Preparation of Clathrin-Coated Vesicles From Arabidopsis thaliana Seedlings

Mosesso, Niccolò; Bläske, Tobias; Nagel, Marie-Kristin; Laumann, Michael; Isono, Erika

doi:10.3389/fpls.2018.01972

Cited by 10 publications

(6 citation statements)

References 30 publications

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“…Further to this, digestion of the cell wall has been shown to result in intracellular aggregation of certain EAPs (Kang et al, 2003). However, the average CCV size closely matches that of biochemically purified CCVs from plant tissues (Mosesso et al, 2018;Reynolds et al, 2014), suggesting that CCVs in both contexts are formed in a similar fashion. A further limitation is accessibility to only the top view of the CCV, which means that hallmark features of the CME, such as the highly curved neck of CCVs, are obstructed by the vesicle itself.…”

Section: Unroofing Metal Replicamentioning

confidence: 84%

“…When EM approaches are combined with protocols for the enrichment of CCVs from plant tissues, as described in detail by Mosesso et al (2018) and Reynolds et al (2014), one can begin to define the molecular anatomy of the plant CCV. The disadvantage of examining CCVs isolated from plant tissues is that the preparation could be a mixture of PM-and trans-Golgi network (TGN)-derived CCV populations.…”

Section: Electron Microscopy Methods; Characterization Of Cme At the Ultrastructural Levelmentioning

confidence: 99%

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Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis

Johnson

Gnyliukh

Kaufmann

et al. 2020

Journal of Cell Science

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Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and inter-cellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how it functions in planta. In order to facilitate the direct quantitative study of plant CME, here we review current routinely used methods and present refined, standardized quantitative imaging protocols which allow the detailed characterization of CME at multiple scales in plant tissues. These include: (i) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultra-structure of clathrin-coated vesicles; (ii) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (iii) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (iv) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples.

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Section: Unroofing Metal Replicamentioning

confidence: 84%

Section: Electron Microscopy Methods; Characterization Of Cme At the Ultrastructural Levelmentioning

confidence: 99%

Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis

Johnson

Gnyliukh

Kaufmann

et al. 2020

Journal of Cell Science

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“…By using the same sample preparation for biochemically isolated CCVs, we could directly compare them to specific CCV populations visualized inside cells. While we found that there were small differences in the sizes of endocytic and isolated CCVs, this is likely due to the fact that the isolated CCV preparations from whole cell lysates contain a mixture of CCVs derived from different origins -clathrin-mediated endocytosis and the early endosome/trans-Golgi network (22,27,28). We then validated our high throughput pseudo 3D analysis by applying it to metal replicas of unroofed cells carrying an inducible loss of endocytic membrane bending.…”

Section: Discussionmentioning

confidence: 77%

User-friendly electron microscopy protocols for the visualization of biological macromolecular complexes in three dimensions: Visualization of planta clathrin-coated vesicles at ultrastructural resolution

Johnson

Kaufmann

Sommer

et al. 2022

Preprint

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Biological systems are the sum of their dynamic 3-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolutions 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 protocols require specialized equipment and personnel to complete them. Here we present novel accessible protocols to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolutions, focusing on Arabidopsis clathrin-coated vesicles (CCVs) - an essential trafficking organelle lacking detailed structural characterization due to their low preservation in classical electron microscopy techniques. First, we establish a protocol to visualize CCVs in unroofed cells using scanning-transmission electron microscopy (STEM) 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 protocol in any electron microscopy lab. Secondly, we demonstrate 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 workflow to screen the ‘pseudo 3D’ morphology of CCVs imaged with 2D modalities. Overall, we present accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.

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“…The data reported here reflects the content of CCVs isolated from undifferentiated, rapidly dividing and expanding Arabidopsis suspension cultured cells under conditions amenable to tissue culture. Future experiments probing CCV content in other cell and tissue types under varying conditions or in different genetic backgrounds might apply our isolation methodology (Reynolds et al, 2014) using seedlings as a sample source as recently described (Nagel et al, 2017;Mosesso et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

Dahhan¹,

Reynolds²,

Cárdenas³

et al. 2021

Preprint

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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 AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) 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 trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis 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.

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Preparation of Clathrin-Coated Vesicles From Arabidopsis thaliana Seedlings

Cited by 10 publications

References 30 publications

Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis

Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis

User-friendly electron microscopy protocols for the visualization of biological macromolecular complexes in three dimensions: Visualization of planta clathrin-coated vesicles at ultrastructural resolution

Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

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