Imaging TRPC1-Akt Interaction In Cell Migration By Fluorescence Resonance Energy Transfer (FRET)

BackgroundCell migration is the cells migrate after received migrate signal or certain substances of gradient concentration. Cell migration requires internal and external factors, external factors such as EGF, IL1signal molecule; Intracellular factors such as signal transduction system, cytoskeleton and molecular motors, various molecules involved in adhesion plaque formation. The process of cell migration is extracellular signaling molecules combine with the receptor of cell surface, start the intracellular signaling molecules, intracellular signaling molecules take the motion information to executive unit of cell migration----the cytoskeleton and molecular motors, and then completed a series migration activities. A variety of intracellular signaling molecules can interact each other, affect the structure, activity and distribution of the cytoskeleton and molecular motor, thus achieve the purpose of accurate adjustment of the cell movement.TRP channels are an important class of non-selective cation channel super-family on the cell membrane, mainly Permeability Ca2+, Na+and so on. All of the TRP channel protein has six transmembrane domains, and formed4tetramers. ion channel hole is formed in5to6inter-segment, and cationic in and out of the cell through the lipid bilayer. Initial, TRP ion channels are limited know to the sensory system, because the channel feel the intracellular environment by a variety of stimuli, participate the pain and temperature sensation, mechanical feel and taste sense development and maintenance of intracellular ion homeostasis and many other life activities.The C-terminus of TRPC molecule begin from TRP box, and it is relevant with phosphatidylinositol-mediated channel gating loci, TRP domain is involved in the regulation of the TRP channel function. The TRPC subfamily includes TRPC1-77subtypes. TRPC1is detected high level of expression in the brain, heart, kidney, lung, skeletal muscle, prostate, skin, testicular and ovarian. TRPC1is recognized as the first mammalian TRP channels, involved in receptor-mediated calcium-dependent smooth muscle and glandular secretion and systolic function, involved in the cell membrane receptor activation of phospholipase C (PLC) mediated calcium ions enter. TRPC1channel is a non-selective cation channels, and the main permeability of ions is Ca2+and Na+. TRPC1participate in many signal path adjust cell growth and apoptosis, dendritic formation, cell cycle regulation, cell migration.TRPC1can be activated by ligand such as exogenous small molecule, chemical compound, inorganic ion and endogenous substance the change of Ca2+concentration, PLC, STIM1, also can be activated by G-protein-coupled receptors or receptor tyrosine kinases mediate PLC signal path, and is considered to be the molecular basis of most likely calcium library manipulation calcium channel and receptor-operated calcium channels. Many studies show that TRPC1interact with phosphatidylinositol, such as TRP protein, PIP2and PIP3are regulated by PI3Kγ and PLC in the process of cell polarization, they are interacted with each other on the cell membrane. Generally speaking, the PLC regulating extracellular calcium ions into intracellular mainly through the TRPC channels formed with intermolecular lipid connection play a role, this protein-lipid complexes involved in TRPC channel positioning and surface expression of the membraneregulation. PIP2hydrolysis can affect the TRP channel’s gating, the mediation mechanism may concern with TRPC molecule C-terminus exists phosphatidylinositol-mediated channel gating sites. PIP2and PIP3-mediated endothelin-1the vascular peptide-1activation rabbit coronary artery myocytes TRPC1/5/6. Activation of native TRPC1/C5/C6channels by endothelin-1is mediated by both PIP3and PIP2in rabbit coronary artery myocytes.Protein kinase B (PKB/Akt) participate in more than one signaling pathway and plays a key regulatory molecules in the signaling pathway, and regulate a variety of downstream molecular, play an important role in cell migration and invasion, cycle regulation, apoptosis and proliferation, tumor angiogenesis. PI3K/Akt pathway is important in tumor cell migration signal transduction pathways, studies have shown that inhibit PI3K by inhibitor Wortmannin significant effect basic fibroblast growth factor (bFGF)-induced rat vascular smooth muscle cell migration, mainly affecting the role of phosphorylated Akt protein.Recent studies have shown that PI3K/Akt pathway is involved in muscle development and regeneration. Inhibiting TRPC1channels or inhibiting Ca2+fluxes decreases PI3K/Akt activation, slows down myoblasts migration and impairs muscle regeneration, on the contrary TRPC1-mediated Ca2+influx enhances PI3K/Akt pathway during muscle regeneration.PI3K/Akt signaling pathway is an important signal pathways in cancer research,it plays a significant role in apoptosis and proliferation, tumor angiogenesis, cell migration and invasion, and cycle regulation. Akt is a serine/threonine kinase which is the most important signaling molecule in downstream of PI3K, approximately constituted by more than480amino acid sequence.The amino acids of Akt molecule consist of a N-end of the PH domain, the intermediate catalytic domain which has enzyme activity and a C-terminal short carboxy-terminal regulatory domain from the N-terminus to the C terminal.PH domain mediated plasma membrane translocation process after Akt activated, it constitute a hydrophobic region whicd contains seven antiparallel β replicates. The intermediate catalytic domain contains ATP binding sites which can catalyze serine/threonine residue groups and phosphorylate the two groups, it is necessary that the phosphorylation of Thr308points for Akt activation;The C-terminal is a hydrophobic proline-rich domain and transmembrane transporter, which contains the second phosphorylation site (Ser473) that can make Akt fully activated. Akt could be activated by a variety of substances in normal and tumor cells,such as epidermal growth factors, hormones, extracellular matrix components and so on, Akt adjusted the downstream100kinds of substrate molecules through phosphorylation and dephosphorylation, thereby regulated cellgrowth, proliferation and apoptosis.Activation of Akt include PI3K-dependent and PI3K-independent modes. PI3K is a intracellular phosphatidylinositol kinase,it can specific activate the third hydroxyl group of the ring in inositol group, and it also have the dual activity of protein kinases and lipid kinases. Akt is a central part of the PI3K/Akt pathway. PI3K/Akt signaling pathway rely on the phosphorylation of Akt protein in the regulation of multiple biological processes. Activation of Akt needs Thr308and Ser473phosphorylated simultaneously in the Akt polypeptide chain.The specific process is:When the extracellular factors such as epidermal growth factor receptor act on the cell membrane receptor tyrosine kinase (RTK), PI3K on the cell membrane is activated after receptor tyrosine kinase and the ligand binded, and it generated second messenger in the plasma membrane PIP3, PIP3can be combined with the intracellular signaling protein Akt and PDK1, PDK2protein, after PIP3and Akt PH domain binded,inactive Akt protein translocated from the cytoplasm to the cell membrane, at the same time, Ser124and Thr450of Akt protein chains are phosphorylated and get catalytic activity. Ser124and Thr450phosphorylation changed Akt protein spatial structure, exposed Thr308and Ser473sites.Meanwhile, PDK1protein and Akt which translocated to the plasma membrane close to each other,PDK1protein catalysed Akt Thr308phosphorylation, and PDK2protein catalysed Akt Ser473phosphorylation.Activated Thr308and Ser473eventually lead to Akt completely activated.Phosphorylation cascade of the downstream target proteins caused by the activated of Akt, result in the corresponding target protein activation or deactivation, thus regulate cell growth and survival, proliferation or apoptosis, cell migration, angiogenesis and other cellular activities and biological effects.In conclusion, TRPC1and Akt may interact in cell migration, but in the process of cell migration the TRPC1and Akt interact with each other is not yet clear, this study use the classical model of epidermal growth factor stimulated and intuced cos-7cells to study cell migration, through fluorescence resonance energy transfer (FRET) technology to explore the interaction between TRPC1and Akt in cell migration.ObjectiveThis experiment use the technology of plasmid construct make two purposes gene TRPC1and Akt are recombined into the the fluorescence expression vector plasmid pECFP-C1and pEYFP-C1, obtained pECFP-C1-TRPC1and pEYFP-C1-Akt two recombinant plasmids. Use Immunofluorescence techniques, recombinant plasmids express fusion fluorescent protein technology and FRET (Fluorescence Resonance Energy Transfer, FRET) technology receptor protein bleaching method, study whether TRPC1and Akt interact with each other in the migration of EGF stimulate and induce cos-7cell.Methods1. Obtain the desired plasmidby molecular cloning recombinant DNA technology Reorganize cDNA TRPC1, Akt and YFP to pECFP-C1, pEYFP-C1and pECFP-C1plasmids respectively by molecular cloning recombinant DNA technology, to obtain pECFP-C1-TRPC1, pEYFP-Cl-Akt and pECFP-Cl-YFP three recombinant plasmids.2. COS-7cells show chemotaxis for EGFCOS-7cells show chemotaxis for EGF, In EGF’s side of the cell membrane formed ruffle. Cut the coverslip a small piece of corner and put it into six-well plate, evenly cultivate COS-7cells on the coverslip. Plus10nM EGF in the corner of the coverslip, make the COS-7cells spot activated by EGF.3. TRPCl and Akt immunofluorescence localization in cos-7cell4. The expression and localization of fluorescence fusion-protein in cos-7cell5. fluorescence fusion-protein translocation after EGF activate cos-7cell6. Laser scanning confocal microscope detect FRETpECFP-C1-TRPC1make protein TRPCl and CFP fusion expression, pEYFP-C1-Akt make protein Akt and YFP fusion expression. To detect whether TRPC1and Akt interact each other is tantamount to study whether the CFP and YFP interact happened FRET. Protein CFP and YFP proteins constitute an energy donor and the energy acceptor, when the two are separated by1.0-10.0nm,the emission spectrum of the donor and absorption spectrum of the acceptor have significant overlap, as the emission spectrum of the donor and emission spectra of the acceptor completely separated, and non-excitation to receptor in the excitation wavelength of the donor. While the donor excitation light excitated,the donor molecule from the ground state moved in the excited state,due to the dipole-dipole interaction, energy excitated by donor molecule will be a non-radiation form passed to the receptor molecules. FRET occurs between donor and receptor will decrease fluorescence intensity caused by the donor and increase fluorescence intensity caused by the acceptor,Meanwhile it will shorten donor fluorescence lifetime and extend acceptor fluorescence lifetime. The condition of FRET:(1)The donor’s fluorescence quantum yield is higher.(2)The donor’s emission spectrum and the receptor’s absorption spectrum can effectively overlap (greater than30%).(3) For the distance between the donor and acceptor in the1.0-10.0nm.Results1. Successfully construct pECFP-C1-TRPC1, pEYFP-C1-Akt and pECFP-C1-YFP plasmids.2. In EGF activated COS-7cells, TRPC1and Akt co-localization.3. Expression fusion fluorescence protein TRPC1and activate COS-7with EGF, TRPC1translocation from the cytoplasm to the cell membrane and juxtamembrane region.4. Expression fusion fluorescence protein Akt and activate COS-7with EGF, Akt distribution did not change.5. Co-expression fusion fluorescence protein and activate cos-7with EGF, TRPC1and Akt translocation from the cytoplasm to the cell membrane, and co-localization in anterior pole of the direction cell migration.6. Co-expression fusion fluorescence protein, not happen FRET and no interaction be between TRPC1and Akt.7. Co-expression fusion fluorescence protein and activate cos-7cell with EGF, TRPC1and Akt occured of FRET, so they are interact with each other.ConclusionThere is a pronounced increase FRET between TRPC1and Akt in cos-7cells stimulated by EGF, supporting the idea that interaction of TRPC1and Akt in cell migration.