Presentation Title

Design of a novel construct for live cell imaging of ezrin-actin association using Förster Resonance Energy Transfer (FRET)

Project Collaborators

Emily E Whitaker (Graduate Student Mentor), Markus Thali (Primary Faculty Mentor)

Abstract

HIV-1 can spread efficiently cell-to-cell via a junction known as a virological synapse (VS). A VS forms when Env (the viral fusogen) on the infected (producer) cell binds to CD4 on the uninfected (target) cell, allowing transfer of viral particles. Sometimes, a VS will result in cell-cell fusion to form one, multinucleated infected cell, (syncytium). Various proteins which localize at the synapse have been found to inhibit fusion, one of which is the protein ezrin.

Phosphorylated ezrin serves to link cytosolic proteins to the actin cytoskeleton and has been shown to accumulate at the producer side of the synapse (presynapse). Due to its known role as a cytoskeletal linker, and the implication of ezrin in fusion inhibition, we theorize that it associates with actin at the HIV-1 presynapse to regulate cytoskeletal tension and prevent fusion of the producer cell membrane with that of the target cell.

To further elucidate the spatial and temporal role of p-ezrin at the VS, we will create a cell line that stably expresses ezrin with an internal mTurquoise2 fluorophore. mTurquoise2 will act as a Förster Resonance Energy Transfer (FRET) donor for mRuby, which is fused to the actin-associated peptide, LifeAct. FRET requires close proximity of the donor and acceptor fluorophores, which we will use to discern when actin and ezrin are associating, and when they are separate. This will allow live analysis of ezrin-actin association during HIV-1 cell-to-cell transmission and cell-cell fusion, which will help uncover the mechanism by which p-ezrin regulates these events.

Primary Faculty Mentor Name

Markus Thali

Graduate Student Mentors

Emily E Whitaker

Status

Undergraduate

Student College

College of Agriculture and Life Sciences

Program/Major

Molecular Genetics

Primary Research Category

Biological Sciences

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Design of a novel construct for live cell imaging of ezrin-actin association using Förster Resonance Energy Transfer (FRET)

HIV-1 can spread efficiently cell-to-cell via a junction known as a virological synapse (VS). A VS forms when Env (the viral fusogen) on the infected (producer) cell binds to CD4 on the uninfected (target) cell, allowing transfer of viral particles. Sometimes, a VS will result in cell-cell fusion to form one, multinucleated infected cell, (syncytium). Various proteins which localize at the synapse have been found to inhibit fusion, one of which is the protein ezrin.

Phosphorylated ezrin serves to link cytosolic proteins to the actin cytoskeleton and has been shown to accumulate at the producer side of the synapse (presynapse). Due to its known role as a cytoskeletal linker, and the implication of ezrin in fusion inhibition, we theorize that it associates with actin at the HIV-1 presynapse to regulate cytoskeletal tension and prevent fusion of the producer cell membrane with that of the target cell.

To further elucidate the spatial and temporal role of p-ezrin at the VS, we will create a cell line that stably expresses ezrin with an internal mTurquoise2 fluorophore. mTurquoise2 will act as a Förster Resonance Energy Transfer (FRET) donor for mRuby, which is fused to the actin-associated peptide, LifeAct. FRET requires close proximity of the donor and acceptor fluorophores, which we will use to discern when actin and ezrin are associating, and when they are separate. This will allow live analysis of ezrin-actin association during HIV-1 cell-to-cell transmission and cell-cell fusion, which will help uncover the mechanism by which p-ezrin regulates these events.