1 / 23

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:

zonta
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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.

More Related