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
