Objective

All living cells need a strict control of levels of proteins and other components at their surfaces. A process called endocytosis is a specialized mechanism that selectively takes material from the cell surface into the cell interior. In plants, the major type of endocytosis depends on the coat protein clathrin that helps to assemble the endocytic membrane vesicles at the plasma membrane. This Clathrin-mediated endocytosis is in plants the major mechanism controlling cell surface levels and thus activities of receptors, transporters, channels, and other proteins with functions in hormone perception, immunity, nutrient uptake and development. Despite the crucial importance of this process, the machinery and plant-specific modifications of Clathrin-mediated endocytosis are largely unknown. The first and decisive step in Clathrin-mediated endocytosis is recognition of the cargos at the plasma membrane by the adaptor (AP) proteins. The AP-2 clathrin adaptor complex was identified in the model plant Arabidopsis and shown to regulate endocytosis of the well-known and developmentally crucial plant cargos, the PIN transporters for the plant hormone auxin and receptor for another plant hormone brassinosteroid, BRASSINOSTEROID INSENSITIVE1 (BRI1). However, fundamental questions still awaiting clarifications: (i) how are cargoes selectively recruited by the AP-2; (ii) what is the identity of the additional regulators of the process, (iii) which cargo posttranslational modifications are required for this recruitment, and (iv) what developmental and physiological roles plays AP-2-mediated cargo recruitment and Clathrin-mediated endocytosis. The project aims at finding proteins associated with the clathrin machinery using immunoprecipitation followed by mass spectrometry proteomics of the AP-2 complex. Next, we will establish high resolution live imaging and electron microscopy methods to study the interaction between adaptor proteins and selected cargos (exemplified by BRI1 receptor and PIN transporters) in vivo. We will use these advanced imaging techniques to follow the dynamics of the plant endocytic machinery including adaptors, newly identified regulators and the cargoes. We will also generate conditional knockout mutants to study the function of the novel clathrin accessory proteins in endocytosis and in the signaling processes including brassinosteroid- and auxin-mediated physiology. This genetic material will also provide a basis for the future broader studies on the role of CME and cargo recruitment in multitude of developmental and physiological processes in plants including defense against pathogens and nutrient uptake. The project will benefit from the complementary expertise of the participating laboratories: (i) proteomics and biochemistry expertise of Russinova lab (VIB-UGent) and (ii) cell biology and state-of-the-art imaging of the Friml lab (IST Vienna). In addition, the related but diversified interests in hormone perception and signaling (Russinova) and hormone transport and polarity (Friml) allow to focus on the favorite topics while maximally sharing the expertise and tools of individual partners.

Project details

Project name: Molecular mechanisms of endocytic cargo recognition in plants
Project reference: I 3630 Internationale Projekte
Funding Scheme: International program
Contribution: EUR 338.814,00
Funding provided by the Austrian Science Fund (FWF)
Duration: From 2018/02/01-2022/01/31
Project leader: Prof. Jiří Friml