Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis
Résumé
Statement: We present detailed novel quantitative imaging protocols and review 15 pharmacological and genetic manipulation to provide standardized experimental approaches 16 for the investigation of plant clathrin-mediated endocytosis at multiple scales. Abstract 18 Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects 19 of plant growth, development, intra-and inter-cellular signaling, nutrient uptake and 20 pathogen defense. Despite these significant roles, little is known about the precise molecular 21 details of how it functions in planta. In order to facilitate the direct quantitative study of plant 22 CME, here we review current routinely used methods and present refined, standardized 23 quantitative imaging protocols which allow the detailed characterization of CME at multiple 24 scales in plant tissues. These include: (i) an efficient electron microscopy protocol for the 25 imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed 26 characterization of the ultra-structure of clathrin-coated vesicles; (ii) a detailed protocol and 27 analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal 28 interplay of endocytosis components during single CME events; (iii) a semi-automated 29 analysis to allow the quantitative characterization of global internalization of cargos in whole 30 plant tissues; and (iv) an overview and validation of useful genetic and pharmacological tools 31 to interrogate the molecular mechanisms and function of CME in intact plant samples. 32 2
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