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Strategic Methods To ALK Inhibitor That Only A Few Know About by zhang qing
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Strategic Methods To ALK Inhibitor That Only A Few Know About |
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Research,Health,Press Release
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We previously established that a kinase dead mutant of NPM-ALK protein could not nucleate AGs . Consistent with this result, we observed that AGs disappear when NPM-ALK NIH3T3 cells are treated with an ALK specific inhibitor that does not modify the level of NPM-ALK but inhib?its its kinase activity .To test whether other ALK fusions nucleate AGs, we stained pre?viously described TPM3- and ATIC-ALK NIH3T3 cells with anti-ALK and anti-phospho-ALK antibodies. Confo?cal microscopy analysis showed that both cell types concentrate the ALK fusion proteins in cytoplasmic foci in their phosphorylated . Altogether, these results indicate that ALK inhibitor is present in its phospho?rylated form in AGs and that AG formation relies on the active tyrosine kinase domain of ALK fusions. It is well established that cytoplasmic foci such as PBs and SGs ac?cumulate mRNAs together with mRNA-binding proteins. As AGs contain the RNA-binding protein AUF1 , we fur?ther asked whether AGs also contained mRNAs. First, to test this hypothesis, we used ethidium bromide to fluorescently label endogenous RNAs in NPM-ALK NIH3T3 cells, as previously de?scribed . When cells were simultaneously stained with anti-ALK antibodies, we observed that ~50% of ALK-containing cytoplasmic foci also concentrate EtBr . Second, PABP was detected in AGs of NPM-ALK 3T3 cells stained both with anti-PABP and anti–phospho-ALK antibodies . Those results were indicative of the presence of mRNAs in AGs. To directly test this hypothesis, we transiently expressed both a lacZ reporter mRNA containing MS2-binding sites in its 3' untranslated region and a yellow fluorescent protein -MS2 fusion protein. When expressed alone, the YFP-MS2 protein was found exclusively in the nucleus, due to its nuclear localization signal . In contrast, coexpressing lacZ- 3'MS2 mRNA and YFP-MS2 allowed YFP-MS2 tethering to lacZ-3'MS2 mRNA and its subsequent export from the nucleus to the cytoplasm where it concentrates in PBs . In NPM-ALK transformed NIH3T3cells, the reporter mRNA is both visualized in PBs revealed by anti-DCP1 antibody and, although at a weaker intensity , in most AGs with a diameter > 0.1 µm . These data are in agreement with our previous findings showing that PBs and AGs are distinct structures , and reveal that AGs and PBs can harbor the same mRNA species. Finally, to know whether mRNAs contained in AGs were polyadenylated, we per?formed in situ hybridization using oligo probe. Concomitant im-munodetection of AGs using anti-phospho-ALK antibodies revealed that most large AGs contained both NPM-ALK and oligo . All together, those different approaches show that AGs concentrate mRNAs that are polyadenylated. To further characterize AG formation, we transiently expressed NPM-ALK as a GFP or Halo fusion protein in NIH3T3 cells. Those fusion proteins conserve their phosphorylation sta?tus as shown by Western blot analysis and nucleate cytoplasmic foci the number, shape, and size of which were identical to those observed in NPM-ALK sta?bly transformed NIH3T3 cells . We then visualized AG formation and movements in the cytoplasm conduct?ing both short and long term time-lapse analysis to track AG movements, as previously described for PBs . Using GFP-NPM-ALK tran?siently transfected cells, we observed that AGs rapidly grow to reach their standard size . Single particle tracking from short-range movies allowed us to classify AGs into three categories according to their roamed distances: <5 µm, 5–10 µm or >10 µm . The latter could be tracked for distances up to 18 µm . To compare AG and PB movements in the same cellular context, we transfected NIH3T3 cells with a GFP-DCP1a construct and observed similar tracking profiles for AGs and PBs, although AGs move globally slightly faster than PBs . Owing to this similarity, we tested whether AGs could associate to the micro?tubule cytoskeleton, as recently described for PBs . Using double staining with anti–a-tubulin and anti-ALK antibodies, we observed AGs dec?orating individual microtubule bundles in NPM-ALK NIH3T3 cells , sug?gesting that AG movements rely on their association with microtubules. This hypoth?esis was tested by treating cells with no-codazole, which binds to tubulin monomers and leads to microtubule destabilization. NIH3T3 cells were transiently transfected with NPM-ALK-GFP, and AG movements were observed before and after microtubule disruption. Short-range time-lapse analysis indicates that nocodazole significantly re?duces AG mobility . Comparison with PBs observed in GFP-DCP1a–expressing NIH3T3 cells shows that nocodazole treatment inhibits both AG and PB movements . Altogether, our results in?dicate that, like PBs, AGs use the microtubule network to roam the cytoplasm.
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