Abstract
The maintenance of the haematopoietic stem cells and the production of platelets thereof is acutely dependent upon the thrombopoietin receptor (TpoR). TpoR dimerizes in the presence of its ligand thrombopoietin leading to the activation of downstream JAK2-STAT signalling. Alternatively, the receptor dimerizes in the presence of activating mutations of JAK2 (JAK2 V617F), calreticulin or TpoR leading to myeloproliferation. These effects are dependent upon the surface expression of the receptor. TpoR is secreted through the Golgi-dependent pathway. Although, an unconventional autophagosome-lysosome mediated traffic has been postulated; questions remained whether the observed lysosomal localization was indicative of degradation or secretion. We fused the pH-sensitive FRET pair of TOLLES-YPet with TpoR (SRAI-TpoR) that showed FRET quenching at low pH environment of lysosomes. Using this construct, we demonstrated the presence of quenched SRAI-TpoR on the cell surface indicating that TpoR could indeed be secreted through low pH compartments. We further demonstrated that the lysosome-autophagosome-mediated secretion of TpoR was promoted by JAK2 V617F mutation, partially utilized by TpoR W515L but abrogated by calreticulin mutations. Finally, we showed that the lysosome-autophagosome mediated secretion was dependent upon Rab1A. Our study conclusively showed the unconventional traffic of TpoR and its modulation by the driver mutations causing myeloproliferation.
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Introduction
The thrombopoietin receptor (TpoR) is a type I cytokine receptor present on the surface of hematopoietic stem cells, megakaryocytes and platelets [1, 2]. TpoR is a single pass transmembrane protein that homodimerizes in the presence of its ligand thrombopoietin (TPO) [3]. The activated receptor initiates signaling through the JAK2/STAT1/3/5 pathway allowing lineage-specific differentiation of stem and progenitor cells. Therefore, activating mutations associated with the receptor (such as TpoR W515L/S505N) or the JAK2 (JAK2 V617F) were found to cause pathological proliferation of progenitor cells, leading to excess production of platelets. This condition has been termed myeloproliferative neoplasm (MPN). Presently, the spectrum of driver mutations of MPN includes point mutations of TpoR, JAK2 as well as +1 frameshift mutations of CALR which is an endoplasmic reticulum chaperone [4]. The activation of the TpoR either by TPO or by the activating mutations require its localization at the cell surface. Therefore, mechanisms of TpoR anterograde traffic that control the levels of the receptor on the cell surface are crucial to its function and the development of myeloproliferative neoplasm. Like most cytokine receptors, TpoR is trafficked through a Golgi-dependent secretion pathway [5]. However, the presence of JAK2 V617F or TpoR mutations such as TpoR P106L has been postulated to favor an unconventional autophagosome-lysosome-dependent secretion pathway [6,7,8]. Although increased localization of TpoR with lysosomes or LC3 positive vesicles has been shown, questions remained whether such localization indicated enhanced TpoR degradation in autophagosomes-lysosomes instead of secretion [9]. Moreover, the mechanism by which TpoR entered the Golgi-independent and autophagosome-lysosome-dependent secretion is unknown.
Golgi-independent unconventional protein secretion pathway has been documented for several proteins as well as the SARS-CoV-2 virus egress [10, 11]. Reports have indicated that proteins lacking signal peptide such as yeast Acb1 (a homolog of Dictyostelium discoideum AcbA), IL1β (interleukin 1 beta), ferritin and IDE (insulin-degrading enzyme) were secreted using GRASP and autophagy-based mechanism [12,13,14,15] suggesting probable convergence of GRASP and autophagy pathways. Recently their role in the unconventional traffic of proteins with signal sequences such as CFTR has been reported [16].
In this article, we used a pH-dependent FRET construct [17] of TpoR (SRAI-TpoR) and demonstrated the presence of TpoR with pH-quenched FRET on the cell surface. We further showed that while JAK2 V617F promoted lysosome-autophagosome mediated secretion, presence of CALR mutations blocked the unconventional traffic of TpoR. Finally, we showed that the unconventional traffic through autophagosomes-lysosomes was dependent upon Rab1A.
Methods
Cell culture and transfection
HEK293 cells, U2OS cells and DU145 cells were cultured in DMEM (Dulbecco Modified Eagle Medium) (Himedia, catalog no. AL007A) with 10% heat-inactivated FBS (Fetal Bovine Serum) and 1% Streptomycin, and Penicillin at 37 °C with 5% CO2. HEL cells and K562 cells were cultured in RPMI-1640 (Roswell Park Memorial Institute 1640) (Himedia, catalog no. AL199A) with 10% heat-inactivated FBS (Fetal Bovine Serum) and 1% Streptomycin, and Penicillin at 37 °C with 5% CO2. HEK293 cells were seeded on 6 well plates (at 3*105 cells/well) while DU145 cells were plated on a 60 mm culture dish (at 6*105 cells/dish) for immunoblotting and transfected with 4 to 6 μg of indicated plasmids using Lipofectamine™ 2000 (Invitrogen, Thermofisher Scientific) according to manufacturer’s instruction. The cells were plated on poly-L-lysine coated coverslips for immunofluorescence. U2OS cells, HEL cells and K562 cells were seeded on ibidi-treat 35 mm imaging dish (ibidi, Germany) for time-lapse imaging and transfected with the mentioned plasmid/siRNA. Cell lines were obtained from National Centre for Cell Science, Pune, India and authenticated at source. HEL cells were a kind gift from the lab of Dr. William Vainchenker, Institut Gustave Roussy, France and authenticated at source.
Inhibitor treatment
For lysosomal and proteasomal inhibition, 13 h post-transfection cells were treated with 100 nM Bafilomycin A1 (MedChemExpress, catalog no.- HY-100558) or 5 μM MG132 (MedChemExpress, catalog no.- HY-13259) for 5 h.
Immunoblotting
Cells were harvested, rinsed with PBS (Phosphate-buffered saline) and resuspended in NP-40 lysis buffer (50 mM Tris pH7.4, 150 mM NaCl, 1 mM EDTA, 15% glycerol, 1% NP40) to which 1 mM PMSF (phenylmethylsulfonyl fluoride) and protease inhibitor cocktail were freshly added. Cells were centrifuged at 10,000 × g for 15 min at 4 °C and the lysate was collected in a fresh tube. An equal amount of protein was loaded onto SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then probed with primary antibodies overnight at 4 °C. The membrane was incubated with HRP-conjugated secondary antibodies and developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fischer, catalog no.-1863096) and imaged by ImageQuant™ Chemiluminescent Imaging system, LAS 500 (GE, USA). Details of the antibodies are given in the supplementary methods.
Endo H and PNGase F digestions
HEK/DU145 cells were harvested 24 h following transfection, and K562 cells were harvested 24 h post electroporation and lysed using NP-40 lysis buffer. Cell lysates were left untreated or digested with Endo H (Endoglycosidase-H) (New England Biolabs, catalog no.-P0702S) or PNGase F (Peptide N-glycosidase F) (New England Biolabs, catalog no.-P0704S) as per the protocol provided by New England BioLabs. 7% SDS-PAGE or 4–15% gradient SDS PAGE (Biorad, cat. no. 4561085) was run to separate the proteins and then the blot was probed with an anti-HA antibody.
Surface pulldown
HEK293/DU145 cells were washed with ice-cold HBSS and then resuspended in 50 μl of 10 mg/ml EZ-LinkTM Sulfo-NHS-S-S-Biotin (0.5 mg/ml final concentration) (Thermo Scientific, catalog no. 21331) following which the cells were incubated for 30 min at 4 °C on a rotor to label the cell surface proteins. After biotinylation, the cells are washed with HBSS containing 5 mM Tris pH7.4 thrice. For surface pulldown, cells were lysed. 50 μl of lysate was kept aside as input and the remaining lysate was incubated with streptavidin agarose beads (Thermo Scientific, catalog no. S951) overnight at 4 °C under rotating conditions. The beads were centrifuged at 2000 × g for 5 min and then washed thrice with NP-40 lysis buffer containing protease inhibitor cocktail and PMSF. The beads were resuspended in 2X Laemmli buffer supplemented with 1 mM DTT and heated for 3 min at 95 °C. The digested proteins were detected by western blot using anti-HA antibody.
Coimmunoprecipitation
HEK cells co-transfected with TpoR along with either CALR del52 H170A or del52 Y109F/D135L were harvested and lysed with NP-40 lysis buffer. 50 µl of the lysate was kept as input and 400 µg in 400 µl of protein was used for co-immunoprecipitation. Anti-Flag or Anti-HA antibody were added to the cell lysate and then incubated for 4 h under rotating conditions at 4 °C. 50 μl of the Protein G PLUS Agarose beads (BioBharti, catalog no. BB-PG001PC) was added to the antibody-bead complex was centrifuged at 2000 × g for 5 min. The supernatant was discarded and the immunoprecipitate was washed with NP-40 lysis buffer, resuspended in 2X Laemmli buffer containing 1 mM DTT and analyzed by western blotting.
Immunofluorescence and confocal microscopy
HEK293 cells, DU145 cells and HEL cells were grown overnight on coverslips coated with poly-L-lysine. 24 h after transfection, the cells were fixed with 4% formaldehyde, then permeabilized and blocked with 0.2% Triton-X100 in PBS containing 10% FBS. The fixed cells were incubated overnight with a primary antibody at 4 °C in a humidified chamber. Following this, the cells were incubated with the required secondary Alexa Fluor-tagged secondary antibody for 2 h at room temperature. Further details of the secondary antibodies may be found in the supplementary methods. The slides were inspected by Leica TCS SP8 Confocal Laser Scanning Microscope equipped with a 63X oil-immersion objective lens. The images were further processed by ImageJ software.
Flow cytometry
Cells were rinsed with PBS, trypsinized and collected. The cells were stained with APC mouse anti-human CD110 antibody (BD PharmingenTM, catalog no. AB_10894008) in the dark for 30 min to mark the surface thrombopoietin receptor. Following PBS wash, the cells were analyzed on a flow cytometer (BD Fortessa X20).
Statistical analysis
Statistical significance was computed using the Student’s t test, ANOVA test and Wilcoxon Signed-Rank test as applicable to the various data sets. Graphs were plotted on MS Excel.
Results
pH-dependent ratiometric FRET shows a lysosome-mediated TpoR secretion
We used a TOLLES-YPet FRET pair and fused it to the N-terminus of TpoR henceforth called SRAI-TpoR (Fig. 1A). Low pH specifies the pH of the lumen of lysosomes which ranges from 4.0 to 5.0. The pH of the endoplasmic reticulum is 7.4 ± 0.2 [18] and the Golgi apparatus is 6.2 ± 0.4 [18]. The pH of autophagosome before fusion with lysosomes is 6.0 [19]. The TOLLES-YPet FRET pair shows FRET quenching at pH ≤5 that is irreversible upon further increase in pH [17]. Thus, TpoR that is resident in low pH compartments (lysosomes) or has passed through the low pH compartments (secretory lysosomes) to reach the cell surface would show FRET quenching. The SRAI-TpoR was expressed in U2OS cell line along with JAK2 WT. U2OS with its large diameter is amenable to live cell imaging of cellular ultrastructure and vesicle traffic. We observed TpoR with strong FRET signal in cells expressing JAK2 WT (Fig. 1B). Additionally, distinct puncta were observed for FRET quenched TpoR (indicated with an arrow). This indicated the presence of TpoR in low pH compartments. Bafilomycin A1, a V-ATPase inhibitor disrupts the fusion of autophagosome and lysosome by preventing acidification of autolysosome [20]. Interestingly, when lysosome acidification was inhibited using Bafilomycin A1, TpoR with strong FRET emission was observed without any FRET quenched puncta (Fig. 1B). Strikingly, when JAK2 V617F was expressed, both FRET and FRET quenched TpoR puncta were prominent. We further analyzed the surface SRAI-TpoR in the presence of JAK2 WT, JAK2 WT + Bafilomycin A1 and JAK2 V617F. We again observed FRET signal at the surface both in the presence and absence of Bafilomycin A1 (Fig. 1C). Strikingly, FRET quenched TpoR was prominently observed at the surface in the presence of JAK2 V617F (Fig. 1C, D). Thus, the SRAI-TpoR construct indicated the presence of receptors that have passed through the low pH compartments to reach the cell surface possibly through a Golgi-independent unconventional secretion.
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.
Effect of JAK2 on the unconventional secretion of TpoR
To further investigate the unconventional secretion of TpoR, HEK293 cells were transfected with control, JAK2 WT or JAK2 V617F. Of note, HEK cells showed low expression of endogenous JAK2. The localization of TpoR in lysosomes and autophagosomes was studied through immunofluorescence and confocal microscopy. The confocal images depict the colocalization of TpoR in the presence of endogenous JAK2, overexpression of JAK2 WT and JAK2 V617F with the markers for lysosome (LAMP1), and autophagosome (LC3), the organelles involved in unconventional protein secretion (Fig. 2A). The percentage of LAMP1+ lysosomes colocalizing with TpoR was found to be higher in JAK2 WT transfected cells when compared to JAK2 V617F or cells with endogenous JAK2 (Fig. 2A, B). Strikingly, the percentage of LC3+ vesicles with TpoR was significantly high in cells expressing JAK2 V617F (Fig. 2A, B). This indicated that TpoR followed both conventional Golgi-dependent and unconventional lysosome-autophagosome-dependent anterograde traffic routes in the presence of JAK2 WT as well as V617F. Furthermore, the Golgi-independent traffic of TpoR was preferred in the presence of JAK2 V617F. TpoR has four sites for N-glycosylation and the addition of complex sugars coincides with Golgi-dependent anterograde traffic of the receptor [21]. Therefore, we performed enzymatic digestion to study the N-glycosylation pattern of TpoR. Endo H is an enzyme that cleaves glycoprotein with high mannose content. Thus, Endo H cleaves protein that has been modified in the endoplasmic reticulum but that has not been modified by the Golgi resident glycosidases and glycosyltransferases. However, PNGase F is an enzyme that cleaves all the N-linked oligosaccharides present in a glycoprotein irrespective of their mannose content. Thus, PNGase F treatment produces proteins devoid of N-glycosylation. Endo H-resistant form corresponding to the complex glycosylated (mature) receptor was observed in the case of JAK2 WT overexpressing cells (Fig. 2C). However, the mature band was absent in the presence of JAK2 V617F. To further explore the N-glycosylation status of surface TpoR, we performed surface immunoprecipitation followed by Endo H digestion of surface TpoR in cells transfected with JAK2 WT or JAK2 V617F (Fig. 2D) and calculated the mature to the immature ratio of surface TpoR. We observed a decrease in the mature-to-immature ratio in the case of cells expressing JAK2 V617F compared to JAK2 WT (Supplementary Fig. 1A). Taken together, the data showed that TpoR trafficked through the autophagosome-lysosome pathway both in the presence of JAK2 WT as well as JAK2 V617F. Additionally, TpoR favored the unconventional pathway in the presence of JAK2 V617F.
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 shows a lower molecular weight as indicated with an arrow head while the complex glycosylated TpoR retains its original position as indicated by asterisk. Representative image of the blot is shown (n = 3). D HEK293 cells transfected with TpoR and JAK2 WT or JAK2 V617F were harvested 24 h after transfection and incubated with 50 μl of 10 mg/ml NHS-S-S-Biotin for 30 min. Cells were then lysed and incubated with streptavidin beads followed by treatment with Endo H. Western blot analysis was performed. Representative image of the blot is shown (n = 3).
Bafilomycin A1 blocks the accumulation of TpoR in LAMP1+ vesicles in the presence of JAK2 V617F
To assess the contribution of autophagosome-lysosome towards TpoR degradation vis-à -vis secretion, we examined the colocalization of TpoR with lysosomes and autophagosomes in cells treated with Bafilomycin A1 and MG132. Bafilomycin A1, a V-ATPase inhibitor disrupts the fusion of autophagosome and lysosome by preventing acidification of autolysosome [20] while MG132 is a proteasomal inhibitor. HEK cells were co-transfected with TpoR and either JAK2 WT or JAK2 V617F followed by treatment with 100 nM Bafilomycin A1 or 5 μM MG132 for 5 h. Immunofluorescence staining for TpoR, LAMP1 and LC3 was performed in these cells. As expected, treatment with Bafilomycin A1 reduced the percentage of lysosomes (LAMP1+) containing TpoR both in the presence of JAK2 WT or JAK2 V617F (Fig. 3A, B). The decrease was greater in JAK2 V617F corroborating our earlier observation that TpoR preferred the unconventional traffic in the presence of JAK2 V617F (Fig. 2B). A similar decrease in the lysosomal localization of TpoR was observed when the cells were treated with MG132. It was earlier shown for the secretion of extracellular vesicle-associated cargo that Bafilomycin A1 treatment increased autophagosome-associated secretion [22]. Therefore, we checked whether such a scenario was true for TpoR. Treatment with Bafilomycin A1 was found to decrease the percentage of LC3+ autophagosomes containing TpoR (Fig. 3A, B). No difference was observed between the percentage of autophagosomes containing TpoR in the case of JAK2 WT or JAK2 V617F. Next, we checked the surface localization of the receptor with Bafilomycin A1 treatment in the presence of JAK2 WT and JAK2 V617F (Fig. 3C). Our results showed no significant change in the surface expression of the receptor. However, TpoR expression increased at the cell surface when treated with MG132 (Fig. 3C). The low levels of localization of TpoR in autophagosomes along with an unaltered surface expression upon Bafilomycin A1 treatment indicated that the unconventional traffic of TpoR was not a mere escape from lysosome-mediated degradation (Fig. 3B, C). To understand whether the receptors reaching the surface after treatment with Bafilomycin A1 were using the Golgi-dependent or independent traffic, we analyzed the surface TpoR for the N-glycosylation pattern. We observed mature N-glycosylated TpoR in the presence of JAK2 V617F that was not observed in untreated cells (Fig. 3D). Mature TpoR was not observed in the case of JAK2 V617F cells treated with MG132 (Fig. 3D). This indicated that blocking the autophagosome-lysosome traffic was perhaps compensated through a Golgi mediated traffic of TpoR in cells expressing 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 image of the flow analysis is shown (n = 3). D HEK293 cells with the indicated conditions was incubated with 50 μl of 10 mg/ml NHS-S-S-Biotin for 30 min. Cells were then lysed and incubated with streptavidin beads overnight at 4 degrees and then treated with Endo H and subjected to immunoblotting.
TpoR preferentially traffics through autophagosome-lysosome in the presence of JAK2 V617F
To further confirm our observations of autophagosome-lysosome mediated secretion of TpoR, we used the ATG5 deficient cell line DU145. DU145 cells harbor a mutation in the splice site of ATG5 leading to premature termination of protein translation and loss of Atg5 dependent autophagosomes [23]. We confirmed the absence of LC3-II in the DU145 cell line and its restoration when supplemented with exogenous ATG5 (Supplementary Fig. 1D). We also confirmed that induction of autophagy did not change the expression of TpoR indicating that restoration of autophagy did not promote the degradation of TpoR (Supplementary Fig. 1D). In DU145 cells, the expression of TpoR in the presence of JAK2 WT or V617F resulted in a comparable co-localization of TpoR in LAMP1+ lysosomes (Fig. 4A, B). To check if exogenous ATG5 could reverse the effect on TpoR localization in lysosomes, we overexpressed ATG5 in DU145. In the presence of exogenous ATG5, the percentage of LAMP1+ lysosomes with TpoR decreased in the presence of JAK2 WT and JAK2 V617F in comparison to cells lacking Atg5 (Fig. 4A, B). This hinted that the autophagosomes routed TpoR to the surface. To understand whether such a scenario is true, we observed the surface expression of TpoR. Indeed, with the restoration of autophagosomes, the surface expression of TpoR increased in the presence of JAK2 V617F (Fig. 4C). In contrast, the surface expression of TpoR in DU145 cells expressing TpoR and JAK2 WT decreased when autophagy was restored (Fig. 4C). Together, our data shows that JAK2 V617F dependent TpoR traffic is biased towards autophagosome-lysosome mediated unconventional secretion.
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).
To further establish this bias, we used a double block system where autophagosome deficient DU145 cells were blocked for ER to Golgi traffic using overexpression of Syntaxin 5. In the presence of Syntaxin 5, the surface expression of TpoR showed a stark decrease in the presence of JAK2 WT and JAK2 V617F (Fig. 4C). This was true even when Atg5 was overexpressed along with Syntaxin 5. Together, our data showed that JAK2 V617F biased the traffic of TpoR through the unconventional autophagosome-lysosome pathway.
CALR mutations block unconventional traffic of TpoR
Like JAK2 V617F, frameshift mutations of calreticulin (CALR) result in cytokine-independent activation of TpoR. The mutations are classified into Type I mutation (CALR del52) and Type II mutation (CALR ins5) depending upon the extent of retention of the CALR WT tail sequence [24]. Mutant CALRs bind to the Asn residue of TpoR and possibly co-traffic to the cell surface [25]. We examined the anterograde traffic pathway followed by TpoR in the presence of CALR mutants by measuring the percentage of lysosomes and autophagosomes showing co-localization of TpoR. The confocal microscopy images depicted that TpoR colocalized with LAMP1+ vesicles in the case of CALR ins5 (Type II mutation) but the colocalization was extremely low in CALR del52 (Type I mutation) expressing cells (Fig. 5A, B). Strikingly, the percentage of LC3+ autophagosomes containing TpoR was negligible in the case of both Type I and II CALR mutations (Fig. 5A, B). The Endo H digestion of TpoR in the presence of CALR ins5 and del52 mutations showed a strong Endo H-resistant band (Fig. 5C) indicating that the receptor followed the Golgi-dependent pathway in the presence of these CALR mutants. Moreover, surface immunoprecipitation showed the presence of Endo H resistant TpoR on the surface (Supplementary Fig. 1E). This was also observed in the SRAI-TpoR construct where TpoR showed intense FRET signal in the presence of CALR del52 (Fig. 5D). The H170 residue in CALR WT has been previously shown to be essential for the structural integrity and chaperone activity of the protein [26]. H170A is the residue involved in zinc binding and mutation in this residue prevents homo-multimerization of CALR del52 and hinders binding to TpoR [27]. The D135L mutant is a lectin-binding deficient CALR del52 which allows homo-multimerization but abrogates the binding of CALR del52 to TpoR [25]. To confirm our results, we used the structural and lectin-binding/chaperone mutations of CALR del52 (del52 H170A and del52 Y109F/D135L). Co-immunoprecipitation of TpoR with these mutants showed that these mutations prevented CALR del52 from interacting with the receptor (Fig. 5E). Furthermore, the mutations restored the lysosomal localization of TpoR (Fig. 5F, G). Thus, abrogation of the interaction of CALR del52 with TpoR could restore the Golgi-independent traffic of the receptor. Together, the data shows that TpoR is restricted to the Golgi-dependent secretion pathway in the presence of Type I and II CALR mutations.
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 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 (right panel) to identify areas with (yellow) and without FRET (red). (right) Overlay of FRET signal with donor TOLLES signal. The scale bar represents 10 μm. E HEK293 cells expressing TpoR along with JAK2 WT and CALR del52 mutants H170A or Y109F/D135L were lysed 24 h after transfected and co-immunoprecipitated using anti-HA antibody followed by immunoblotting with an anti-Flag antibody for the CALR del52 double mutants. F The subcellular localization of TpoR was analyzed by confocal microscopy. HEK293 cells were transfected with TpoR along with JAK2 WT and CALR del52 mutants H170A or Y109F/D135L and stained for organelle marker lysotracker (green) and antibodies against HA tag for TpoR (red). The scale bar represents 10 μm. Arrowhead represents colocalization of TpoR with lysotracker. G The signal intensities of TpoR puncta and lysotracker in (F) 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 = 209–1295 for lysotracker.
Unconventional secretion of TpoR requires RAB1A
Rab proteins are small GTPases that modulate vesicular traffic by recruiting effector molecules at target membranes upon their activation [28]. We hypothesized that the traffic of TpoR through Golgi-dependent and autophagosome-lysosome-dependent routes may involve distinct Rab proteins. Various Rabs are known to help in the maintenance of Golgi ultrastructure and vesicle traffic including Rab 6 [28]. Additionally, a recent report indicated a unique localization of Rab 1A on autophagosomes [29]. Therefore, we checked whether Rab 1A and Rab 6 could differentiate TpoR undergoing autophagosome-lysosome and Golgi traffic respectively. Confocal imaging of TpoR in the presence of JAK2 WT and JAK2 V617F showed extensive colocalization with Rab 1A vesicles (Fig. 6A). However, such colocalization was not observed in the presence of CALR del52 or CALR ins5. Rab 1B showed a similar trend, although the extent of localization of TpoR in Rab 1B vesicles was lower compared to Rab 1A. Thus, autophagosome-lysosome dependent traffic of TpoR may be dependent upon Rab 1A. On the other hand, TpoR showed extensive colocalization with Rab 6 vesicles in the presence of CALR del52 and ins5 with much reduced colocalization in the presence of JAK2 WT or V617F. This confirmed our earlier observation that CALR del52 and CALR ins5 restricted TpoR to the Golgi-dependent secretion (Fig. 5). To further confirm whether Rab 1A could indeed traffic TpoR through autophagosome-lysosomes, we used a siRNA mediated knockdown of Rab 1A. Knockdown of Rab 1A was previously reported to induce Golgi fragmentation that could be reversed by the simultaneous overexpression of Rab 1B [29]. We also observed similar Golgi fragmentation in the presence of siRab 1A (Supplementary Fig. 1F). To ensure that we perturb Rab1A without disturbing the Golgi ultrastructure, we used the overexpression of Rab 1B along with siRab 1A. Since TpoR showed a bias towards autophagosome-lysosome dependent traffic in the presence of JAK2 V617F, we verified whether the same could be reversed by the knockdown of Rab 1A. Following knockdown of Rab 1A, the localization of TpoR in lysosomes showed a non-significant reduction. However, Rab 1A knockdown caused a significant decrease in the localization of TpoR in autophagosomes (Fig. 6B, C). This indicated that TpoR utilized Rab 1A vesicles for localization in autophagosomes. Finally, we looked at the glycosylation pattern of the surface localized TpoR in the presence of JAK2 V617F alone on in combination with siRab 1A. We observed the presence of a complex glycosylated Endo H resistant TpoR on the cell surface specifically upon knockdown of Rab 1A (Fig. 6D). This indicated that Rab 1A was essential for Golgi-independent traffic of TpoR. Moreover, similar to previous result (Fig. 3E), blocking of autophagosome-lysosome mediated traffic diverted TpoR towards Golgi-dependent secretion.
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 791–1208 for LC3. D HEK293 cells transfected with TpoR along with JAK2V617F and scrambled siRNA or siRNA against Rab1A were harvested 24 h after transfection and incubated with 50 ul of 10 mg/ml NHS-S-S-Biotin for 30 min. Cells were then lysed and incubated with streptavidin beads overnight at 4 degrees and then treated with Endo H and subjected to immunoblotting. Representative image of the blot is shown (n = 3).
TpoR W515L shows a partial Golgi-independent secretion
We next analyzed the effect of TpoR W515L driver mutation on the traffic of the receptor. We again generated a SRAI-TpoR W515L construct and observed for FRET quenching to detect lysosome resident receptor or receptors that have trafficked through lysosomes. We observed the presence of high FRET intensity puncta in U2OS cells transfected with SRAI-TpoR W515L (Fig. 7A). We also observed puncta that showed FRET quenching indicating a lysosomal localization or lysosomal traffic of the receptor. We further analyzed the SRAI-TpoR W515L FRET signal at the cell surface (Fig. 7B). We observed both FRET signal and FRET quenched signal at the cell surface indicating that both Golgi dependent and independent routes may be involved in the traffic of TpoR W515L. Next, we used immunofluorescence followed by confocal microscopy to check the presence of TpoR W515L in autophagosomes and lysosomes. While nearly 77% of the LC3 puncta colocalized with TpoR WT, 56% of the LC3 puncta showed colocalization with TpoR W515L (Fig. 7C, D). This hinted towards a partial Golgi-independent secretion of TpoR W515L. We further tested using Endo H and PNGase F digestion of TpoR W515L from whole cell lysate and surface immunoprecipitates (Fig. 7E). We observed a predominance of high mannose immature TpoR in both the whole cell lysate and the cell surface indicating the traffic of TpoR W515L through the Golgi-independent pathway. Finally, using live cell imaging, we observed that TpoR W515L showed a partial co-localization with Rab 1 A and Rab 6 with little to no co-localization in Rab 1B (Fig. 7F). Together, these data indicate towards a partial Golgi-independent traffic of TpoR W515L.
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. HEK293 cells were transfected with TpoR W515L along with JAK2 WT and stained for organelle marker (green; LAMP1 or LC3) and antibodies against anti-HA tag for HA-TpoR W515L (red). The scale bar represents 10 μm. Arrowhead represents colocalization of TpoR W515L in LAMP1/LC3 vesicles. D The signal intensities of TpoR W515L puncta and LAMP1 or LC3 puncta in (C) were calculated as gray values and the percentage colocalization of TpoR W515L 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, no. of vesicles n = 915 for LAMP1 and n = 390 for LC3. E HEK293 cells expressing TpoR W515L along with JAK2 WT were lysed 24 h post-transfection and treated with Endo H or PNGase F and subjected to Western blot analysis. HEK293 cells transfected with TpoR W515L and JAK2 WT were harvested 24 h after transfection and incubated with 50 μl of 10 mg/ml NHS-S-S-Biotin for 30 min. Cells were then lysed and incubated with streptavidin beads followed by treatment with Endo H. Western blot analysis was performed. F The subcellular localization of TpoR W515L was analyzed by live cell imaging and confocal microscopy. U2OS cells were transfected with TpoR W515L 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.
TpoR traffic in haematopoietic cell lines is dependent upon the presence of MPN driver mutations
We used the HEL cell line that expresses endogenous JAK2 V617F. We introduced the SRAI-TpoR construct into HEL cells and observed the presence of both FRET and FRET quenched signal (Fig. 8A). Next, we treated the HEL SRAI-TpoR WT cells with Bafilomycin A1 to prevent lysosomal acidification and observed a complete loss of FRET quenched puncta (Fig. 8A). This indicated that JAK2 V617F promoted the traffic of TpoR WT through acidic lysosomes. We next probed through live-cell imaging the localization of TpoR in Rab 1A, Rab 1B and Rab 6 vesicles in HEL cells (Fig. 8B). We observed strong co-localization of TpoR WT with Rab 1A along with negligible co-localization with Rab 6. This was again indicative of a preference towards autophagosome-lysosome mediated traffic of TpoR in HEL cells. To confirm these results, we performed immunofluorescence followed by confocal microscopy to observe the localization of TpoR in autophagosomes and lysosomes. Similar to our earlier observation with JAK2 V617F (Fig. 2A, B), HEL cells showed high degree of co-localization of TpoR in autophagosomes and lysosomes (Fig. 8C). Interestingly, the percentage of autophagosomes with TpoR localization decreased significantly upon knockdown of Rab 1A (siRab 1A + Rab 1B) (Fig. 8D). This indicated that the observed JAK2 V617F induced Golgi-independent traffic of TpoR in HEK and U2OS cell lines was indeed representative of the scenario in haematopoietic cells. Finally, we used the K562 cell line to overexpress SRAI-TpoR WT along with CALR del52 (Supplementary Fig. 2A). K562 cells produced faint SRAI donor-acceptor signals. Nevertheless, we were able to detect FRET signal in K562 cells in the presence of CALR del52 (Supplementary Fig. 2B). Moreover, Endo H digestion of the cell lysate of K562 cells expressing TpoR and CALR del52 confirmed the presence of mature glycosylated TpoR confirming our previous results that showed a Golgi-dependent traffic of the receptor in the presence of CALR del52.
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 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. D The signal intensities of TpoR puncta and LAMP1 or LC3 puncta in C 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 = 250–500 for LAMP1and 100–250 for LC3.
Taken together, our data establishes the autophagosome-lysosome mediated traffic of TpoR. Moreover, we showed that the autophagosome-lysosome mediated traffic as marked by Rab 1A was preferred in the presence of JAK2 V617F, partially utilized by TpoR W515L but was blocked by CALR mutations.
Discussion
The traffic of TpoR to the cell surface is crucial to the receptor-associated signaling pathways and the production of platelets [5]. Testament to this was the occurrence of TpoR mutations associated with hereditary thrombocytopenia (TpoR R102P and TpoR P106L) that resulted in abnormally low platelet numbers [30, 31]. Moreover, the platelet surface TpoR was necessary to scavenge serum thrombopoietin (TPO) thereby maintaining platelet homeostasis [32]. How the receptor reached the surface has therefore been of great interest. TpoR was documented to be secreted to the surface through the Golgi-dependent anterograde pathway. In fact, Golgi glycosidases and glycosyltransferases were found to be crucial to the surface expression and stability of the receptor [21]. TpoR was reported to be degraded by proteasome-lysosome machinery in the context of JAK2 V617F leading to lower cell surface expression of TpoR [6]. Moreover, it was observed that JAK2 WT protected TpoR from lysosomal degradation. Unconventional traffic of TpoR was suggested to occur in response to JAK2 V617F; although lysosome-autophagosomal localization of TpoR was also observed with JAK2 WT [33]. Yet questions remained whether the observed localization in the autophagosome-lysosomal compartment was an indicator of TpoR degradation or a true secretion event. Such a lysosome-mediated TpoR degradation was recently documented in the context of CALR mutations [9].
With the help of the pH-sensitive FRET construct SRAI-TpoR, we established the autophagosome-lysosome-mediated unconventional secretion of TpoR. TpoR was found to utilize the unconventional secretion pathway in the presence of both JAK2 WT and V617F. However, TpoR preferred the autophagosome-lysosome-mediated secretion in the presence of JAK2 V617F. In the presence of Bafilomycin A1, the FRET puncta observed in the cells could be derived from Golgi as well as from lysosomes with increased pH. However, surface TpoR FRET signal which was uniquely high in Bafilomycin A1 treated condition compared to JAK2 V617F indicated that in the presence of Bafilomycin A1, the surface TpoR originated from the Golgi. Further experiments highlighted this scenario where in the presence of JAK2 V617F, Bafilomycin A1 was able to induce an Endo H resistant TpoR (Golgi trafficked) on the cell surface that was not observed in control JAK2 V617F cells. Interestingly, when the unconventional secretion pathway of TpoR was blocked using Bafilomycin A1 or ATG5 deficiency, the receptor was found to undergo Golgi-dependent secretion. This showed that targeting the unconventional secretion in JAK2 V617F would not be a viable therapeutic approach. CALR mutants restricted the traffic of TpoR to the Golgi-dependent secretion. It may be recalled that CALR mutants favored the stability of the TpoR [25]. Thus, the low prevalence of the receptor in lysosome-autophagosomes was an indication of mutant CALR-induced stabilization of TpoR. TpoR W515L was previously reported to utilize the Golgi-dependent secretion pathway [34, 35]. We observed a partial Golgi-independent traffic of TpoR W515L whereby FRET quenched puncta and Endo H sensitive TpoR were both detected on the surface. Moreover, our data on TpoR W515L glycosylation agreed with the previous reports [34]. Taken together, while TpoR preferred the Golgi-independent traffic in the presence of JAK2 V617F mutation, Golgi-independent traffic was partially utilized by TpoR W515L and completely blocked by CALR mutations.
The Golgi-dependent and independent traffic routes necessitate the utilization of different Rabs and associated activator proteins and kinesins. Therefore, we tested if we could parse out the two secretion routes using specific Rabs. In our experiments, Rab 1A appeared to be relevant for autophagosome-lysosome mediated traffic of TpoR while Rab 6 marked the Golgi-dependent traffic vesicles of TpoR. Knockdown of Rab 1A decreased the localization of TpoR in lysosomes in the presence of JAK2 V617F. Although the decrease was statistically significant, it did not eliminate the unconventional secretion. This may be attributed to the degeneracy amongst the Rab proteins where other Rabs could compensate for the loss of Rab 1A. Notwithstanding, Rab 1A appears to be a marker of the unconventional traffic vesicles of TpoR. It remains to be seen whether Rab 1A acts as a general marker for cargo undergoing unconventional secretion. Our results further hint at possible therapeutic interventions by targeting Rab GTPases specifically Rab 6 in the case of CALR del52.
Taken together, we established the unconventional traffic of TpoR through autophagosome-lysosome mediated anterograde traffic. We showed that in the presence of JAK2 V617F, the Rab 1A mediated unconventional traffic was preferred over Rab 6-mediated Golgi-dependent secretion pathway. TpoR W515L utilized both Rab 1A and Rab 6 mediated traffic. Finally, the CALR mutations restricted the TpoR to the Golgi-dependent traffic marked by Rab 6.
Data availability
All data has been shown in the article.
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Acknowledgements
We acknowledge Atushi Miyawaki (Univ. of Tokyo, Japan) for the TOLLLES-YPet (SRAI) FRET parent construct. We acknowledge Stefan N. Constantinescu (LICR, Brussels) for the HA-TpoR, CALR del52 H170A and CALR del52 Y109F/D135L constructs. We acknowledge Nava Segev (Uni. of Illinois, USA) for helping us secure the plasmids of Rab 1A and Rab 1B created by Mitsunori Fukuda (Tohoku University, Japan). We acknowledge Suzanne R Pfeffer (Stanford, USA) for gifting the Rab 6 construct. We acknowledge Min Goo Lee (Yonsei University College of Medicine, Seoul) for HA-Stx5. We acknowledge IIT Delhi CRF and SATHI for the microscopy facility. SC, SS, AS, FA and SN acknowledge their respective fellowships from DBT, IIT Delhi, DBT, DBT and UGC. AR acknowledges funding from DBT/Wellcome Trust India Alliance (IA/I/19/1/504285), SERB (CRG/2023/003853) and IIT Delhi start-up grant.
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AR conceptualized the project, secured the funding, designed the experiments, analyzed the data and wrote the original draft. SC conceptualized the project, designed and performed the microscopy and biochemistry experiments, analyzed the data, performed ImageJ analysis and wrote the manuscript. SS operated the flow cytometer, analyzed the flow data and performed the ImageJ analysis for quantification of images. AS produced crucial plasmids (CALR constructs) for the project. FA produced crucial reagents (TpoR mCherry construct) and performed the ImageJ analysis for quantification of images. SN produced crucial plasmids (CALR constructs) for the project. All authors reviewed and edited the final manuscript.
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Chowdhury, S., Shrivastva, S., Singh, A. et al. Autophagosome-lysosome mediated secretion of the thrombopoietin receptor is modulated by distinct driver mutations of myeloproliferative neoplasm. Leukemia 39, 2181–2195 (2025). https://doi.org/10.1038/s41375-025-02676-6
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DOI: https://doi.org/10.1038/s41375-025-02676-6
