230 likes | 372 Views
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:
E N D
Sub-cellular Localization of Arabidopsis thaliana SEC8 In Polarly-Growing Cells Kory S. Herrick John E. Fowler, Ph.D.
Axon and dendrite growth sites in neurons. Polarized Growth
What is the exocyst? • Complex of eight proteins: • SEC3, SEC5, SEC6, SEC8, SEC10, SEC 15, EXO70, & EXO84.
Question • Is the exocyst complex associated with polarized cellular growth in plants?
Hypothesis A. thaliana SEC8 localizes asymmetrically to regions of the plasma membrane where polarized growth occurs.
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.
Hypothesis Prediction GFP AtSEC8
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.
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.
Produce GFP-AtSec8 Fusions Hygr Kan pCamLAT52 AtSec8 GFP LAT52
Experiment 1 Progress • Seeds harvested • Next steps: • Plant on hygromycin media, and select for transformed plants. • Characterize GFP-AtSEC8 localization in pollen.
Experiment 2 • Transient transformation of GFP- AtSEC8 and GFP-only control plasmids into maize and A. thaliana cells. • Biolistic bombardment.
Transformed maize cell: • Transmitted light: • Fluorescent light:
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?
Future Experimentation • Transiently transform GFP-AtSec8 into A. thaliana cells. • Characterize GFP-AtSec8 localization in stably-transformed plant.
Acknowledgments • John E. Fowler, Ph.D. • Kevin Ahern, Ph.D. • National Science Foundation. • Howard Hughes Medical Institute. • Center for Gene Research and Biotechnology.