Download Atsec8 Gene Product Localization

Sub-cellular Localization
of Arabidopsis thaliana
SEC8 In Polarly-Growing
Cells
Kory S. Herrick
John E. Fowler, Ph.D.
Polarized Growth
Polarized Growth
 Axon and dendrite
growth sites in
neurons.
What is the exocyst?
 Complex of eight proteins:
 SEC3, SEC5, SEC6, SEC8, SEC10, SEC 15,
EXO70, & EXO84.
Vesicle docking and exocytosis
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
General Overview
Two experiments:
1. Long-term stable expression of GFPlabeled SEC8 in A. thaliana.
2. Short-term transient expression of GFPlabeled SEC8 in maize and Arabidopsis
thaliana.
Experiment 1 Overview
1. Produce GFP-AtSec8 constructs.
2. Clone GFP-AtSec8 and GFP only control
into pCamLAT52 Arabidopsis pollen
vector.
3. Stable transformation into A. thaliana.
4. Look for GFP under fluorescence
microscope.
Produce GFP-AtSec8 Fusions
Plasmid sequencing
Stable transformation with
Agrobacterium.
Agrobacterium transformation
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.
Biolistics Particle Delivery
Transformed maize cell:
 Transmitted light:
 Fluorescent light:
Experiment 2 Conclusions
1. GFP-tagged SEC8 did not demonstrate
asymmetrical localization in maize.
2. Distribution suggests random
intracellular diffusion
3. Problem with A. thaliana SEC8 in maize?
Successful A. thaliana
transformation
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.