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A Proximity dependent labeling approach to define flotillin-2 interacting proteins within Membrane Domains of HeLa cells

ABSTRACT: The formation of plasma membrane microdomains, known as lipid rafts, is driven by specific interactions among cholesterol, sphingolipids, and certain proteins such as flotillins. Flotillins are known to oligomerize and associate with defined lipid assemblies, thereby promoting the coalescence and stabilization of these membrane rafts. Acting as scaffolding molecules, flotillins play critical roles in various cellular processes, including the spatial organization of signaling complexes and membrane compartmentalization. To identify flotillin-associated proteins within membrane rafts in vivo, we employed the BioID proximity labeling technique to analyze the flotillin-2 interactome in living HeLa cells. Flotillin-2 was fused to the promiscuous biotin ligase BirA* at either the N- or C-terminus and expressed in HeLa cells, enabling biotinylation of proximal proteins. Biotinylated proteins were enriched on streptavidin affinity beads and subsequently identified via LC-MS analysis. This investigation of the flotillin-2 interactome provides new insights into the molecular composition of membrane rafts and offers a valuable framework for understanding the mechanisms underlying raft formation, regulation, and associated cellular functions in native membranes.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Michał Tracz

LAB HEAD: Michał Tracz

PROVIDER: PXD065739 | Pride | 2025-08-22

REPOSITORIES: Pride

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			Action	DRS
	250131_ZCYT_AHR_10_HDMSe.raw.zip	Raw
	250131_ZCYT_AHR_1_HDMSe.raw.zip	Raw
	250131_ZCYT_AHR_2_HDMSe.raw.zip	Raw
	250131_ZCYT_AHR_3_HDMSe.raw.zip	Raw
	250131_ZCYT_AHR_4_HDMSe.raw.zip	Raw

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Project description:The simultaneous overexpression of flotillins 1 and 2 is observed in many cancers and considered as a marker of poor prognosis, notably in breast cancer. Flotillin-overexpression was shown to participate in the invasive behavior of cancer cell leading to metastasis. To better characterize the specific contribution of flotillin upregulation in the acquisition of cellular invasive properties, we stably expressed at high level exogenous flotillin 1 (HA-tagged) and flotillin 2 (mCherry tagged) in the non-tumoral mammary epithelial MCF10A human cells. The obtained cell line was named MCF10AF1F2. As a control, we generated in parallel the MCF10AmCherry cell line, stably expressing mCherry alone. We compared by RNA-sequencing (RNA-seq) the expression profiles of MCF10AmCh cells and MCF10AF1F2 cells. This allows to identify the gene expression changes and the downstream signaling pathways associated with flotillin upregulation. Using two differential gene expression analysis pipelines (Tuxedo and DSeq2), we identified 232 upregulated and 570 downregulated genes (802 genes in total, fold change 2.8, P<0.05) in MCF10AF1F2 cells compared with control cells. Gene ontology (GO) cluster analysis) revealed that flotillin upregulation was associated with deregulation of genes involved in extracellular matrix, cell adhesion, migration, differentiation, apoptosis and signaling pathways, particularly MAP kinases. Because these data suggested that MCF10AF1F2 cells underwent epithelio to mesenchymal transition (EMT), we compared the list of genes differentially in MCF10AF1F2 cells with the EMT transcriptional signature previously established by a meta-analysis (Gröger et al al. PloSOne 2012, https://doi.org/10.1371/journal.pone.0051136). One third (22/67) of the upregulated genes and more than half (39/62) of the downregulated genes in this EMT signature overlapped with the genes deregulated upon flotillin overexpression in MCF10A cells. Using complementary approaches, we confirmed that upregulation of flotillins un non-tumoral mammary epithelial cells is sufficient to promote EMT.

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A Proximity dependent labeling approach to define flotillin-2 interacting proteins within Membrane Domains of HeLa cells

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