1. RIC1-GFP Pollen Morphology, Growth patterns, and Brefeldin A Geoff Harlow & Mike Oliveira 3/15/12

2. Introduction Cell Polarity in Plants Pollen growth RIC1 and its role in the ROP1 signalling network 3. Why is cell polarity important? Explains how cells make concerted efforts to mechanically achieve survival Serves as a model for mechanical construction that has stood the test of time (millions of years!) 4. Examples of Cell Polarity & Shape Formation in Plants Pavement Cell Root Hairs Trichomes Pollen Tubes Frontiers of scientific inquiry revealed through imaging! 5. Pavement Cells 6. Root Hairs 7. Trichomes 8. ROP1 GTPICR1 / RIP1RIC3ROP1 RIC4GDPSEC3Ca2+actin disassembly+F-actin assemblyPolar exocytosis 9. Why was RIC1 selected? Interacts with ROP1 bound to GTP o ROP1: Rho-GTPase of plants 224 amino acids Localizes to plasma membrane Contains CRIB domain o CRIB: cdc42/Rac-interactive bindingServes as good reporter for tip growth 10. Research Question Research Question: o What is the effect of BFA on Pollen Tube Growth? Hypothesis: o BFA will inhibit pollen tube growth and RIC1 expression at the tip due to inhibition of exocytosis 11. What is Brefeldin A? Fungal metabolite Blocks transport from ER GolgiRough ERGolgiNucleus Vesicles Cell Membrane 12. Methods (1) RIC1 fused with GFP (2) RIC1-GFP inserted into pollen genome (3)Pollen grown on slides containing agarose gel o o o o o o18% Sucrose 5% Boric Acid 1M MgSO4 1M Ca(NO3)2 1M CaCl2 +Brefeldin A @ 1uM, 5uM, 10uM (4) Incubated 3hrs @ 30 degrees C. (5) Visualized using Leica SP2 confocal microscope (6) Analyzed using MATLAB 13. Image Analysis Methodology Image Image Image Image Image ImageInput: Video DataThreshold the imageRemove noiseFind edges and create line of symmetryMorphological image closing to create solid regionFind tip and define search regionTUBE AREA TUBE INTENSITYConnected ComponentsTIP INTENSITYGrowth Normalized Area (Arean / Area1) Tip Intensity Normalized Intensity ( IntTIP / IntTUBE ) 14. Image Analysis - Finding Tube Region Input ImageThresholdRemove NoiseCC/Close Image Gaps 15. Image Analysis - Finding Tube Region Input ImageOutput Image 16. Image Analysis Tip Tracking Input ImageFind Symmetry/TipDefine Search AreaFinal Image 17. Growth Input Data: o Tube mask images Extract area in each image Normalize area to first image Plot Normalized Area vs. Time 18. Tip Tracking RIC1 Expression Input Data: o Segmented tube images o Segmented tip images Determine average intensity o Total pollen tube o Pollen tube tip Normalize intensity o Tip / Tube Plot Normalized Intensity vs. Time 19. Results - Control 20. Results - 1uM BFA 21. Results - 5uM BFA 22. Results - 10uM BFA No growth detected, but interesting observationsWhat is this? 23. Z-Stack of 10uM BFA Treated Pollen Grain (A) 24. Z-Stack of 10uM BFA Treated Pollen Grain (B) 25. Results - Growth 1.2Normalized Area1.151.1Control1.05 11 uM BFA0.955 uM BFA0.9 0.85 0.8 050100150200Time (s)250300350 26. Results Tip Intensity Normalized Intensity1.3 1.2 1.1 1Control 1 uM BFA 5 uM BFA0.9 0.8 0.7 0.6 0.5 0.4 0.3 050100150200Time (s)250300350 27. Results Growth and RIC1 Expression Control1 uM BFA1.2 1 0.8 0.6 0.4 0.21.4Normalized Area/Intensity1.4Normalized Area/IntensityNormalized Area/Intensity1.45 uM BFA1.2 10.8 0.6 0.4100200300400Time (s)Normalized Area Normalized Intensity1 0.8 0.6 0.4 0.20.2 01.20100200 Time (s)3004000100200 Time (s)300400 28. Discussion Questions Does the data support that RIC1 actually oscillates? Are there lag and go phases of total pollen growth? What is the cashew shape inside of the pollen grains? 29. Conclusions RIC1 appears to oscillate, but its not definitive 1uM BFA o RIC1 does not seem to localize at the membrane 5uM BFA o Appears to inhibit growth o RIC1 does not seem to localize at the membrane 10uM BFA o appears to inhibit growth o Produces cashew shaped RIC1 aggregates centrally 30. Future Experiments Other drugs that inhibit/enhance exocytosis: o Wortmannin o Latrunquilin B o Jasplakinolide Other potential genes to label: o o o oSEC3 ICR1 RIP1 PMEi Optimizing growth conditions: o Reduce time between incubation interruption and visualization 31. References Yuan Qin, Zhenbiao Yang. Rapid tip growth: Insights from pollen tubes. Seminars in Cell & Developmental Biology, Volume 22, Issue 8, October 2011, Pages 816-824 Yan Zhang, Junmin He, David Lee, and Sheila McCormick. Interdependence of Endomembrance Trafficking and Actin Dynamics during Polarized Growth of Arabidopsis Pollen Tubes. Plant Physiol. Vol. 152, 2010, Pages 22002210 http://arabidopsis.org/servlets/TairObject?id=34472 &type=gene 32. Appendix: MATLAB Functions Software Function Read images Threshold an image Remove small clusters Close region gaps Find connected componentsMATLAB Function imread() graythresh() bwareaopen() imclose() bwconncomp()