Amphipathic Fluorophores for Probing Cell Membrane

Properties

Demetrius CummingsUniversity of California, Riverside

Outline…

• Background

• Experimental process

• Results and future goals

Background…

• Prestin

• Enhances electromotility

• Sound reception

• Other properties?

http://med.stanford.edu/ohns/research/

Fluorophores…

• Fluorescent component; multiple types

• Often used in diagnostics: tissues, macromolecules, diseases

http://exobio.ucsd.edu/MOD/detection.htm

Plasma Membrane…the

“Fluid Mosaic”

(Dowhan & Bogdanov, 2005)

•Amphipathic structure

The Goals…

• Synthesize an amphipathic dye for the purpose of binding to the plasma membrane.

• Monitor the photophysical properties of the dye and HEK cells in various conditions.

The Process…(part 1)

Dye preparation Utilize HEK cells

Analysis: photophysicalproperties

Combine dye and cells

Analysis: photophysicalproperties

Analysis: photophysicalproperties

Dye preparation Utilize HEK cells

Dye Synthesis…

Nucleophilic Aromatic Substitution…

•Halide-dependent

•Yields…--chloro- = 37%--bromo- = 97%

The Process…(part 2)

Dye preparation Utilize HEK cells

Analysis: photophysicalproperties

Combine dye and cells

Analysis: photophysicalproperties

Analysis: photophysicalproperties

Results…

Water (more polar solvent) V.S. Hexane (less polar solvent)

The Process…(part 3)

Dye preparation Utilize HEK cells

Analysis: photophysicalproperties

Combine dye and cells

Analysis: photophysicalproperties

Analysis: photophysicalproperties

• Low absorbance

• High incidence of light scattering

The Process…(part 4)

Dye preparation Utilize HEK cells

Analysis: photophysicalproperties

Combine dye and cells

Analysis: photophysicalproperties

Analysis: photophysicalproperties

•Blue shift evident in HEK cells with dye

•Location: less polar environment

Results…

Summary…• The efficiency and mechanism of the

dye are halide-dependent.

• The absorbance varies directly with dye-solvent and cell-solvent concentration; the specific wavelengths of absorbance remains constant.

• Blue shift of the dye-HEK cell mix indicates binding to the less polar environment.

Future Research…

• Continue fluorescence analysis of HEK cells: excitation, emission, anisotropy

• Effect of dye on cells?

• Apply same procedure to cells transfected with prestin

Acknowledgements…

• Dr. Bahman Anvari

• Dr. Valentine Vullev

• Dr. Feng Qian

• Dr. Valentine Vullev’s lab

• Program coordinators