Endocytic trafficking underlies processes essential for plant growth and development, including the perception of and response to abiotic and extracellular stimuli, post‐Golgi and exocytic ...trafficking, and cytokinesis. Protein adaptors and regulatory factors of clathrin‐mediated endocytosis that contribute to the formation of endocytic clathrin‐coated vesicles are evolutionarily conserved. Yet, work of the last ten years has identified differences between the endocytic mechanisms of plants and Opisthokonts involving the endocytic adaptor TPLATE complex, the requirement of actin during CME, and the function of clathrin‐independent endocytosis in the uptake of plant‐specific plasma membrane proteins. Here, we review clathrin‐mediated and ‐independent pathways in plants and describe recent advances enabled by new proteomic and imaging methods, and conditional perturbation of endocytosis. In addition, we summarize the formation and trafficking of clathrin‐coated vesicles based on temporal and structural data garnered from high‐resolution quantitative imaging studies. Finally, new information about the cross‐talk between endocytosis and other endomembrane trafficking pathways and organelles will also be discussed.
Endocytic trafficking underlies processes essential for plant growth and development. This review (a) provides an overview of plant clathrin‐mediated and ‐independent endocytosis based on recent advances enabled by cutting‐edge proteomic and imaging methods and conditional perturbation of endocytosis, yielding high‐resolution structural and temporal details, and (b) discusses the cross‐talk between endocytosis and other endomembrane trafficking pathways and organelles.
In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of ...functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.
Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking ...of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.
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.
Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their ...physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the “pseudo 3D” morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.
Planta clathrin-coated vesicles are essential trafficking organelles but have not undergone detailed characterization. Here the authors present accessible methods to visualize them in three dimensions with nanometer resolution in cells or biochemically isolated preparations. The presented methods focus on creating 3D representations of single vesicles at high resolution, or, by utilizing deep learning, provide a high-throughput robust screening approach to analyze the 3D morphology of vesicles from lower resolution images..
The sub-compartmentalization of plant cells has necessitated the development of transport mechanisms which deliver protein and membrane cargos in a spatially and temporally controlled fashion between ...the various endomembrane compartments within the cell as well as the cell periphery. These transport pathways are essential to several subcellular processes and organismal responses including cell growth and expansion, the construction of de novo plasma membrane and cell wall during cytokinesis, construction of the cell wall, and the delivery and recycling of transporters and receptors located at the interface between the plant cell and its environment. Consequently, disruption of the proteins or lipid homeostasis facilitating these transport pathways results in a range of developmental defects, including seedling lethality. Many of the key principles of endomembrane transport (cargo recognition and recruitment of vesicle coat proteins through adaptors, membrane deformation, vesicle scission, vesicle transport, and vesicle docking and fusion) are evolutionarily conserved across Eukaryotes and specifically between land plants and members of the Opisthokont supergroup, which includes yeast and animals. Yet, since the evolution of plants, yeast, and animals from the last eukaryotic common ancestor, proteins facilitating membrane trafficking have expanded, diverged in function, or become lost in specific lineages, resulting in evolutionarily divergent vesicle transport mechanisms. The work described in this thesis characterizes evolutionarily conserved membrane trafficking pathways in plants and identifies key points of evolutionarily divergence between plants and Opisthokonts. First, the proteomic and biochemical characterization of a heterogenous mixture of clathrin coated vesicles (CCVs) from undifferentiated, suspension cultured Arabidopsis cells yields the surprising enrichment of the vesicle adaptor, AP-4, with plant CCVs and the low abundance and depletion from purified CCVs of the known clathrin-mediated endocytosis adaptor, TPLATE complex. Secondly, I include biochemical data supporting the peripheral association of the TPLATE subunit of the Jotnarlog and endocytic adaptor, TPLATE complex, with enriched clathrin coated vesicles relative to the evolutionarily conserved endocytic adaptor, AP-2, which is more tightly associated. Additional data describing the assembly and interaction network of the Stomatal Cytokinesis Defective complex, an evolutionarily divergent regulator of an evolutionarily conserved small GTPase, RabE1, which functions in post-Golgi trafficking.
The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm ...maleimide-PEG (Mal-PEG) to beta-cross-linked (betaXL-Hb) hemoglobin (Hb) tetramers through the Cys beta93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three betaXL-Hb to one Mal-PEG. Thermal denaturation studies showed that the cross-linked and PEGylated species were more stable than native Hb. Cross-linking under oxy-conditions produced a high oxygen affinity Hb species (P50 = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P50 = 9.67 Torr). Inside-out PEGylation can be used to produce a hemoglobin-based oxygen carrier and potentially for other multiprotein complexes.
The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside‐out PEGylation, has been used here to conjugate a multiarm ...maleimide‐PEG (Mal‐PEG) to β‐cross‐linked (βXL‐Hb) hemoglobin (Hb) tetramers through the Cys β93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three βXL‐Hb to one Mal‐PEG. Thermal denaturation studies showed that the cross‐linked and PEGylated species were more stable than native Hb. Cross‐linking under oxy‐conditions produced a high oxygen affinity Hb species (P50 = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P50 = 9.67 Torr). Inside‐out PEGylation can be used to produce a hemoglobin‐based oxygen carrier and potentially for other multiprotein complexes.
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