#244

Plasma membrane targeting and polar growth in pollen and seedlings

The pollen tube is a single polarized cell whose growth is highly dependent upon cytoskeleton, a tip-focus calcium gradient, and protein and membrane delivery and recycling at the apical plasma membrane (PM). Chemical disruption of exocytosis, endocytosis or protein interactions that affect polar PM localization can be detected with GFP-RIP1, a marker that displays focused tip localization in pollen tubes. ROP1-interacting partner 1 (RIP1) is localized to and recruits the GTPase ROP1 to the pollen tube tip. This high throughput screen uses a 384-well microtiter plate format that is applicable to germinating pollen or cultured cells. Arrayed chemicals are distributed via a Beckman Biomek robot utilizing a 96-well pin tool. For germinating pollen, the screen involves several tiers. PRIMARY SCREEN: The primary screen detects compounds that inhibit pollen germination which is dependent upon tip-focused vesicle and protein targeting and recycling. Among the possibilities, germination inhibitors could affect exocytosis, endocytosis, protein targeting, or interacting ROP or calcium signaling pathways. The primary screen is rapid (15 sec/well) and automated using the Atto Pathway confocal microscope. SECONDARY SCREEN: The germination inhibitors are diluted serially and pollen of tobacco expressing GFP-RIP1 (or other markers) is geminated in their presence. Scoring is automated on the Atto Pathway by multiple image capture (GFP and transmitted). Bioactive chemicals that result in mis-localization are examined at higher resolution using conventional LSCM. GFP-RIP1 is localized to the apical PM via the endomembrane system, so chemicals that affect the exocytosis/endocytosis of GFP-RIP1 or its interaction with ROP would result in mis-localization and loss of growth polarity. Our initial screen of a library of bioactive chemicals demonstrated that we can detect useful chemicals. In addition, several chemicals of known activity in this library inhibited pollen germination including cytochalasins, cyclohexamide, and several ionophores. None resulted in mis-localization indicating the high specificity of the screen. SUMMARY STATISTICS: In a primary screen of 2016 compounds from the Microsource Spectrum library, we recovered 115 chemicals (5.7% of the total) that resulted in complete inhibition of pollen germination. These 115 compounds were diluted and examined for mis-localization of GFP-RIP1 and loss of growth polarity. Four of the chemicals (0.2% of the 2016 total) were determined to result in mis-localization of RIP1-GFP and loss of growth polarity (pollen tube tip swelling). THIS IS A NEW SCREENING APPROACH AND THESE CHEMICALS HAVE NOT BEEN FULLY CHARACTERIZED YET. HOWEVER, THE EXAMPLE SHOWN, WHICH AFFECTS POLLEN AND SEEDLING POLARITY MARKERS, INDICATES THE NATURE OF THE CHEMICALS WE HAVE RECOVERED. WE WILL PROVIDE UPDATES AS OUR CHARACTERIZATION BECOMES MORE COMPLETE.

NSF: 2010-0520325

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In total, 1 compound from 1 library is annotated. Out of these, 1 has a score of 1 or higher, and 0 are annotated as inactive.