars within boxes indicate median values for each population of cells; outer bars indicate 5th/95th percentile; and outer points indicate outliers. The statistical significance of rescue measurements for populations of Mlph mutant transfected cells relative to positive (wild-type Mlph) and negative (Slp1) controls is shown in Table 1.

View larger version (117K): [in this window] [in a new window]   Fig. 6. Depletion of Mlph together with interacting proteins Rab27a and MyoVa, but not EB1, causes clustering of wild-type melanosomes. Wild-type melan-ink melanocytes were transfected with the siRNA oligonucleotides that specifically prevent the synthesis of the indicate proteins. Transfected cells were fixed 72 hours later and stained with the indicated specific antibodies to allow correlation of depletion of protein to melanosome distribution. Panels A, C, E, G, I, K and M are fluorescence images showing the expression and distribution of the indicated protein in transfected cells and panels B, D, F, H, J, L and N are transmitted light images showing the distribution of pigment granules in transfected cells. Bar, 20 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 7. siRNA-mediated depletion of individual Rab27a-Mlph-MyoVa complex components affects the overall stability of the complex. Pools of wild-type melan-ink melanocytes were transfected with the indicated siRNA oligonucleotide, harvested 72 hours later and lysates subjected to immunoblotting using the indicated antibodies. Transfections were carried out in triplicate and the migration of molecular-weight standards is indicated on the left of each blot.

View larger version (36K): [in this window] [in a new window]   Fig. 8. Depletion of Rab27a and MyoVa, but not EB1, prevents Mlph-mediated rescue of melan-ln melanosome transport defects. Melan-ln melanocytes were sequentially transfected first with siRNA to allow depletion of the indicated proteins and second with plasmid DNA allowing expression of Myc-tagged Mlph as described in Materials and Methods. Cells were then stained with antibodies allowing detection of the overexpressed Myc-Mlph (panels A,C,G,K) and siRNA-targeted protein to confirm depletion in each cells (panels D,H,L). Panels E, I and M are merged fluorescent images showing Myc-Mlph (green) and siRNA-targeted protein (red) and B, F, J and N are transmission images showing the distribution of melanosomes. Arrows indicate colocalisation of Mlph protein with the cytoplasmic surface of melanosomes. Panel O shows quantification of the extent of rescue of melanosome transport by the Mlph in melan-ln cells siRNA-depleted of Mlph-interacting proteins using the indicated oligonucleotides. Rescue efficiency for populations of transfected cells was determined as described in Hume et al. (Hume et al., 2006). Shaded boxes indicate 25th/75th percentile; solid and dotted bars within boxes indicate median and mean values for each population of cells; outer bars indicate 5th/95th percentile; and outer points indicate outliers. The statistical significance of rescue measurements for populations of transfected cells relative to positive and negative controls is shown in Table 2. Bar, 20 µm.

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