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First published online 20 November 2007
doi: 10.1242/jcs.014217

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Myosin VI and its interacting protein LMTK2 regulate tubule formation and transport to the endocytic recycling compartment

¹ Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 2XY, UK
² Departments of Neuroscience and Neurology, The Institute of Psychiatry, Kings College London, London, SE5 8AF, UK
³ MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

View larger version (23K): [in this window] [in a new window]   Fig. 1. Mapping of the interaction domains in LMTK2 and myosin VI. The interaction between myosin VI and LMTK2, which was discovered in a yeast two-hybrid screen, was verified using a mammalian two-hybrid assay. (A) Domain organisation of LMTK2 showing the kinase domain (grey), the two N-terminal putative transmembrane domains (vertical zig-zag lines), the PxxP motifs (black bars), and the binding sites for p35 and PP1C/Inh2. To map the binding site for myosin VI on LMTK2, CHO cells were co-transfected with the myosin VI tail fused to the Gal4 DNA-binding domain in the bait vector, LMTK2-deletion fragments fused to the activating domain in the prey vector and two other plasmids, pG5luc and pRL-CMV, expressing the inducible reporter and the co-reporter, respectively. Relative luciferase activity was measured using the Dual Luciferase Reporter Assay System kit (Promega). The deletion fragments used in this mammalian two-hybrid assay are depicted on the left and the corresponding amino acid (AA) numbers, the strength of the interaction (++, + or –) are shown, in brackets the actual luciferase activity relative to negative control are shown for a representative experiment. Fragment 567-773 is the minimal LMTK2 fragment that is still able to interact with myosin VI. (B) Myosin-VI-domain organisation. LI, alternatively spliced 31 aa large insert; SI, 9 aa small insert, RRL, GIPC/opineurin-binding motif; WWY, Dab2/LMTK2-binding motif. (C) Binding of myosin VI to LMTK2 requires the WWY motif. The mammalian two-hybrid assay was used to test binding of the LMTK2 fragment (451-1095) against wild-type myosin-VI-tail LI or the tail containing a W1192L mutation (WWYWLY). Data are given as the mean ± s.d. of three independent experiments. (D) LMTK2 binding to myosin VI does not require the large insert in the myosin VI tail. Binding of the LMTK2 fragment (451-1095) was tested against the myosin VI tail containing the 31 aa insert (MyoVI-LI-tail) or the tail lacking the insert (Myo6-NI-tail). Data are given as the mean ± s.d. of four independent experiments.

View larger version (44K): [in this window] [in a new window]   Fig. 2. LMTK2 binds myosin VI in vitro and in vivo. (A) LMTK2 binds directly to purified myosin VI tail. Pull-down assays were performed using in vitro-translated LMTK2 fragment and GST-tagged myosin VI tail. [35S]-labelled in-vitro-translated LMTK2-451-1095 was incubated with 5 µg of either GST alone (lane 2), GST-tagged myosin VI tail (lane 3) or GST-tagged myosin VI tail with the W1192L mutation (WWYWLY, lane 4). Lane 1 shows 5% of the input used for the pull down assays. Mutation of the WWY motif within the myosin VI tail dramatically reduces LMTK2 binding. (B) (C) Co-immunoprecipitation of LMTK2 and myosin VI from HeLa cells. (B) HeLa cells, untransfected (lanes 1, 2, 4) or transfected with GFP-tagged myosin VI (lane 3), were lysed and myosin VI was immunoprecipitated using polyclonal antibodies against the myosin VI tail (lanes 2-3). As a control immunoprecipitation was performed using non-immune rabbit IgG (lane 4). Lane 1 shows the whole-cell lysate, which is equivalent to 5% of the input used for each immunoprecipitation. The immunoprecipitated protein complexes were blotted with antibodies against LMTK2. Note the abnormal mobility of LMTK2 and its fragments on SDS-PAGE as described previously (Kawa et al., 2004). (C) Endogenous LMTK2 was immunoprecipitated from HeLa cells transfected with GFP–myosin-VI, and the immunoprecipitated complexes were blotted with anti-myosin VI tail antibodies. Lane 1 shows 5% of the whole-cell lysate used for each immunoprecipitation; lane 3 shows the negative control immunoprecipitation with non-immune rabbit IgG.

View larger version (62K): [in this window] [in a new window]   Fig. 3. LMTK2 and myosin VI colocalise in cultured cells. (A) HeLa cells were co-transfected with untagged LMTK2 and GFP-tagged myosin VI, and labelled with a (a,a') polyclonal antibody against LMTK2 and (b,b') a monoclonal antibody against GFP. Panels a-c are confocal z-stacks, panels a'-c' are magnifications of a single confocal slice of the boxed regions in a-c. (B) Wide field images are shown of RPE cells that were transfected with LMTK2-GFP and labelled with (d,d') monoclonal antibody against GFP and (e,e') polyclonal antibody against myosin VI. Panels d'-f' are magnifications of the boxed regions in d-f. Bars, 10 µm.

View larger version (82K): [in this window] [in a new window]   Fig. 4. LMTK2 is present in the endocytic compartments. (a-c) HeLa cells transfected with LMTK2-GFP were loaded with Tf–Alexa-Fluor-555 for 15 minutes, fixed and processed for immunofluorescence with anti-GFP antibody. (d-j) HeLa cells transfected with untagged LMTK2 were immunolabelled for (d,g) LMTK2 and (e) Rab5 or (h) EEA1. (k-m) HeLa cells stably expressing GFP-Rab11 were transfected with untagged LMTK2 and labelled with antibodies to LMTK2 and GFP. Merged images show LMTK2 colocalisation with the marker proteins in yellow. Single confocal slices (a-c,k-m) and wide field images (d-j) are shown. Insets represent enlarged images of the boxed regions. Bar, 10 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Depletion of myosin VI and LMTK2 changes the morphology and distribution of transferrin-positive endosomes. (A-C) HeLa cells were transfected twice with siRNA targeting myosin VI or LMTK2, or with non-specific control siRNA. (A,B) Two days after the second transfection the cells were harvested and processed for (A) western blotting with antibodies against LMTK2, myosin VI and -tubulin. In parallel experiments, cells were immunolabelled for (B) EEA1 and Vps26 or (C) loaded with Tf–Alexa-Fluor-555 before fixation. Insets represent enlarged images of the boxed regions. All images are confocal z-projections. Bars, 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Depletion of myosin VI or LMTK2 inhibits delivery of TfR to the Rab11-positive perinuclear recycling compartment. (A) HeLa cells stably expressing GFP-Rab11 were transfected with siRNA targeting myosin VI or LMTK2 and processed for immunofluorescence with antibodies against (a,d,g) TfR and (b,e,h) GFP, and labelled with DAPI to visualise nuclei. Merged images are shown in c, f and i. Bar, 10 µm. (B) HeLa cells transfected with siRNA targeting myosin VI or LMTK2 were pulsed with Tf–Alexa-Fluor-647 at 37°C for 30 minutes, washed and incubated at 37°C in the presence of excess of unlabelled transferrin. The amount of Tf–Alexa-Fluor-647 per cell was determined by FACS analysis. Data are presented as the mean ± s.e. from three independent experiments, each performed in duplicate.

View larger version (56K): [in this window] [in a new window]   Fig. 7. Depletion of myosin VI and LMTK2 leads to reduction in Rab11-positive tubule formation. HeLa cells stably expressing GFP-Rab11 were transfected with siRNAs targeting myosin VI or LMTK2 and processed for immunofluorescence with anti-GFP antibodies. (A) A cell displaying a representative GFP-Rab11 distribution is shown for mock-transfected cells and for cells transfected with siRNA targeting myosin VI or LMTK2. Whereas multiple tubules emanate from the juxtanuclear region in mock-transfected cells (arrows), an almost complete lack of tubules was observed in both knockdowns. (B) Quantification of the number of tubules in mock-transfected or KD cells is shown. At least 500 cells were counted for the control group and for each RNAi experiment, and scored as either containing or not containing tubules. The results are expressed as percentage of cells containing tubules (mean ± range from two independent experiments, each performed in triplicate). Bar, 10 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 8. Depletion of myosin VI and LMTK2 leads to reduction in EHD1- and EHD3-positive tubule formation. (A,B) HeLa cells stably expressing (A) GFP-EHD1 or (B) GFP-EHD3 were treated with siRNA targeting myosin VI or LMTK2, fixed and mounted for immunofluorescence. Representative examples of (A) GFP-EHD1 or (B) GFP-EHD3 distribution are shown for each KD and control cell. (C) To quantify the number of EHD3 positive tubules in KD and control cells at least 200 randomly chosen cells were counted for each condition and scored as having either no, few (<15) or multiple (>15) tubules. The results are given as percentage of cells for each category and are expressed as the mean ± s.d. from three independent experiments, each performed in triplicate. (D,E) The number of tubules was counted and the length of individual tubules was measured in 25 cells for each knockdown. Cells without tubules were not taken into account. Branches were considered as separate tubules. The histograms depict the (D) number of tubules per cell and the (E) length of individual tubules. In (A) confocal z-stacks and in (B) wide-field images are shown. Bars, 10 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 9. Possible roles of LMTK2 and myosin VI in the delivery of cargo from the early endosome to the ERC. After internalisation, endocytic vesicles are delivered to and fuse with the early endosome (EE), where proteins destined for degradation are sorted and transported to the lysosome. Proteins moving back to the cell surface can either take a fast recycling route directly from the EE or a slower route via the ERC. A number of proteins including Rab11, Rab11-FIP2, EHD3, Rab4 and rabenosyn 5 have been shown to regulate trafficking between the EE and the ERC. Our results add myosin VI (an actin motor protein) and its binding partner LMTK2 (a protein kinase) to this list of proteins required for the delivery of cargo to the ERC. For the exit of receptors from the ERC the motor protein myosin Vb, and also Rab11 and EHD1 are required.