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Visualizing Autophagy in Nutrient Starved Cells Via Transfection of an LC3 Dual Fluorescence Plasmid

Bryan Gutman, Renee Saneholtz, and Dr. Heather Coan, Department of Biology, Western Carolina University, 1 University Way, Cullowhee, NC 28723

Autophagy is an intracellular process used to degrade and recycle unneeded or damaged cellular material. The autophagy pathway is vital in maintaining cellular homeostasis during times of stress. Tracking activation of this pathway can help us better understand how cells respond to a variety of factors such as biomaterials, therapeutics, and damage. LC3 is a protein that is modified and incorporated into autophagosomes. By incorporating the LC3 gene fused with fluorescent genes into cells, activation and progression of autophagy can be tracked. This experiment centers around perfecting the methodology of transfecting human embryonic kidney (HEK-293) cells with plasmids containing LC3. Six well dishes were seeded with 200,000 cells/well and transfected with the plasmid vector via liposomes. LC3 is fused to fluorescent genes mApple and green fluorescent protein (GFP) to verify transfection and track LC3 via fluorescence microscopy. Using two different fluorescent proteins is essential in tracking the progression of autophagy. Early stages are indicated by the presence of both green (GFP) and red (mApple) puncta in autophagosomes. When autophagosomes fuse with lysosomes, the low pH environment denatures GFP. The lack of green puncta is an indicator that autophagy has progressed to later stages. We tracked autophagy in healthy/untreated cells and in nutrient-starved cells using this methodology. Transfection was achieved but was inconsistent. We believe that the size of the plasmid limits our ability to dependably transfect. Future experiments will refine the methodology to produce a more efficient delivery of the LC3 plasmid in order to explore the role of keratin in the autophagy pathway.




Additional Abstract Information

Presenters: Bryan Gutman, Renee Saneholtz

Institution: Western Carolina University

Type: Poster

Subject: Biology

Status: Approved


Time and Location

Session: Poster 2
Date/Time: Mon 3:00pm-4:00pm
Session Number: 2651