Autophagosome-lysosome mediated secretion of the thrombopoietin receptor is modulated by distinct driver mutations of myeloproliferative neoplasm

Figures

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  Fig. 1. pH-dependent ratiometric FRET of SRAI-TpoR. A Schematic representation of SRAI-TpoR construct. B U2OS cells were transfected with JAK2 WT, JAK2 WT+ Bafilomycin A1, JAK2 V617F. Imaging of TOLLES-YPet signal was performed under a confocal microscope. (Left) Ratiometric FRET (Intensity of TOLLES/ Intensity of YPet) approach was used to generate the rainbow RGB images. The ratiometric FRET image (left panel) has been represented; overlay of the donor TOLLES signal (red) and FRET signal (green) has been represented (middle and right panels) to identify areas with (yellow) and without FRET (red). (center) Overlay of FRET signal with donor TOLLES signal; (right) Inset shows an enlarged view with arrowheads indicating puncta with quenched FRET signal. The scale bar represents 10 μm. C Imaging of TOLLES-YPet signal was performed at the cell surface. Overlay of FRET signal (green) with donor TOLLES signal (red) is shown. Arrowhead indicates quenched FRET signal. The scale bar represents 10 μm. D Data represents percentage of FRET puncta ± SEM. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 10–12 for each case.

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  Fig. 2. Effect of JAK2 on unconventional secretion of TpoR. A The subcellular localization of TpoR was analyzed by confocal microscopy. HEK293 cells were transfected with TpoR alone or TpoR along with JAK2 WT and JAK2 V617F and stained for organelle marker (green; LAMP1 or LC3) and antibodies against anti-HA tag for TpoR (red). The scale bar represents 10 μm. Arrowhead represents colocalization of TpoR in LAMP1/LC3 vesicles. B The signal intensities of TpoR puncta and LAMP1 or LC3 puncta in (A) were calculated as gray values and the percentage colocalization of TpoR was obtained in each case. Data represents percentage of colocalization ±SEM. Error bars represent SEM. p-value is indicated in each graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 5–10 for each case. C HEK293 cells expressing TpoR alone or TpoR along with JAK2 WT and JAK2 V617F were lysed 24 h post-transfection and treated with Endo H or PNGase F and subjected to Western blot analysis. In the input lane, the upper band represents a mixture of complex glycosylated mature TpoR as well as high mannose containing immature TpoR. Following Endo H digestion, the high mannose containing immature TpoR…

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  Fig. 3. Accumulation of TpoR in LAMP1+ vesicles in the presence of JAK2 V617F. A The subcellular localization of TpoR was analyzed by confocal microscopy. HEK293 cells were transfected with TpoR along with JAK2 WT and JAK2 V617F and 13 h post-transfection cells were treated with 100 nM Bafilomycin A1 or 5 μM MG132 for 5 h. Cells were stained for organelle marker (green; LAMP1 or LC3) and antibodies against HA tag for TpoR (red). The scale bar represents 10 μm. Arrowhead represents colocalization of TpoR in LAMP1/LC3 vesicles. B The signal intensities of TpoR puncta and LAMP1 or LC3 puncta in (A) were calculated as gray values and the percentage colocalization of TpoR was calculated in each case. Data represents percentage of colocalization ± SEM. Error bars represent SEM. p-value is indicated in each graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 5–10 for each case, no. of vesicles n = 19–780 for LAMP1 and n = 25–238 for LC3. C HEK293 with the indicated conditions was stained with CD110-APC. Cells were washed and analyzed under flow cytometer. Percentage of CD110+ (TpoR) cells and median fluorescence intensity (MFI) is indicated in each case. Representative…

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  Fig. 4. TpoR preferentially traffics through autophagosome-lysosome in the presence of JAK2 V617F. A The subcellular localization of TpoR was analyzed by confocal microscopy. DU145 cells expressing TpoR along with JAK2 WT, JAK2 V617F or Atg5 and stained for organelle marker (green; LAMP1) and antibodies against HA tag for TpoR (red). The scale bar represents 10 μm. Arrowheads indicate colocalization of TpoR in LAMP1 lysosomes. B The signal intensities of TpoR puncta and LAMP1 or LC3 puncta in (A) were calculated as gray values and the percentage colocalization of TpoR was calculated in each case. Data represents percentage of colocalization ±SEM. Error bars represent SEM. p-value is indicated in each graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 5–10 for each case, no. of vesicles n = 556–608 for LAMP1. C Cell surface expression of TpoR was investigated by flow cytometry using CD110-APC antibody against surface TpoR. Median fluorescence intensity (MFI) is indicated in each case. Representative image of the flow analysis is shown (n = 3).

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  Fig. 5. CALR mutations block unconventional traffic of TpoR. A The subcellular localization of TpoR was analyzed by confocal microscopy. HEK293 cells were transfected with TpoR along with JAK2 WT and CALR ins5 or del52 and stained for organelle marker (green; LC3) or lysotracker and antibodies against HA tag for TpoR (red). The scale bar represents 10 μm. Arrowheads indicate colocalization of TpoR with lysotracker. B The signal intensities of TpoR puncta and lysotracker or LC3 puncta in A were calculated as gray values and the percentage colocalization of TpoR was calculated in each case. Data represents percentage of colocalization ±SEM. Error bars represent SEM. p-value is indicated in each graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 5–10 for each case, no. of vesicles n = 19–209 for lysotracker and n = 38–143 for LC3. C HEK293 cells expressing TpoR along with JAK2 WT and CALR ins5 or del52 were lysed 24 h post-transfection and treated with Endo H or PNGase F and subjected to Western blot analysis. Representative image of the blot is shown (n = 3). D U2OS cells were transfected with SRAI-TpoR, JAK2 WT, CALR del52. Imaging of TOLLES-YPet signal was…

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  Fig. 6. Unconventional secretion of TpoR requires RAB1A. A The subcellular localization of TpoR was analyzed by live cell imaging and confocal microscopy. U2OS cells were transfected with TpoR tagged with GFP and JAK2WT or JAK2V617F or CALR ins5 or CALR del52. Rab1A, Rab1B or Rab6 tagged with mCherry. The scale bar represents 10 μm. Arrowhead shows colocalization of TpoR with respective Rab. B The subcellular localization of TpoR was analyzed by confocal microscopy. HEK cells were transfected with TpoR along with JAK2 V617F and scrambled siRNA or siRNA against Rab1A. Rab1B was overexpressed to ensure Golgi integrity. Samples were stained for organelle marker (green; LAMP1 or LC3) and antibodies against HA tag for TpoR (red). The scale bar represents 10 μm. Arrowhead shows colocalization of TpoR with LAMP1 and LC3. C The signal intensities of TpoR puncta and LAMP1 or LC3 puncta in (B) were calculated as gray values and the percentage colocalization of TpoR was calculated in each case. Data represents percentage of colocalization ±SEM. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 5–10 for each case, no. of vesicles n = 686–841 for LAMP1and…

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  Fig. 7. TpoR W515L shows a partial Golgi-independent secretion. A U2OS cells were transfected with TpoR W515L + JAK2 WT. Imaging of TOLLES-YPet signal was performed under a confocal microscope. (Left) Ratiometric FRET (Intensity of TOLLES/ Intensity of YPet) approach was used to generate the rainbow RGB images. The ratiometric FRET image (left panel) has been represented; overlay of the donor TOLLES signal (red) and FRET signal (green) has been represented (middle and right panels) to identify areas with (yellow) and without FRET (red). (center) Overlay of FRET signal with donor TOLLES signal; (right) Inset shows an enlarged view with arrowheads indicating puncta with quenched FRET signal. The scale bar represents 10 μm. B Imaging of TOLLES-YPet signal was performed at the cell surface. Overlay of FRET signal (green) with donor TOLLES signal (red) is shown. Arrowhead indicates quenched FRET signal. The scale bar represents 10 μm. Data represents percentage of FRET puncta ±SEM. p-value is indicated in each graph. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No. of cells n = 10–12 for each case. C The subcellular localization of TpoR W515L was analyzed by confocal microscopy.…

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  Fig. 8. TpoR traffic in haematopoietic cell lines is dependent upon the presence of JAK2 V617F. A HEL cells were transfected with TpoR alone or TpoR treated with Bafilomycin A1. Imaging of TOLLES-YPet signal was performed under a confocal microscope. (Left) Ratiometric FRET (Intensity of TOLLES/ Intensity of YPet) approach was used to generate the rainbow RGB images. The ratiometric FRET image (left panel) has been represented; overlay of the donor TOLLES signal (red) and FRET signal (green) has been represented (middle and right panels) to identify areas with (yellow) and without FRET (red). (center) Overlay of FRET signal with donor TOLLES signal; (right) Inset shows an enlarged view with arrowheads indicating puncta with quenched FRET signal. The scale bar represents 10 μm. B The subcellular localization of TpoR was analyzed by live cell imaging and confocal microscopy. HEL cells were transfected with TpoR tagged with GFP and JAK2WT. Rab1A, Rab1B or Rab6 tagged with mCherry. The scale bar represents 10 μm. Arrowhead shows colocalization of TpoR W515L with respective Rab. C The subcellular localization of TpoR was analyzed by confocal microscopy. HEL cells were transfected with TpoR…