Endocytosis is a fundamental cellular process by means of which cells internalize extracellular and plasma membrane cargos for recycling or degradation. It is important for the establishment and maintenance of cell polarity, subcellular signaling and uptake of nutrients into specialized cells, but also for plant cell interactions with pathogenic and symbiotic microbes. Endocytosis starts by vesicle formation at the plasma membrane and progresses through early and late endosomal compartments. In these endosomes cargo is sorted and it is either recycled back to the plasma membrane, or degraded in the lytic vacuole. This book presents an overview of our current knowledge of endocytosis in plants with a main focus on the key molecules undergoing and regulating endocytosis. It also provides up to date methodological approaches as well as principles of protein, structural lipid, sugar and microbe internalization in plant cells. The individual chapters describe clathrin-mediated and fluid-phase endocytosis, as well as flotillin-mediated endocytosis and internalization of microbes. The book was written for a broad spectrum of readers including students, teachers and researchers.

This second edition details techniques for the study of cargo trafficking through endosomes. New and updated chapters guide readers through methods and protocols on the structural aspects of plant endosomes, combined biochemical, omics, imaging approaches to study the dynamics and contents of endosomal compartments. Additional chapters are dedicated to the analysis of lipids on endosomes and the identification and analysis of lipid binding proteins and lipid-binding domains relevant for the study of plant endosomes. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Plant Endosomes: Methods and Protocols, Second Edition aims to ensure successful results in the further study of this vital field.

Endocytosis is a fundamental biological process, which is conserved among all eukaryotes. It is essential not only for many physiological and signalling processes but also for interactions between eukaryotic cells and pathogens or symbionts. This book covers all aspects of endocytosis in both lower and higher plants, including basic types of endocytosis, endocytic compartments, and molecules involved in endocytic internalization and recycling in diverse plant cell types. It provides a comparison with endocytosis in animals and yeast and discusses future prospects in this new and rapidly evolving plant research field. Readers will find an overview of the state-of-the-art methods and techniques applied in plant endocytosis research.

Endocytosis and endosomal trafficking control the abundance and distribution of key membrane proteins at the cell surface and in other cellular compartments. The Endosomal Sorting Complex Required for Transport (ESCRT) mediates the formation of vesicles to sequester ubiquitinated membrane proteins (cargo) into late endosomes en route to the vacuole for degradation. We previously found that the late-acting ESCRT components, ISTL1 and LIP5 are important for plant development, proper defense responses, and multivesicular endosome formation in root cells. Using genetic, confocal, and electron microscopy approaches, I found a unique role for ISTL1 and LIP5 in endosomal trafficking in tapetal cells, which surround and nourish developing pollen grains within anthers. Together, ISTL1 and LIP5 are required in the tapetum to maintain plasma membrane localization of ABCG9, a transporter important for pollen wall development. Plants lacking ISTL1 and LIP5 showed defects in exocytosis of the sec-RFP secretion marker in anthers. Pollen in the istl1 lip5 mutant developed abnormal pollen wall patterns, leading to partial pollen lethality. Finally, istl1 lip5 tapetal cells exhibited ultrastructural defects in both multivesicular endosome morphology and trans-Golgi Networks, indicating that besides endosomal sorting, late-acting ESCRTs have a role in the early endosomal pathway and/or exocytosis. The ESCRT machinery mediates negative membrane deformation (i.e. membrane bending away from the cytoplasm). Exactly how the ESCRT proteins orchestrate membrane deformation and fission is not well known. The current models for how intralumenal vesicles form assume that vesicles are formed one at a time. However, in plants intralumenal vesicles form as concatenated networks, establishing a novel mechanism to entrap cargo more efficiently. In Chapter 3, I use electron tomography to analyze vesicle network architecture from plant endosomes. I developed quantitative tools to evaluate the complexity of individual vesicle networks and applied them to compare intralumenal vesicle networks from different cell types. In addition, I developed rules and tools to recapitulate how these intralumenal vesicle networks form and infer the activity of budding sites on the endosomal surface.

Membrane proteins are essential determinants of many biological processes in plants. They function in metabolic processes, signal transduction, transport of small molecules and polymers across endo- and plasma membranes, and intercompartmental trafficking of proteins, lipids, and cell wall components. During these integrative processes, dynamic interactions of membrane proteins with other membrane or soluble components are thought to provide a high degree of flexibility that usually characterizes higher plants. This concept is supported by the recent release of a first, partial Arabidopsis interactome by the Arabidopsis Interactome Mapping Consortium (http://www.sciencemag.org/content/333/6042/601.full.htm). The Arabidopsis interactome reveals a strong enrichment of a few network communities, including those for transmembrane transport and vesicle trafficking. Strikingly, the large transmembrane transport community shares a high amount of proteins with the vesicle trafficking community suggesting a strong physical and functional overlap and interaction.