Sub-cellular Localization Sub-cellular Localization of of Arabidopsis thalianaArabidopsis thaliana

SEC8 In Polarly-Growing SEC8 In Polarly-Growing CellsCells

Kory S. HerrickKory S. Herrick

John E. Fowler, Ph.D.John E. Fowler, Ph.D.

Polarized Growth Polarized Growth

Polarized GrowthPolarized Growth

Axon and dendrite Axon and dendrite growth sites in growth sites in neurons.neurons.

What is the exocyst?What is the exocyst? Complex of eight proteins:Complex of eight proteins:

SEC3, SEC5, SEC6, SEC8, SEC10, SEC 15, SEC3, SEC5, SEC6, SEC8, SEC10, SEC 15, EXO70, & EXO84.EXO70, & EXO84.

Vesicle docking and exocytosisVesicle docking and exocytosis

QuestionQuestion

Is the exocyst complex associated with Is the exocyst complex associated with polarized cellular growth in plants?polarized cellular growth in plants?

HypothesisHypothesis

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

StrategyStrategy

Generate plasmid constructs encoding Generate plasmid constructs encoding Green Fluorescent Protein (GFP) fused to Green Fluorescent Protein (GFP) fused to the the AtSec8AtSec8 gene. gene.

Transformation of GFP-Transformation of GFP-AtSec8AtSec8 fusions fusions into polarly-growing cells.into polarly-growing cells.

Characterization of gene product Characterization of gene product localization using fluorescence localization using fluorescence microscopy.microscopy.

Hypothesis PredictionHypothesis Prediction

General OverviewGeneral OverviewTwo experiments:Two experiments:

1.1. Long-term stable expression of GFP-Long-term stable expression of GFP-labeled SEC8 in labeled SEC8 in A. thalianaA. thaliana..

2.2. Short-term transient expression of GFP-Short-term transient expression of GFP-labeled SEC8 in maize and labeled SEC8 in maize and Arabidopsis Arabidopsis thalianathaliana..

Experiment 1 OverviewExperiment 1 Overview

1.1. Produce GFP-Produce GFP-AtSec8AtSec8 constructs. constructs.

2.2. Clone GFP-Clone GFP-AtSec8AtSec8 and GFP only control and GFP only control into pCamLAT52 Arabidopsis pollen into pCamLAT52 Arabidopsis pollen vector.vector.

3.3. Stable transformation into Stable transformation into A. thalianaA. thaliana..

4.4. Look for GFP under fluorescence Look for GFP under fluorescence microscope.microscope.

Produce GFP-AtSec8 FusionsProduce GFP-AtSec8 Fusions

Plasmid sequencingPlasmid sequencing

Stable transformation with Stable transformation with Agrobacterium.Agrobacterium.

AgrobacteriumAgrobacterium transformation transformation

Experiment 1 ProgressExperiment 1 Progress

Seeds harvestedSeeds harvested Next steps: Next steps:

Plant on hygromycin media, and select for Plant on hygromycin media, and select for transformed plants.transformed plants.

Characterize GFP-AtSEC8 localization in pollen.Characterize GFP-AtSEC8 localization in pollen.

Experiment 2Experiment 2

Transient transformation of GFP- AtSEC8 Transient transformation of GFP- AtSEC8 and GFP-only control plasmids into maize and GFP-only control plasmids into maize and and A. thalianaA. thaliana cells. cells. Biolistic bombardment.Biolistic bombardment.

Biolistics Particle DeliveryBiolistics Particle Delivery

Transformed maize cell:Transformed maize cell:

Transmitted light:Transmitted light:

Fluorescent light:Fluorescent light:

Experiment 2 ConclusionsExperiment 2 Conclusions

1.1. GFP-tagged SEC8 did not demonstrate GFP-tagged SEC8 did not demonstrate asymmetrical localization in maize.asymmetrical localization in maize.

2.2. Distribution suggests random Distribution suggests random intracellular diffusionintracellular diffusion

3.3. Problem with Problem with A. thalianaA. thaliana SEC8 in maize? SEC8 in maize?

Successful Successful A. thalianaA. thaliana transformationtransformation

Future ExperimentationFuture Experimentation

Transiently transform GFP-AtSec8 into Transiently transform GFP-AtSec8 into A. A. thalianathaliana cells. cells.

Characterize GFP-AtSec8 localization in Characterize GFP-AtSec8 localization in stably-transformed plant. stably-transformed plant.

AcknowledgmentsAcknowledgments

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

Biotechnology.Biotechnology.