Diffusion of CaM and CaMK-II

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Diffusion of CaM and CaMK-II

• Andrew Harrell
• Dr. Waxham Lab
• University of Texas Medical School

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Ficks Diffusion Model












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Fluorescence

• Excitation of a molecule to a higher energy state
  by photon energy.
• Subsequent lowering of energy state, accompanied
  by an emission of radiation.
• Ultraviolet -gt Visible light.

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Fluorescent Correlation Spectroscopy

• Uses multi-photon laser excitation to induce
  fluorescence.
• Fluorescent intensity is recorded as a function
  of time.
• A correlation curve is created, which relates
  fluorescence at a particular time to fluorescence
  at other times.

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FCS Apparatus

• Laser light (? 780 nm) chosen to maximize dye
  activity.

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Data Collection

• Measure the fluorescent intensity as a function
  of time.
• Computer calculates the correlation function vs.
  (a time delay).

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Correlation Curves

• Wavelength 780 nm chosen to maximize activity of
  the Alexa-488 dye.
• D(CaM) 75.00
• D(CaMKIICaM) 15.78
• ( )

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Determining Diffusion Constants

• Interpolate along the curve to find G(0).
• G(0) is inversely proportional to the
  concentration.
• Determine the x-coordinate of the point on the
  best-fit curve whose corresponds to half
  of G(0).
• The time is called .
• Based on a Gaussian approximation to the
  excitation volume, and the two-photon excitation
  method, we know that

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Procedural Concerns

• Bleaching
• Possibility that molecules will be chemically
  altered by the light, in a way which prevents
  future fluorescence.
• Two-photon excitation helps to avoid bleaching.
• Determining the size of the activity volume
• 3-D Gaussian approximation vs. solution to
  Maxwells equations

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Related Topics

• Fluorescence Recovery After Photobleaching (FRAP)
  method.
• Opposite of FCS uses an intense pulse to
  photobleach all of the molecules in a certain
  volume and then observes fluorescent molecules as
  they diffuse back into the region.
• Measuring simultaneous fluorescence of multiple
  molecules

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Acknowledgements

• Dr. Waxham lab director
• Hugo Sanabria supervisor
• Matt Swulius provided images
• Ben Goins thesis material

Questions???