Although it is known that proteins are delivered to and recycled from the plasma membrane (PM) via endosomes, the nature of the compartments and pathways responsible for cargo and vesicle sorting and cellular signaling is poorly understood. To define and dissect specific recycling pathways, chemical effectors of proteins involved in vesicle trafficking, especially through endosomes, would be invaluable. Thus, we identified chemicals affecting essential steps in PM/endosome trafficking, using the intensely localized PM transport at the tips of germinating pollen tubes. The basic mechanisms of this localized growth are likely similar to those of non-tip growing cells in seedlings. The compound endosidin 1 (ES1) interfered selectively with endocytosis in seedlings, providing a unique tool to dissect recycling pathways. ES1 treatment induced the rapid agglomeration of the auxin translocators PIN2 and AUX1 and the brassinosteroid receptor BRI1 into distinct endomembrane compartments termed “endosidin bodies”; however, the markers PIN1, PIN7, and other PM proteins were unaffected. Endosidin bodies were defined by the syntaxin SYP61 and the V-ATPase subunit VHA-a1, two trans -Golgi network (TGN)/endosomal proteins. Interestingly, brassinosteroid (BR)-induced gene expression was inhibited by ES1 and treated seedlings displayed a brassinolide (BL)-insensitive phenotype similar to a bri1 loss-of-function mutant. No effect was detected in auxin signaling. Thus, PIN2, AUX1, and BRI1 use interactive pathways involving an early SYP61/VHA-a1 endosomal compartment.

Endomembrane trafficking relies on the coordination of a highly complex, dynamic network of intracellular vesicles. Understanding the network will require a dissection of cargo and vesicle dynamics at the cellular level in vivo. This is also a key to establishing a link between vesicular networks and their functional roles in development. We used a high-content intracellular screen to discover small molecules targeting endomembrane trafficking in vivo in a complex eukaryote, Arabidopsis thaliana . Tens of thousands of molecules were prescreened and a selected subset was interrogated against a panel of plasma membrane (PM) and other endomembrane compartment markers to identify molecules that altered vesicle trafficking. The extensive image dataset was transformed by a flexible algorithm into a marker-by-phenotype-by-treatment time matrix and revealed groups of molecules that induced similar subcellular fingerprints (clusters). This matrix provides a platform for a systems view of trafficking. Molecules from distinct clusters presented avenues and enabled an entry point to dissect recycling at the PM, vacuolar sorting, and cell-plate maturation. Bioactivity in human cells indicated the value of the approach to identifying small molecules that are active in diverse organisms for biology and drug discovery.

Since the introduction of chemical genomics to plant biology as a tool for basic research, the field has advanced significantly. There are now examples of important basic discoveries that demonstrate the power and untapped potential of this approach. Given the combination of protein and small-molecule complexity, new phenotypes can be described through the perturbation of cellular functions that can be linked to growth and developmental phenotypes. There are now clear examples of overcoming functional redundancy in plants to dissect molecular mechanisms or critical pathways such as hormone signaling and dynamic intracellular processes. Owing to ongoing advances, including more sophisticated high-content screening and rapid approaches for target identification, the field is beginning to move forward. However, there are also challenges to improve automation, imaging, and analysis and provide chemical biology resources to the broader plant biology community.

Interactions between plant cells and the environment rely on modulation of protein receptors, transporters, channels, and lipids at the plasma membran

The inhibitor endosin 16 defines a distinct pathway for endomembrane sorting via regulation of the small GTPase RabA that is essential for the establishment of

Chemical genomics (i.e. genomics scale chemical genetics) approaches capitalize on the ability of low molecular mass molecules to modify biological processes. Such molecules are used to modify the activity of a protein or a pathway in a manner that it is tunable and reversible. Bioactive chemicals resulting from forward or reverse chemical screens can be useful in understanding and dissecting complex biological processes due to the essentially limitless variation in structure and activities inherent in chemical space. A major advantage of this approach as a powerful addition to conventional plant genetics is the fact that chemical genomics can address loss-of-function lethality and redundancy. Furthermore, the ability of chemicals to be added at will and to act quickly can permit the study of processes that are highly dynamic such as endomembrane trafficking. An important aspect of utilizing small molecules effectively is to characterize bioactive chemicals in detail including an understanding of structur...