Clathrin-independent endocytosis contributes to uptake of glucose into BY-2 protoplasts (original) (raw)
Related papers
2019
In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.
Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants
2022
Clathrin-mediated endocytosis (CME) is an essential cellular internalisation pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. In plants, the evolutionarily ancient TSET/TPLATE complex (TPC) plays an essential, but not well-defined role in CME. Here, we show that two highly disordered TPC subunits, AtEH1 and AtEH2 function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, early-, and late-stage endocytic accessory proteins. Importantly, clathrin forms ordered assemblies within the condensate environment. Biomolecular condensation therefore acts to promote dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, the disordered region sequence properties of AtEH1 regulate the material properties of the endocytic cond...
Evolutionary unique mechanistic framework of clathrin-mediated endocytosis in plants
In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME follows the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the surface but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mecha...
Clathrin Mediates Endocytosis and Polar Distribution of PIN Auxin Transporters in Arabidopsis
The Plant Cell, 2011
Endocytosis is a crucial mechanism by which eukaryotic cells internalize extracellular and plasma membrane material, and it is required for a multitude of cellular and developmental processes in unicellular and multicellular organisms. In animals and yeast, the best characterized pathway for endocytosis depends on the function of the vesicle coat protein clathrin. Clathrinmediated endocytosis has recently been demonstrated also in plant cells, but its physiological and developmental roles remain unclear. Here, we assessed the roles of the clathrin-mediated mechanism of endocytosis in plants by genetic means. We interfered with clathrin heavy chain (CHC) function through mutants and dominant-negative approaches in Arabidopsis thaliana and established tools to manipulate clathrin function in a cell type-specific manner. The chc2 single mutants and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis as well as in internalization of prominent plasma membrane proteins. Interference with clathrin-mediated endocytosis led to defects in constitutive endocytic recycling of PIN auxin transporters and their polar distribution in embryos and roots. Consistent with this, these lines had altered auxin distribution patterns and associated auxin transport-related phenotypes, such as aberrant embryo patterning, imperfect cotyledon specification, agravitropic growth, and impaired lateral root organogenesis. Together, these data demonstrate a fundamental role for clathrin function in cell polarity, growth, patterning, and organogenesis in plants. 2004). Such CCVs were found at the plasma membrane, trans-Golgi network, endosomes, and lysosomes, where they effect endocytosis, protein sorting, and degradation (Kirchhausen, 1 These authors contributed equally to this work.
Proceedings of the National Academy of Sciences, 2010
Endocytosis performs a wide range of functions in animals and plants. Clathrin-coated vesicle (CCV) formation is an initial step of endocytosis, and in animal cells is largely achieved by dynamins. However, little is known of its molecular mechanisms in plant cells. To identify dynamin-related proteins (DRPs) involved in endocytic CCV formation in plant cells, we compared the behaviors of two structurally different Arabidopsis DRPs, DRP2B and DRP1A, with those of the clathrin light chain (CLC), a marker of CCVs, at the plasma membrane by variable incidence angle fluorescent microscopy (VIAFM). DRP2B shares domain organization with animal dynamins whereas DRP1A is plant-specific. We show that green fluorescent protein (GFP)-tagged DRP2B and DRP1A colocalized with CLC tagged with monomeric Kusabira Orange (mKO) in Arabidopsis cultured cells. Time-lapse VIAFM observations suggested that both GFP-DRP2B and GFP-DRP1A appeared and accumulated on the existing mKO-CLC foci and disappeared at the same time as or immediately after the disappearance of mKO-CLC. Moreover, DRP2B and DRP1A colocalized and assembled/disassembled together at the plasma membrane in Arabidopsis cells. A yeast twohybrid assay showed that DRP2B and DRP1A interacted with each other. An inhibitor of clathrin-mediated endocytosis, tyrphostin A23, disturbed the localization of DRP1A, but had little effect on the localization of DRP2B, indicating that DRP1A and DRP2B have different molecular properties. These results suggest that DRP2B and DRP1A participate together in endocytic CCV formation in Arabidopsis cells despite the difference of their molecular properties.
Proceedings of the National Academy of Sciences of the United States of America, 2010
Endocytosis performs a wide range of functions in animals and plants. Clathrin-coated vesicle (CCV) formation is an initial step of endocytosis, and in animal cells is largely achieved by dynamins. However, little is known of its molecular mechanisms in plant cells. To identify dynamin-related proteins (DRPs) involved in endocytic CCV formation in plant cells, we compared the behaviors of two structurally different Arabidopsis DRPs, DRP2B and DRP1A, with those of the clathrin light chain (CLC), a marker of CCVs, at the plasma membrane by variable incidence angle fluorescent microscopy (VIAFM). DRP2B shares domain organization with animal dynamins whereas DRP1A is plant-specific. We show that green fluorescent protein (GFP)-tagged DRP2B and DRP1A colocalized with CLC tagged with monomeric Kusabira Orange (mKO) in Arabidopsis cultured cells. Time-lapse VIAFM observations suggested that both GFP-DRP2B and GFP-DRP1A appeared and accumulated on the existing mKO-CLC foci and disappeared at the same time as or immediately after the disappearance of mKO-CLC. Moreover, DRP2B and DRP1A colocalized and assembled/disassembled together at the plasma membrane in Arabidopsis cells. A yeast twohybrid assay showed that DRP2B and DRP1A interacted with each other. An inhibitor of clathrin-mediated endocytosis, tyrphostin A23, disturbed the localization of DRP1A, but had little effect on the localization of DRP2B, indicating that DRP1A and DRP2B have different molecular properties. These results suggest that DRP2B and DRP1A participate together in endocytic CCV formation in Arabidopsis cells despite the difference of their molecular properties.
Visualising endocytosis in plants: past, present, and future
Journal of Microscopy, 2020
Chris Hawes had a lively fascination for the immensely complex organization of the endomembrane system, including the process of endocytosis. This is the method by which eukaryotic cells internalise membrane proteins, lipids, carbohydrates, and cell wall enzymes from the cell surface through membrane bound vesicles. Endocytosis occurs progressively, starting with early membrane deformation, scission, and finally the release of the vesicle into the cytoplasm. Next to secretion, endocytosis allows the cell to control the proteome composition of its inner and outer surface membrane and as such, its communication with the outside world. Whereas endocytosis was initially considered theoretically impossible in plants due to their high turgor pressure, it is now established as essential for plant life. Furthermore, endocytosis remains a highly active field of research, both in yeast, animal, and plant model systems. Over the past three decades, the tools and techniques used to visualise, quantify, and characterise endocytosis have resulted in an increasingly higher spatiotemporal understanding of this process. Here we provide a brief history of plant endocytosis research from the time when Chris Hawes was investigating the process, to the current state-of-the-This article is protected by copyright. All rights reserved. 2 art in the field. We will end this chapter with a discussion on some promising future developments for plant endocytosis research. This article is protected by copyright. All rights reserved. 3 A brief history of endocytosis in plants Chris Hawes had an intense fascination for understanding the intracellular organisation of a cell. Specifically, Chris was intrigued by the immensely complex organization of the endomembrane system, which covers the anterograde trafficking pathways from the endoplasmic reticulum to the Golgi apparatus, to the trans-Golgi network and to the plasma membrane, as well as the retrograde transport route, which recycles material from the Golgi apparatus back to the endoplasmic reticulum. Moreover, he also studied the endocytic pathway, which involves the delivery of material from the cell surface via endosomes and multi-vesicular bodies to the lytic vacuole. He approached his endomembrane research questions largely through imaging, and was very much interested in endocytosis, a process on which he co-authored a book 1. Endocytosis is the process by which eukaryotic cells internalise extracellular molecules and parts of their plasma membrane through the formation of endocytic vesicles. This allows cells to regulate the turnover of plasma membrane proteins (cargos), including cell wall enzymes, receptors, and transporters. Endocytosis is progressive, starting with membrane bending leading to the formation of an invagination, to scission, which releases the internalized vesicle into the cytoplasm (Figure 1a).
Sucrose-inducible Endocytosis as a Mechanism for Nutrient Uptake in Heterotrophic Plant Cells
Plant and Cell Physiology, 2005
The capacity of plant heterotrophic organs to transport and accumulate incoming nutrients (mostly in the form of sucrose) directly impacts their final size, crop productivity and nutritional value. Endocytosis as a mechanism for nutrient uptake in heterotrophic cells was investigated using suspension culture cells of sycamore (Acer pseudoplatanus L.) and the endocytic inhibitors wortmannin and LY294002. Time course analysis of sucrose uptake in intact walled cells revealed a two-phase process involving an initial 90 min wortmannin-and LY294002insensitive sucrose uptake period, followed by a prolonged phase of rapid sucrose accumulation which was greatly inhibited by the two endocytic inhibitors. Walled cells were assessed for their capacity to incorporate the fluorescent endocytosis marker lucifer yellow-CH (LY) in the presence or absence of sucrose. Rates of sucrose and LY accumulation were virtually identical, as was their response to wortmannin. In addition, LY incorporation increased as a function of external sucrose concentration. When sucrose was substituted by other sugars or amino acids, uptake of LY greatly diminished, indicating that sucrose itself is the primary signal of endocytosis. Microscopic observations revealed the formation of vesicles containing LY and its eventual accumulation on the vacuole when sucrose was present in the incubation medium. These results demonstrate the existence of a sucrose-inducible endocytic process as a viable mechanism for solute transport into the vacuole of storage cells.