JCS014217 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Depletion of LMTK2 does not lead to defects in SEAP secretion and VSVG trafficking. (A) To assess the localisation of LMTK2 at the Golgi complex, GFP-tagged LMTK2 was expressed in HeLa cells that were double labelled with antibodies against GFP and the Golgi matrix protein GM130. The merged image and the enlarged image of the boxed area show very little colocalisation between LMTK2 and GM130. (B) To measure protein secretion in cells lacking myosin VI or LMTK2, a stable cell line expressing a secreted form of alkaline phosphatase (SEAP) was transfected twice with siRNA specifically targeting myosin VI or LMTK2. 48 hours after the last transfection the activity of SEAP secreted into the culture medium was measured in a calorimetric assay using para-nitrophenyl phosphate as a substrate (Warner et al., 2003). Whereas absence of myosin VI leads to a dramatic reduction in SEAP secretion, no change in secretion was observed in LMTK2 KD cells. The results are given as the mean ± s.d. from three independent experiments. (C) Finally we used a thermoreversible folding mutant of the viral glycoprotein VSV-G, fused to GFP at its cytoplasmic tail, as a reporter molecule to assess membrane transport from the Golgi complex to the plasma membrane. Mock or siRNA-treated cells were transfected with ts045 VSVG-GFP (a gift from R. Duden, University of London) on day 4. The cells were incubated at 37°C overnight and then for 24 hours at 39°C. On the day of the assay (day 6 of knockdown) 100 mg/ml cycloheximide was added and the cells were shifted to 19°C for 2 hours to allow folding and trafficking of VSV-G from the ER to the Golgi complex. To allow exit of VSV-G from the Golgi complex and its transport to the plasma membrane, the cells were shifted to 32°C for 50 minutes. After fixation the VSV-G at the cell surface was detected by immunofluorescence using a monoclonal antibody to its luminal domain. The bar graph shows the percentage of cells containing VSV-G at the cell surface. In myosin VI KD cells there is a 50% reduction in the number of cells displaying VSV-G at their cell surface, whereas there is no change in export rates of VSV-G in LMTK2 KD compared with control cells. The results given as the mean ± range from two independent experiments.

• Supplemental Figure S2 -

  Fig S2. Localisation of myosin VI in early endocytic pathway. RPE cells were transiently transfected with GFP-myosin VI and double labelled with monoclonal antibodies against GFP and polyclonal antibodies against GIPC (a-c), or directly processed for immunofluorescence with polyclonal antibodies against myosin VI and monoclonal antibodies against Rab5 or EEA1 (d-i). Merged images (c, f and i) show myosin VI in green, markers in red and overlap in yellow. Bar, 10 mm

• Supplemental Figure S3 -

  Fig S3. Transferrin accumulates in enlarged early endosomes in myosin VI and LMTK2 KD cells. HeLa cells treated with corresponding siRNA were loaded with Tf−Alexa-Fluor-555 before fixation and immunolabelled for EEA1. Merged images (c, f and i) show Tf−Alexa-Fluor-555 in red, EEA1 in green and overlap in yellow. Bar, 10 mm

• Supplemental Figure S4 -

  Fig. S4. Depletion of LMTK2 or myosin VI does not affect sorting in the endocytic pathway and EGF receptor degradation. (A) To investigate the effect of LMTK2 or myosin VI depletion on the late endocytic pathway, we visualised the steady-state localisation of lysosomes with a monoclonal antibody against LAMP1 in KD and control cells, which were loaded for 20 minutes with Tf−Alexa-Fluor-555 before fixation. No accumulation of the lysosomal marker LAMP1 in enlarged transferrin-positive endosomes was observed. (B) To further investigate trafficking from endosomes to lysosomes we measured ligand-induced EGFR degradation in myosin VI or LMTK2 KD and control cells. The cells were serum-starved overnight, incubated in medium with 100 mg/ml cycloheximide for 2 hours before stimulation with 100 ng/ml EGF for 3 hours. The cell lysates were blotted with antibodies to EGFR and α-tubulin. In KD cells we observed no delay in EGFR degradation, which is consistent with intact membrane trafficking pathways from early transferrin-positive endosomes to late endosomes/lysosomes.

• Supplemental Figure S5 -

  Fig S5. Transferrin trafficking in myosin VI and LMTK2 knockdown cells. (A-B) To assess the fast recycling pathway, the cells were incubated on ice with 50 mg/ml Tf−Alexa-Fluor-647 for 30 minutes and then allowed to internalise Tf for 60 minutes at 16°C (A) or for 5 minutes at 37°C (B) in the presence of Tf−Alexa-Fluor-647. After washing the cells were incubated at 37°C for different times in the presence of 100 mg/ml unlabelled transferrin. The amount of Tf−Alexa-Fluor-647 remaining in the cell was quantified by FACS analysis. (C,D) To measure the amount of internalised transferrin, HeLa cells treated with corresponding siRNA were incubated in medium with 50 mg/ml Tf−Alexa-Fluor-647 on ice for 30 minutes and then at 37°C for different times in the continuous presence of Tf-Alexa-647. The amount of internalised transferrin was determined by FACS analysis. (C) Uptake curve from a representative experiment. (D) Relative amount of internalised transferrin after 20 minutes (measured in eight experiments). (E) To measure the total amount of TfR, the cells were permeabilised with saponin and incubated with APC-conjugated anti-TfR antibody at 4°C for 30 minutes, washed and fixed. The total amount of TfR was determined by FACS analysis. The graphs represent the mean ± s.e. from three (A,B), eight (D) or four (E) independent experiments.