Molecular mechanisms of endocytic cargo recognition in plants

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.

For plants to grow their cells need to sense signals around them. For example, cells need to sense growth signals and they need to take up nutrients from soils to develop properly. This sensing and nutrient uptake is controlled by proteins on the cell surface. To control the plants sensitivity to these signals, these signal receptor proteins are removed from the cell surface by a process called endocytosis. Critically, endocytosis of these receptors relies on adaptor proteins which bind these receptor proteins into endocytosis events. Thus, if we identify how these adaptors select specific cargos, we can begin to understand how plants respond to different signals. How endocytosis functions in plants has not historically been well characterized. A major reason for this has been due to a lack of imaging methods. Therefore, in this project we developed several new imaging protocols which allowed us to image endocytosis at unprecedented scales. Using these methods, we were able to address questions about the mechanism driving plant endocytosis, for example: During endocytosis, a small area of the cell surface is bent inwards to create a sphere which carries the cargo inside the cell. In cell biology it has long been established that this bending is driven by a protein called actin, so it was expected that actin would be required for plant endocytosis. However, using our new imaging methodologies we showed that this prediction was incorrect, and demonstrated that plant endocytosis functions in a different manner compared to other biological models. To investigate how receptors are selected for endocytosis, and thus how endocytosis regulates plant growth, we focused on studying a major growth signal receptor - BRI1. We identified a specific protein sequence in this receptor which resembled a well-known mammalian cargo motif for endocytic cargo adaptors. Once we confirmed that this sequence was indeed mediating the interaction of BRI1 with an endocytic adaptor protein (AP2), we used our imaging methodologies to confirm that this relationship was functional and identified a bona fide plant cargo recognition mechanism. To investigate adaptor proteins, we investigated the role of a predicted plant-specific adaptor, the TPLATE complex. During our investigations we found that the TPLATE complex was localized outside of endocytosis events, which suggested that it does not actually functioning as an endocytic cargo adaptor. Using a range of biochemical, advanced imaging and biophysical approaches, we instead found that the TPLATE complex was critical for the bending of the cell surface during endocytosis. This identified the first part of the unique plant endocytosis bending machinery. Together, our results from this project have led to a significant updating of plant endocytosis models and have provided many insights into the regulation of proteins which regulate plant growth and development.