Protein trafficking through the endomembrane system of plants is a highly dynamic and transient process. Protein trafficking plays a role in several signal transduction and developmental pathways such as gravitropic response, plant pathogen resistance, cell pattern formation and autophagy. One of the most dynamic compartments of the endomembrane system is the endosome. The endosome is comprised on proteins that transit to and from the plasma membrane, the trans-Golgi network, and the prevacuolar compartment. Given the highly dynamic nature of this compartment, chemical genomics is a useful approach to slow down trafficking of proteins or inhibit trafficking through specific compartments. Since high-throughput cellular phenotyping is not available in Arabidopsis thaliana, tobacco pollen was used as a model system for membrane cycling to identify putative trafficking inhibitors for Arabidopsis. Tobacco pollen displays polarized growth that is dependent on the delivery and recycling of protein to the apical tip, and tobacco pollen is highly amenable to high-throughput screening. Utilizing tobacco pollen, over 46,000 natural and synthetic compounds were assayed and 378 were found to inhibit pollen germination and/or morphology. Of these 378, 365 were novel compounds. These 378 were then screened on three highly characterized Arabidopsis plasma membrane protein known to cycle through endosomal compartments, the brassinosteroid receptor BRI1:BRI1:GFP, and two PINFORMED auxin efflux proteins PIN2:PIN2:GFP and PIN2:PIN1:GFP. With just these three markers, 129 compounds from the pollen screen disrupted the normal localization patterns of at least one of these proteins. To examine a specific physiological response, these 129 were screened for effects on root gravitropic response. Sixteen compounds were identified as strongly disrupting gravity response. From these, the compound Endosidin2 (ES2) was characterized. ES2 disrupted several specific plasma membrane markers without affecting any internal protein markers, with the exception of the auxin homeostasis protein PINFORMED5, which localizes to the endoplasmic reticulum. ES2 specifically inhibits endosomal recycling to the plasma membrane without affecting endocytosis. ES2 allows for specific interrogation into the effects of endosomal recycling without affecting any other internal compartments as Brefeldin A does, the only other compound known to inhibit endosomal recycling.
Recent advances in computer vision and image analysis have enabled biological screens of large volumes. Such high-throughput assays increase the likelihood and scope of discovery ultimately leading to functional analysis of a gene or protein. To increase the efficiency of high-throughput screens, computational tools are essential expedite image analysis, models to make sense of the extracted data, and biological assays to characterize the mutation or small molecule. Chemical genomics, the use of small molecules to inactivate proteins, is an advantageous approach when studying a conserved process such as endomembrane trafficking. Endomembrane trafficking spans kingdoms and is important in defense, development, stress response and other vital physiological process. We have taken numerous approaches to study the quantitative behavior of the dynamic endomembrane system to accelerate discovery and better understand these complex phenomena. First, We identified six small molecules altering endomembrane trafficking in tobacco pollen; we characterized their effect on trafficking dynamics using video tracking facilitated by commercially available software giving us insight into intrinsic quantitative properties of the endomembrane system. Next, we wanted to create an automated tool to enable automatic phenotypic screening in Arabidopsis. EndoQuant is an automated computational tool for automatic sub cellular phenotypic analysis. Once this data was collected we developed a model to predict the biological being disrupted based on the cellular phenotype of a fluorescent marker. Using Gaussian Mixture Model, we were able to successfully predict that a subset of small molecules was disrupting endocytic recycling, leading to better experimental design and faster discovery of bioactive molecules. One particular biologically active molecule in Arabidopsis drastically reduced the root length of seedlings. This was found to be a result of cellulose deposition, most likely due to the miss localization of the cellulose synthesis machinery in response to disrupted trafficking membrane. We have analyzed real time membrane dynamics, automated phenotypic analysis, created a predictive model to link a phenotype with a biological process and characterized a small molecule disrupting the transport of a vital protein complex. These tools and methodologies will augment and accelerate the discovery process and our understanding of endomembrane trafficking in plant cells.
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