Superresolution Analysis Of Interactions Between LRRC8 Members And Between LRRC8A And ANO1,IP3 Receptor And Caveolin-1

Volume-regulatedchloridechannel（VRCC）,alsoknownas volume-sensitive outwardly rectifying anion channel（VSOR）,is a member of the large family of chloride channels.VRCC is named because it can be activated by changes in cell volume,resulting in a chloride current with outward rectification characteristics.VRCC is widely distributed and exists in almost all vertebrate cells.Since its discovery in the 1980s,many experiments have proved that VRCC plays an important role in the regulation of cell volume,proliferation,differentiation of cells,apoptosis,tumor drug resistance,neurotoxicity of cerebral ischemia,insulin resistance and other physiological and pathological processes.In 2014,two groups independently reported that the LRRC8（leucine-rich repeat-containing 8）family of proteins is the molecular basis of VRCC.In addition,Bestrophin and TMEM16A（Anoctamins-1）are also considered likely to participate in the formation of VRCC.LRRC8 family of proteins constitute the main molecular basis of VRCC,which has a total of 5 members:LRRC8A-E.Different combinations of these members form different types of VRCCs,with LRRC8A likely constituting the pore region of VRCC.At present,the mechanism by which VRCC is activated during cell swelling is not clear.Previous studies have shown that intracellular micro-domain Ca²⁺and cell membrane lipid raft structure play important roles in the activation of VRCC.Based on this,this thesis was dedicated to the analysis of the structural basis of the local microenvironment created for the activation of VRCC using super-resolution microscopy STORM technology.Stochastic Optical Reconstruction Microscopy（STORM）and Structured Illumination Microscopy（SIM）are two of the super-resolution microscope technologies invented in recent years.STORM utilizes an optically switchable fluorescent molecule for high-precision positioning with a resolution of theoretically 10-20 nm,which is 10 times the resolution of conventional light microscopy.SIM utilizes moire fringes produced by high-frequency stripe illumination to separate the high-frequency and low-frequency signals of a sample so that the ultrafine structure of the cell can be clearly seen and its resolution theoretically reaches about 100 nm,and because of the very high temporal resolution of the SIM,up to 0.6 sec/frame,super-resolution imaging at the live-cell level become possible.Kv7/KCNQ potassium channel family contains 5 members,KCNQ1-5.Among these,KCNQ2 and KCNQ3 can form heterotetramers in physiological state,while KCNQ2 and KCNQ4 can not.Distinguishable ultrastructural interactions between KCNQ2-4 have been realized recently using STORM technology.Taking advantage of this new development,we in the first part of this study,used the established KCNQ intermolecular interactions as a template,established the super-resolution experiment platform and data analysis method for the analysis of the intermolecular interactions using STORM.Using this platform,in the second part of our study,we analyzed the co-localization of LRRC8 family protein subunits as well as the interactions between LRRC8A and the proteins reportedly important for membrane microdomain and Ca²⁺kinetics:ANO1,IP3R and caveolin.Part 1 Establishment of super-resolution microscopy experiment platform and data analysis methodObjective:To establish stable and reliable STORM and SIM experiment platforms and data analysis methods.Methods:（1）A total of 300 ng of KCNQ2+KCNQ3 and KCNQ2+KCNQ4 cNDA were transfected respectively in HEK293A cells.（2）SORM-related staining and STORM imaging process was performed.（3）OriginPro8 and Metlab and other softwares were used for data analysis.（4）DRGneuronsfromratwereisolatedandcultured,immunocytochemical staining was performed on these neurons,and tubulin was labeled for SIM imaging.（5）Endoplasmic reticulum and cell membranes of wild-type HEK293A were specifically labeled with fluorescent protein-carrying virus,and then dynamic motion of cell membrane and endoplasmic reticulum was observed by SIM system in combination with a microperfusion system.Results:（1）KCNQ2 and KCNQ3 show significant co-localization,but no co-localization of KCNQ2 and KCNQ4 was observed.（2）The microtubules of DRG neurons were clearly observed,the resolution is significantly improved compared to wide field imaging.（3）The dynamic changes of endoplasmic reticulum and cell membrane were clearly observed.Conclusions:（1）We have established a stable and reliable STORM experimental platform and experimental protocol,and with this the data processing methods using OriginPro8,Metlab,Adobe Illustrator CS6 and other softwares.（2）We have established a stable SIM experimental platform and developed the experiment protocols for fixed cells as well as for live cells.Part 2 Interactions between LRRC8 members and between LRRC8A and ANO1,IP3 receptor and caveolin-1Objective:To analyze the co-localization of LRRC8 subunits and to study the interactions between LRRC8A and ANO1,IP3 receptor and caveolin-1.Methods:（1）HEK293 cell line with endogenous LRRC8A-/-,LRRC8D-/-,LRRC8E-/-being deleted was created(HEK293A^{LRRC8A-/-,LRRC8D-/-,LRRC8E-/-});similarly,HEK293A^LRRC8A-/-,HEK293A^ANO1-/-and HEK293A^{LRRC8A-/-,ANO1-/-}cell lines were also created.（2）A total of 300 ng of either LRRC8A+LRRC8D or LRRC8A+LRRC8E were transfected into HEK293A^{LRRC8A-/-,LRRC8D-/-,LRRC8E-/-}cells;HEK293A cells that were not transfected were served as controls.STORM staining,imaging,data analysis were performed on these cells.（3）RFP-tagged LRRC8A plasmid was transfected into HEK293A^LRRC8A-/-cells,and similarly GFP-tagged ANO1 plasmid was transfected into HEK293A^ANO1-/-cells;immunocytochemistry using confocal microscope was performed to test the specificity of antibodies for LRRC8A and ANO1.A total of 300 ng of LRRC8A and ANO1 plasmids were transfected into HEK293A^{LRRC8A-/-,ANO1-/-}cells,and again cells that were not transfected were served as controls.STORM staining,imaging and data analysis were performed on these cells.（4）Interaction between LRRC8A and inositol 1,4,5-trisphosphate receptor（IP3R）,and interaction between ANO1 and IP3R was studied using STORM in wild-type HEK293 cells.（5）Interaction between LRRC8A and caveolin-1 was studied using STORM in wild-type HEK293A cells and in HEK293A^LRRC8A-/-cells.（6）Effect ofβ-cyclodextrin on interaction between LRRC8A and caveolin-1 was studied using STORM in wild type HEK293A cells,HEK293A^LRRC8A-/-cells,and HEK293A^LRRC8A-/-+LRRC8A cells.Results:（1）In HEK293 cells lacking endogenous LRRC8A and LRRC8D,LRRC8E(HEK293A^{LRRC8A-/-,LRRC8D-/-,LRRC8E-/-}),clear interaction was visible between LRRC8A and LRRC8D,and between LRRC8A and LRRC8E when these subunits were transfected into the HEK293 cells and studied under STORM experiments..（2）The fluorescence signals from RFP-LRRC8A and from anti-LRRC8A antibody has good correlation,and similarly the fluorescence signals from GFP-ANO1 and from anti-ANO1 antibody has good correlation,indicating that the specificity of LRRC8A and ANO1 antibody is reliable.（3）In STORM experiments,LRRC8A and ANO1 show good co-localization.（4）Both LRRC8A and ANO1 show strong co-localization with IP3R.（5）LRRC8A and caveolin-1 appear to be well co-localized.（6）When the expression of caveolin-1 was decreased after treatment withβ-cyclodextrin,the co-localization between caveolin-1 and LRRC8A disappeared.Conclusions:（1）LRRC8A interacts with LRRC8D and LRRC8E to form a heteromultimer channel of VRCC.（2）ANO1 interacts with LRRC8A.（3）LRRC8A interacts IP3R,suggesting that IP3R and intracellular microdomain Ca²⁺may be involved in the activation of VRCC.（4）LRRC8A interacts with caveolin-1 which is destroyed when the lipid raft was dissolved.However,further studies are needed to determine if caveolin-1 is involved in the activation of VRCC.