In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.

• Keywords
• Golgi, endocytosis, exocytosis, microscopy, proteomics, vesicle,
• MeSH
• Biological Transport MeSH
• Endocytosis MeSH
• Mass Spectrometry methods   MeSH
• Proteome * analysis   metabolism   MeSH
• Proteomics * methods   MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Proteome * MeSH

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.

• Keywords
• Arabidopsis, cytoskeleton, mitochondrial protein import, phospholipase D alpha1, proteomics, quality control, vesicular transport,
• MeSH
• Arabidopsis metabolism   MeSH
• Endocytosis MeSH
• Phospholipase D genetics   metabolism   MeSH
• Gene Ontology MeSH
• Glucosinolates biosynthesis   MeSH
• Plant Roots metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Proteome metabolism   MeSH
• Proteomics * MeSH
• Synaptotagmin I metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Protein Transport MeSH
• Uncoupling Protein 1 metabolism   MeSH
• Chromatography, High Pressure Liquid MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phospholipase D MeSH
• Glucosinolates MeSH
• Mitochondrial Proteins MeSH
• PLDA1 protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• Proteome MeSH
• Synaptotagmin I MeSH
• SYT1 protein, Arabidopsis MeSH Browser
• Uncoupling Protein 1 MeSH

• Keywords
• mitogen-activated protein kinase (MAPK), phosphoproteomics, proteomics, shot-gun proteomics, signaling, validation,
• Publication type
• Journal Article MeSH

The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes-termed herein as dancing-endosomes-which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth.

• Keywords
• Arabidopsis thaliana, development, endosomes, interaction, root hair, spinning disc microscopy, structured illumination microscopy, trafficking,
• Publication type
• Journal Article MeSH

LY294002 is a synthetic quercetin-like compound, which, unlike wortmannin, is more specific inhibitor of phosphatidylinositol 3-kinase (PI3K). It inhibits endocytosis and vacuolar transport. We report here on the proteome-wide effects of LY294002 on Arabidopsis roots focusing on proteins involved in vesicular trafficking and stress response. At the subcellular level, LY294002 caused swelling and clustering of late endosomes leading to inhibition of vacuolar transport. At the proteome level, this compound caused changes in abundances of proteins categorized to 10 functional classes. Among proteins involved in vesicular trafficking, a small GTPase ARFA1f was more abundant, indicating its possible contribution to the aggregation and fusion of late endosomes triggered by LY294002. Our study provides new information on storage proteins and vacuolar hydrolases in vegetative tissues treated by LY294002. Vacuolar hydrolases were downregulated, while storage proteins were more abundant, suggesting that storage proteins were protected from degradation in swollen multivesicular bodies upon LY294002 treatment. Upregulation of 2S albumin was validated by immunoblotting and immunolabeling analyses. Our study also pointed to the control of antioxidant enzyme machinery by PI3K because LY294002 downregulated two isozymes of superoxide dismutase. This most likely occurred via PI3K-mediated downregulation of protein AtDJ1A. Finally, we discuss specificity differences of LY294002 and wortmannin against PI3K, which are reflected at the proteome level. Compared with wortmannin, LY294002 showed more narrow and perhaps also more specific effects on proteins, as suggested by gene ontology functional annotation.

• MeSH
• Molecular Sequence Annotation MeSH
• Arabidopsis cytology   drug effects   metabolism   MeSH
• Biological Transport MeSH
• Chromones pharmacology   MeSH
• Phosphatidylinositol 3-Kinases metabolism   MeSH
• Stress, Physiological MeSH
• Phosphoinositide-3 Kinase Inhibitors * MeSH
• Plant Roots cytology   drug effects   metabolism   MeSH
• Morpholines pharmacology   MeSH
• Arabidopsis Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Proteome genetics   metabolism   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• trans-Golgi Network metabolism   MeSH
• Transport Vesicles metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one MeSH Browser
• Chromones MeSH
• Phosphoinositide-3 Kinase Inhibitors * MeSH
• Morpholines MeSH
• Arabidopsis Proteins MeSH
• Proteome MeSH