TMK1-mediated Auxin Regulation Of Clathrin In Arabidopsis

Clathrin-mediated endocytosis(CME),a major endocytic pathway in eukaryotic cells,directly regulates the uptake of extracellular nutrients,the abundance of plasma membrane(PM)proteins,and the transduction between extra-and intracellular signals.CME pathway is dependent on the function of clathrin and its adaptor protein(AP)complexes.The function of clathrin depends on a triskelion‐shaped complex that consists of three heavy chains(CHCs)and three light chains(CLCs).AP complexes mainly include heterotetramer AP-2 and heterooctamer TPC(TPLATE complex),which functions to recruit clathrin to the PM.Auxin and SA(salicylic acid)differentially regulate the recruitments of PM-and TGN/EE(trans-Golgi network/early endosome)-association of CLCs and CHCs,respectively.Therefore,inhibition of endocytosis of the clathrin-mediated auxin-efflux transporter PIN-FORMED2(PIN2),thereby influencing on polar auxin transport and thus root tip gravitropism.Auxin-regulated clathrin depends on the cell surface receptor ABP1(auxin binding protein 1),but not the auxin nuclear receptor TIR1/AFB(transport inhibitor response 1/auxin binding F-box protein).SA-regulated clathrin function is independent to ABP1.The latest evidence shows that ABP1 and its upstream regulator TMK1(transmembrane kinase 1)functions together to mediate cell surface signals and regulate rapid auxin response.Therefore,this thesis mainly studies whether TMK family participates in auxin-regulated function of clathrin,and reveals the regulatory mechanism of signal pathway on auxin response(such as gravitropism).Arabidopsis TMK family includes four members: TMK1,TMK2,TMK3,and TMK4,belonging to the LRR-RLK(leucine rich repeat-receptor like protein kinase)family.TMK2 is only expressed in flowers and siliques.In contrast,the other three members are present in many tissues.TMKs show protein-function redundancy,yet they were also shown to regulate different biological processes individually.For example,TMK1 mediates auxin signal transduction pathway to regulate the development of apical hook and gravitropism;TMK4 is involved in auxin synthesis.In this study,laser scanning confocal microscopy,combined with genetic,biochemical,cytological and pharmacological approaches were used to identify the effects of loss function of TMK1,TMK3,and TMK4 on auxin-regulated clathrin function and PIN2-mediated gravitropism.The main results in this study are as follows:(1)Physiological experiments and live-cell imaging analysis showed that under gravity stimulation,the signals of auxin markers and clathrin were asymmetrically distributed on both sides of root tips in WT(wild type)and tmk3 and tmk4 single mutants,while the asymmetric distribution was impaired in tmk1 mutants,indicating that TMK1 mediates the signals of auxin and clathrin to regulate root tip gravitropism.(2)Cell biological experiments and biochemical analysis showed that auxin differentially regulates the membrane-associated recruitments of CLC and CHC in WT and tmk3 and tmk4 single mutants,but not tmk1 mutants,indicating that auxin regulation of clathrin dependents on TMK1.(3)The results of live-cell imaging showed that auxin has no effect on the membrane-associated recruitments of AP-2 and TPC subunits in WT and tmk single mutants,indicating that TMK1-mediated auxin regulation of clathrin was independent to AP-2 and TPC.(4)Pharmacological analysis showed that the uptake of FM4-64 and PIN2 endocytosis are inhibited in WT,tmk3,and tmk4 mutants after auxin treatments,but not for tmk1 mutants,indicating that auxin inhibition of PM endocytosis depends on TMK1.(5)Live-cell imaging showed that under gravity stimulation,asymmetric polar distribution of PIN2 on both sides of epidermal root cells was displayed in WT and tmk single mutants,while such asymmetric distribution was impaired in tmk1 mutants,demonstrating that TMK1 is involved in PIN2-mediated gravitropsim.(6)Immunofluorescence experiments and live-cell imaging analysis showed that SA differentially regulates the membraneassociated recruitments of CLC and CHC in WT and tmk single mutants and the regulatory signals of WT and tmk single mutants were similar,indicating that SAregulated clathrin function is independent to TMK.(7)Pharmacological analysis showed that SA inhibits the endocytosis of PIN2 and RCI2 A PM protein in both WT and the tmk single mutants,indicating that the inhibition of the endocytosis of PM protein by SA is independent to TMK.In summary,TMK1 was shown essential for auxin-regulated clathrin recruitment and therefore CME process.Such regulation plays a critical role in the establishment of an asymmetric distribution of PIN2 and therefore an auxin gradient for root gravitropism.