FAB1 Regulates Clathrin Plasma Membrane Recruitments In Arabidopsis Thaliana(L.)

Phosphoinositides(PIs),which is phosphorylated derivatives of phosphatidylinositol(Ptd Ins),play an important role in cellular processes by recruiting effector proteins,such as vesicle trafficking,membrane trafficking,cytoskeleton organization,nuclear signaling,and stress responses.Clathrin-mediated endocytosis(CME)is one of endocytic pathways for plasma membrane(PM)proteins in eukaryote cells.Previous studies had shown that PI(4,5)P₂ and PI(3,4)P₂ regulated the membrane recruitment of clathrin and the maturation of clathrin-coated pits(CCP),respectively,and thus affect the CME process.Recent studies have shown that FAB1,the synthetic gene of PI(3,5)P₂,mediates the endocytosis of the plasma membrane protein BOR1(Borate receptor 1,BOR1).However,whether FAB1 is involved in the regulation of clathrin membrane recruitments and there exists a relationship between the FAB1 and clathrin-mediated endocytosis pathway are poorly understood.To explore the relationship between the FAB1 and CME,this study used genetic,cytological,and pharmacological strategies,combined with confocal live-cell imaging to dissect FAB1regulation of PM recruitment of clathrin,adaptor protein(AP2 and TPC)complexes,and dynamin-related proteins,and PM protein internalization in fab1b/fab1c/fab1d(abbreviated as fab1b/c/d)triple mutants.In addition,the maintenance of trichome morphology is a general function of FAB1in Arabidopsis.The detailed results were as follows:(1)Genetic studies showed that loss of FAB1B,FAB1C,and FAB1D functions caused defects in trichome development and morphology,including lower density,collapse,and more branch;GUS staining results showed that FAB1B and FAB1C are highly expressed in trichome;FAB1B and FAB1C were introduced into the fab1b/c/d triple mutants,respectively,and the collapse phenotype of trichome was partially restored.These results suggest that FAB1 plays a critical role during trichome development and maintenance of shape.(2)Cell biology studies showed that loss of FAB1B,FAB1C,and FAB1D function attenuated the internalization of the endocytic tracer FM4-64;Under the treatment of BFA,the endocytosis rate of PIN2 was significantly reduced in fab1b/c/d triple mutants relative to the wild type.These results suggest that genetic impairment of FAB1 function inhibits PM protein internalization.(3)Live-cell imaging analysis showed that,the PM abundance of CLC3(Clathrin light chains3),CHC2(Clathrin heavy chain 2),AP-2 subunits,the TPC subunits,and dynamin was reduced in root epidermal cells of fab1b/c/d triple mutants.In addition,the results of immunolocalization and Western Blot also showed that the abundances of CLC3?CHC2 and AP2?were significantly decreased in the PM.These results indicate that FAB1 affect PM recruitments of the clathrin and clathrin-related protein,and then CME.(4)Yeast two-hybrid assays indicated that FAB1B,FAB1C and FAB1D did not interact with CLC3,CHC2 and AP2?.This result suggests that FAB1 regulates the membranes recruitments of clathrin is not through direct interactions,instead could be depend on one or several unknown effectors.In summary,this study can conclude:FAB1 regulates trichome development and maintain their morphology;FAB1 participates in the PM recruitment of clathrin?adaptor proteins and TPC,thereby regulating the CME process.In addition,there is not direct interaction between the FAB1 and CME or CME-related genes.These results together provide a new insight in the molecular mechanism underlying FAB1 regulation of clathrin recruitment to the PM and have a great importance for a deeper understanding of CME regulation in plants.