Role of Lipid Nanodomains and F-actin in CD36 Signal Transduction

• Author / Creator

  Khaing, Swai Mon

• CD36, a multi-ligand plasma membrane receptor, has been implicated in immunity, metabolism and angiogenesis. We have recently demonstrated that CD36 nanoclustering at the plasma membrane is key to the initiation of CD36 signaling. In endothelial cells (ECs), the binding of thrombospondin-1 (TSP-1, an endogenous extracellular matrix anti-angiogenic factor) to CD36 nanoclusters activates an associated Src family kinase, Fyn, leading to ECs apoptosis, hence, inhibiting angiogenesis. Our project centralized in elucidating the mechanisms of CD36-Fyn enrichment on the lipid nanodomains and actin cytoskeleton during TSP-1 induced signaling in ECs.
  We hypothesized that lipid nanodomains play a role in bringing together CD36-Fyn to F-actin regions through adaptor molecules which forms a signaling platform. Using microscopy methods to visualize Fyn, various fluorescent lipid biosensors and F-actin (Phalloidin-AF647), we determined that Fyn is enriched on F-actin area at sites of phosphatidylinositol 4,5-bisphosphate enrichment (PIP2). During TSP-1 stimulation on Human Microvascular Endothelial Cells (HMEC), the CD36-Fyn-F-actin enrichment shift to domains containing PI(3,4,5)P3, suggesting a role for the phosphoinositide 3-kinase in signaling. To test the role of PI3K in Fyn activation and in CD36 nanocluster enhancements, we employed pharmacological inhibition of PI3K (LY294002) to arrest the production of PIP3 on the plasma membrane and depletion of membrane PI(4,5)P2, a precursor for PI(3,4,5)P3 using ionomycin which activates the PLC pathway. Using Immunoblotting and super-resolution fluorescence microscopy (TIRF-PALM), we determined that PI3K is important for Fyn activation and in CD36 nanocluster enhancements in CD36-Fyn signaling upon stimulation with TSP-1. Additionally, we employed a unique optogenetic tool (LARIAT) to facilitate in understanding the role of lipid nanodomains in CD36-Fyn signaling. Upon clustering of CD36 molecules using LARIAT, Fyn activation enhanced within these clusters and this activation is reduced by treatment with LY294002 which further supported our hypothesis that engagement with PI3K (lipid nanodomains PI(4,5)P2 and PI(3,4,5)P3) play a significant role in Fyn activation and CD36 nanoclustering. With this, we proposed a model in which CD36 nanoclusters are located within PI(4,5)P2 domains and upon TSP-1 stimulation, PI3K is engaged, producing PI(3,4,5)P3 within the CD36 nanoclusters and enhances the nanoclusters and downstream Fyn activation. Furthermore, we characterized the potential adaptor proteins involved in connecting F-actin to lipid nanodomains and/or CD36 nanoclusters using BioID proximity dependent biotinylation, fractionation of cortical F actin and G actin and mass spectrometry (MS). The MS screen has narrowed down proteins that are biotinylated, adjacent to CD36 and enriched in F actin. There were nine potential candidate proteins identified and they are as follows; Alpha-actinin-4, RasGTPase-activating protein binding protein 1, E3 ubiquitin-protein ligase UBR4, PDZ and LIM domain protein 7, Erythrocyte band 7 integral membrane protein, Filamin-A, Plectin, RasGTPase-activating-like protein IQGAP1 and Talin-1. Altogether, our investigation provided novel insights into understanding the activity of CD36 signaling and organization of CD36 on the plasma membrane supported by the cortical F-actin.

• Subjects / Keywords

  • CD36, Lipid Nanodomains, F-actin

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3SX64S0J

• License

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