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Sub-cellular Localization of Arabidopsis thaliana SEC8 In Polarly-Growing Cells

Sub-cellular Localization of Arabidopsis thaliana SEC8 In Polarly-Growing Cells. Kory S. Herrick John E. Fowler, Ph.D. Polarized Growth. Axon and dendrite growth sites in neurons. Polarized Growth. What is the exocyst?. Complex of eight proteins:

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Sub-cellular Localization of Arabidopsis thaliana SEC8 In Polarly-Growing Cells

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  1. Sub-cellular Localization of Arabidopsis thaliana SEC8 In Polarly-Growing Cells Kory S. Herrick John E. Fowler, Ph.D.

  2. Polarized Growth

  3. Axon and dendrite growth sites in neurons. Polarized Growth

  4. What is the exocyst? • Complex of eight proteins: • SEC3, SEC5, SEC6, SEC8, SEC10, SEC 15, EXO70, & EXO84.

  5. Vesicle docking and exocytosis

  6. Question • Is the exocyst complex associated with polarized cellular growth in plants?

  7. Hypothesis A. thaliana SEC8 localizes asymmetrically to regions of the plasma membrane where polarized growth occurs.

  8. Strategy • Generate plasmid constructs encoding Green Fluorescent Protein (GFP) fused to the AtSec8 gene. • Transformation of GFP-AtSec8 fusions into polarly-growing cells. • Characterization of gene product localization using fluorescence microscopy.

  9. Hypothesis Prediction GFP AtSEC8

  10. General Overview Two experiments: • Long-term stable expression of GFP-labeled SEC8 in A. thaliana. • Short-term transient expression of GFP-labeled SEC8 in maize and Arabidopsis thaliana.

  11. Experiment 1 Overview • Produce GFP-AtSec8 constructs. • Clone GFP-AtSec8 and GFP only control into pCamLAT52 Arabidopsis pollen vector. • Stable transformation into A. thaliana. • Look for GFP under fluorescence microscope.

  12. Produce GFP-AtSec8 Fusions Hygr Kan pCamLAT52 AtSec8 GFP LAT52

  13. Plasmid sequencing

  14. Stable transformation with Agrobacterium.

  15. Agrobacterium transformation

  16. Experiment 1 Progress • Seeds harvested • Next steps: • Plant on hygromycin media, and select for transformed plants. • Characterize GFP-AtSEC8 localization in pollen.

  17. Experiment 2 • Transient transformation of GFP- AtSEC8 and GFP-only control plasmids into maize and A. thaliana cells. • Biolistic bombardment.

  18. Biolistics Particle Delivery

  19. Transformed maize cell: • Transmitted light: • Fluorescent light:

  20. Experiment 2 Conclusions • GFP-tagged SEC8 did not demonstrate asymmetrical localization in maize. • Distribution suggests random intracellular diffusion • Problem with A. thaliana SEC8 in maize?

  21. Successful A. thaliana transformation

  22. Future Experimentation • Transiently transform GFP-AtSec8 into A. thaliana cells. • Characterize GFP-AtSec8 localization in stably-transformed plant.

  23. Acknowledgments • John E. Fowler, Ph.D. • Kevin Ahern, Ph.D. • National Science Foundation. • Howard Hughes Medical Institute. • Center for Gene Research and Biotechnology.

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