20.109(S07):sample journal article summary

We know what properties we'd like sensors to have. In no particular order:

• it should be bright
• it should respond linearly and quickly to large range of inputs, [Ca2+] for today
• it should be sensitive to even subtle, single cellular stimuli
• it should be inexpensive
• it should not be disruptive to other cellular activities
• it should be easy to use

You've read that genetically encoded calcium sensors have great benefits but how well do they compare to synthetic (aka chemical) sensors like Fura-2, Fluo4-FF or X-Rhod-5F?

Consider the comparison made in the article by Pologruto, Yasuda, and Svoboda J Neurosci (2004)24:9572. These authors

1. try to correlate fluorescence with cellular activity by comparing fluorescence and chemical indicator (finding: fluorescence is nonlinear indicator at low activity levels)
2. try to correlate fluorescence with [Ca2+] (finding: complex relationship)
3. compare readout in cells with in vitro values since other CaM exist in cells and may influence sensitivity (finding: diffusion not influenced by CaM-binding proteins).

terms

• F
  • fluorescence from indicator (for GECI and for chemical indicators)
  • factors influencing F
    • Ca2+ fluctuation…so waited until reached equilibrium, defined F0 as baseline, average F for 200 msec after equilibration and before stimulation
    • photobleaching of indicator…measured as ~40% after 50 minutes
    • noise in PMT…measured “dark” noise for 50 msec with shutter closed then subtracted mean
• phi
  • degree to which fluorescence is saturated
• Rf
  • dynamic range of the indicator
  • = Fmax/Fmin
  • previously experimentally determined
• Kd
  • dissociation constant of Ca2+ from indicator
  • previously experimentally determined as concentration of Ca2+ for 1/2 phi
• n
  • Hill coeff, measure of coopertivity
  • also need to define “alpha” as scaling term and “beta” as non-specific term to solve for phi in terms of Kd, [Ca2+] and n

results

1. single stimuli (pg 9574)
   • “In response to a single action potential, the synthetic indicator produced robust, rapid onset fluorescence changes….In contrast, [two GECI] produced only very small fluorescence responses; these were detected above the noise only when averaging over many (8-16) trials.”
   • Fig 2A
2. variable patterns of stimuli (pg 9575)
   • “both [chemical indicators] respond to Ca2+ elevations sufficiently quickly to follow the stimulus patterns reliably. In contrast, GECI power spectra did not reveal a clear peak above the noise at the stimulus frequency , even under the most favorable conditions. Thus, unlike synthetic indicators, GECIs respond too slowly to follow individual action potentials within a burst.”
   • Fig 3
3. as quantitative measure of Ca2+ (pg 9575)
   • “GECIs have idiosyncratic and complex fluorescence saturation curves, making their use for quantitative [Ca2+] imaging problematic.”
   • Fig 5
4. interaction of GECI with CaM-binding proteins in cell (pg 9576)
   • “Because CaM (and hence GECI) properties are changed by interactions with CaM-binding proteins, assessing GECI mobility is important for the interpretation of GECI signals”
   • “In all cases, after bleaching, fluorescence recovered to >95% of the baseline fluorescence.”
   • “We conclude that GECIs are mostly freely diffusible”