Event Title

Quantifying the Localization of the Muscarinic M2 Acetylcholine Receptor within the Endoplasmic Reticulum using Image Cross-correlation Spectroscopy

Mentor 1

Valerica Raicu

Location

Union Wisconsin Room

Start Date

29-4-2016 1:30 PM

End Date

29-4-2016 3:30 PM

Description

The muscarinic M2 acetylcholine receptor (M2R) is a G-protein coupled receptor which modulates many physiological functions in the central and peripheral nervous system and is a potential pharmaceutical target in the treatment of Alzheimer's disease, schizophrenia, Parkinson's disease, and chronic obstructive pulmonary disease. M2R has been found to form complexes, or oligomers, consisting of multiple individual receptors (protomers). It is not known at this time whether these oligomers are present at both the plasma membrane and internal membranous structures. We tagged M2Rs with fluorescent proteins in order to determine the distribution of oligomeric variants and the binding interfaces of protomers, and unexpectedly found the M2Rs more concentrated in intracellular structures in addition to the cellular membrane. In order to better understand the phenomena of M2R “bright spots” within the cell, we stained the most likely structure to house them, the endoplasmic reticulum (ER), with a red fluorescent dye selective for sulphonylurea receptors that are prominent in the ER. Using a spectrally-resolved two-photon laser scanning microscope developed in our lab, fluorescence intensity maps were obtained for both ER fluorescent signal and M2R fluorescence signal after deconvolution of the spectrally resolved data. In order to quantify the co-localization of the M2Rs and ER, intracellular regions-of-interest (ROIs) within separate images corresponding to fluorescently-tagged M2R and ER, respectively, were compared using a process called image cross-correlation spectroscopy (ICCS). ICCS is a powerful bioimaging technique based on the measurement and analysis of fluctuations in the intensity of fluorescence micrographs, which relates the dynamics of these fluctuations to the number of fluorescent molecules in the observation volume. A spatial correlation function is computed for each spectral image when compared against itself (i.e., autocorrelation) as well as for each pair of M2R and ER images when compared against each other (i.e., cross-correlation), representing the extent of signal overlap as a function of spatial lag. After the removal of white noise, the amplitudes of these correlation functions give direct information about the percentage of co-localized molecules from each species. ICCS analysis performed on rough ROI selections yielded comparative information as to the extent of M2R and ER co-localization, and various image processing methods were applied in order to improve analysis and reach quantifiable localization and concentration.

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Apr 29th, 1:30 PM Apr 29th, 3:30 PM

Quantifying the Localization of the Muscarinic M2 Acetylcholine Receptor within the Endoplasmic Reticulum using Image Cross-correlation Spectroscopy

Union Wisconsin Room

The muscarinic M2 acetylcholine receptor (M2R) is a G-protein coupled receptor which modulates many physiological functions in the central and peripheral nervous system and is a potential pharmaceutical target in the treatment of Alzheimer's disease, schizophrenia, Parkinson's disease, and chronic obstructive pulmonary disease. M2R has been found to form complexes, or oligomers, consisting of multiple individual receptors (protomers). It is not known at this time whether these oligomers are present at both the plasma membrane and internal membranous structures. We tagged M2Rs with fluorescent proteins in order to determine the distribution of oligomeric variants and the binding interfaces of protomers, and unexpectedly found the M2Rs more concentrated in intracellular structures in addition to the cellular membrane. In order to better understand the phenomena of M2R “bright spots” within the cell, we stained the most likely structure to house them, the endoplasmic reticulum (ER), with a red fluorescent dye selective for sulphonylurea receptors that are prominent in the ER. Using a spectrally-resolved two-photon laser scanning microscope developed in our lab, fluorescence intensity maps were obtained for both ER fluorescent signal and M2R fluorescence signal after deconvolution of the spectrally resolved data. In order to quantify the co-localization of the M2Rs and ER, intracellular regions-of-interest (ROIs) within separate images corresponding to fluorescently-tagged M2R and ER, respectively, were compared using a process called image cross-correlation spectroscopy (ICCS). ICCS is a powerful bioimaging technique based on the measurement and analysis of fluctuations in the intensity of fluorescence micrographs, which relates the dynamics of these fluctuations to the number of fluorescent molecules in the observation volume. A spatial correlation function is computed for each spectral image when compared against itself (i.e., autocorrelation) as well as for each pair of M2R and ER images when compared against each other (i.e., cross-correlation), representing the extent of signal overlap as a function of spatial lag. After the removal of white noise, the amplitudes of these correlation functions give direct information about the percentage of co-localized molecules from each species. ICCS analysis performed on rough ROI selections yielded comparative information as to the extent of M2R and ER co-localization, and various image processing methods were applied in order to improve analysis and reach quantifiable localization and concentration.