MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells.
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PROVIDER: PRJNA779151 | ENA |
REPOSITORIES: ENA
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Project description:The lipid raft resident protein, MAL2, has been implicated as contributing to the pathogenesis of several malignancies, including breast cancer, but the underlying mechanism for its effects on tumorigenesis is unknown. Here, we show that MAL2-mediated lipid raft formation leads to HER2 plasma membrane retention and enhanced HER2 signaling in breast cancer cells. We employed proximity ligation assays (PLA) to define physical interactions between HER2 and MAL2 in lipid rafts and super-resolution structured illumination microscopy (SR-SIM imaging) to determine the structural organization of the HER2/Ezrin/NHERF1/PMCA2 protein complex. Formation of this protein complex maintains low intracellular calcium concentrations in the vicinity of the plasma membrane. Importantly, we also observed enhanced HER2-MAL2 protein interactions in lipid rafts in trastuzumab-resistant breast cancer cells. Our findings suggest that MAL2 is crucial for lipid raft formation, HER2 signaling and HER2 membrane stability in breast cancer cells, suggesting MAL2 as a potential therapeutic target.
2021-12-15 | GSE188513 | GEO
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| S-EPMC8762588 | biostudies-literature
Project description:Proteomics of membrane protrusions from activated neutrophils
2022-11-14 | MSV000090706 | MassIVE
Project description:This upload concerns 2 independent but related experiments that were searched together: a) To reveal proteins that consistently localise to actin-rich protrusions across different human migratory cell-lines, we profiled the distribution of cellular proteins between protrusions and cell-bodies in a panel of 5 established human cell-lines (see experimental design). Cell lines were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to the filter overnight. The next day, the media on top of the cells was refreshed, and media was also added to the bottom chamber in order to open the pores and allow formation of protrusions. Cells were allowed to form protrusions for 2 hrs before being fixed with ice-cold methanol. Cells were then rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of the filters in 2% SDS, 100mM Tris pH 7.5 b) To analyse the temporal dynamics of protein localisation to cell protrusions, we carried out a time-course spatial proteomics analysis of protrusions and cell-bodies in MDA-MB231 cells. Cells were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was refreshed, and the filters were either left with no media in the bottom chamber (closed pores), or media added to the bottom chamber (open pores) for different lengths of times (1, 2, 4 , & 8 hrs) to induce protrusion formation. Cells were then fixed with ice-cold methanol, rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of each filter by 2% SDS, 100mM Tris pH 7.5.
2020-10-20 | PXD021239 | Pride
Project description:We used a fractionation scheme to isolate protrusions and cell bodies from control or APC knockdown fibroblasts, which were induced to extend protrusions in response to addition of lysophosphatidic acid (LPA). Four replicate protrusion and cell body samples for each cell type were analyzed by RNA-Seq. To identify RNAs enriched in protrusions, a protrusion/cell body ratio was calculated. We find that ca. 7% of detected RNAs were enriched in protrusions of control cells. Knockdown of APC reduced the protrusion enrichement of a subset of these RNAs, indicating that they are dependent on APC for their localization. Localization of the remaining RNAs was not significantly affected upon APC knockdown suggesting that their localization is mediated by an APC-independent mechanism.
2017-10-11 | GSE82229 | GEO
Project description:We generated fibroblast lines overexpressing beta-globin constructs under a doxycycline-inducible promoter. These constructs contain the beta-globin coding sequence followed by either a control 3'UTR (HBB) or the Pkp4 3'UTR. The Pkp4 3'UTR carries a protrusion-targeting element and is expected to compete with localization of endogenous protrusion-localized mRNAs. We used a fractionation scheme to isolate protrusions and cell bodies from HBB and Pkp4 UTR-expressing fibroblasts, which were induced to extend protrusions in response to addition of lysophosphatidic acid (LPA). 4 replicate protrusion and cell body samples for each cell type were analyzed by RNA-Seq. To identify RNAs enriched in protrusions, a protrusion/cell body ratio was calculated. We find that ca. 4.5% of detected RNAs were enriched in protrusions of control HBB cells. Overexpression of the Pkp4 UTR reduced the protrusion enrichement of a subset of these RNAs, while the degree of localization of the remaining RNAs was not significantly affected.
2017-10-11 | GSE82230 | GEO
Project description:Deciphering the complex biology of cell migration is key to understand human-related disorders. During angiogenesis, endothelial cells engage in coordinated migration events to form new blood vessels from parental counterparts. However, while subcellular localisation of mRNAs and localised translation are fundamental steps between gene transcription and protein activity, whether local regulation of gene expression controls the complex morphogenetic process of angiogenesis has never been addressed. Here, we set out to investigate the cytoplasmic distribution of mRNAs in migratory endothelial cells. We isolated RNA from endothelial cell protrusions from their cell bodies and used RNA-sequencing to identify asymmetrically distributed transcripts. These studies identified a set of transcripts enriched in endothelial cell protrusions over cell bodies, which included classically protrusion-enriched mRNAs and other intriguing transcripts encoding proteins implicated in cell migration.
2020-09-07 | GSE133055 | GEO
Project description:3' mRNA-seq of protrusions and cell bodies of nt or LARP6 siRNA transfected MDA-MB231 cells. These data show that Ribosomal protein (RP) -mRNAs localization to cell protrusions is mediated by LARP6.
2022-10-03 | E-MTAB-8472 | biostudies-arrayexpress
Project description:3' mRNA-seq of protrusions and cell bodies of a panel of normal and malignant cell lines. These data show that Ribosomal protein (RP) -mRNAs localization to cell protrusions is a conserved phenomenon.
2020-10-30 | E-MTAB-8470 | biostudies-arrayexpress
Project description:Transcriptome analysis of specific mRNA interacting with KIF1C-GFP in HeLa Cells. RNA localization and local translation are involved in a wide range of cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC dependent manner, with important roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with many APC-dependent mRNAs and is required for their localization. Live cell imaging of localizing mRNAs further revealed rapid, active transport of single mRNAs over long distances, and showed that this requires both microtubules and KIF1C. Moreover, two color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3’UTR of localized mRNAs being sufficient to trigger KIF1C-dependent RNA transport and localization. These results reveal a general RNA transport pathway in mammalian cells, in which the KIF1C motor transports RNAs to cytoplasmic protrusions. Interestingly, KIF1C also transported its own mRNA suggesting a possible feedback loop acting at the level of mRNA transport.
2020-11-13 | GSE161316 | GEO