TY - GEN AB - Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development by controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scissin machinery in plants, but the precise roles of these proteins in this process is not fully understood. Here, we characterised the roles of Plant Dynamin-Related Proteins 2 (DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to Dynamins’ recruiters, like Endophilin and Amphiphysin, in the CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the Dsh3p1,2,3 triple-mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggests that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME. One Sentence Summary In contrast to predictions based on mammalian systems, plant Dynamin-related proteins 2 are recruited to the site of Clathrin-mediated endocytosis independently of BAR-SH3 proteins. AU - Gnyliukh, Nataliia AU - Johnson, Alexander J AU - Nagel, Marie-Kristin AU - Monzer, Aline AU - Hlavata, Annamaria AU - Isono, Erika AU - Loose, Martin AU - Friml, Jiří ID - 14591 T2 - bioRxiv TI - Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in plants ER - TY - THES AU - Gnyliukh, Nataliia ID - 14510 KW - Clathrin-Mediated Endocytosis KW - vesicle scission KW - Dynamin-Related Protein 2 KW - SH3P2 KW - TPLATE complex KW - Total internal reflection fluorescence microscopy KW - Arabidopsis thaliana SN - 2663-337X TI - Mechanism of clathrin-coated vesicle formation during endocytosis in plants ER - TY - JOUR AB - Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin–mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells. AU - Johnson, Alexander J AU - Dahhan, Dana A AU - Gnyliukh, Nataliia AU - Kaufmann, Walter AU - Zheden, Vanessa AU - Costanzo, Tommaso AU - Mahou, Pierre AU - Hrtyan, Mónika AU - Wang, Jie AU - Aguilera Servin, Juan L AU - van Damme, Daniël AU - Beaurepaire, Emmanuel AU - Loose, Martin AU - Bednarek, Sebastian Y AU - Friml, Jiří ID - 9887 IS - 51 JF - Proceedings of the National Academy of Sciences TI - The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis VL - 118 ER - TY - JOUR AB - Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and inter-cellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how it functions in planta. In order to facilitate the direct quantitative study of plant CME, here we review current routinely used methods and present refined, standardized quantitative imaging protocols which allow the detailed characterization of CME at multiple scales in plant tissues. These include: (i) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultra-structure of clathrin-coated vesicles; (ii) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (iii) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (iv) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples. AU - Johnson, Alexander J AU - Gnyliukh, Nataliia AU - Kaufmann, Walter AU - Narasimhan, Madhumitha AU - Vert, G AU - Bednarek, SY AU - Friml, Jiří ID - 8139 IS - 15 JF - Journal of Cell Science SN - 0021-9533 TI - Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis VL - 133 ER -