5D imaging approaches reveal the formation of distinct intracellular cAMP spatial gradients

Thomas C. Rich; Naga Annamdevula; Kenny Trinh; Andrea L. Britain; Samuel A. Mayes; John R. Griswold; Joshua Deal; Chase Hoffman; Savannah West; Silas J. Leavesley

doi:10.1117/12.2253164

17 February 2017 5D imaging approaches reveal the formation of distinct intracellular cAMP spatial gradients

Thomas C. Rich, Naga Annamdevula, Kenny Trinh, Andrea L. Britain, Samuel A. Mayes, John R. Griswold, Joshua Deal, Chase Hoffman, Savannah West, Silas J. Leavesley

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Proceedings Volume 10070, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIV; 100700R (2017) https://doi.org/10.1117/12.2253164
Event: SPIE BiOS, 2017, San Francisco, California, United States

Abstract

Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions. Several lines of evidence suggest that the distribution of cAMP within cells is not uniform. However, to date, no studies have measured the kinetics of 3D cAMP distributions within cells. This is largely due to the low signal-tonoise ratio of FRET-based probes. We previously reported that hyperspectral imaging improves the signal-to-noise ratio of FRET measurements. Here we utilized hyperspectral imaging approaches to measure FRET signals in five dimensions (5D) – three spatial (x, y, z), wavelength (λ), and time (t) – allowing us to visualize cAMP gradients in pulmonary endothelial cells. cAMP levels were measured using a FRET-based sensor (H188) comprised of a cAMP binding domain sandwiched between FRET donor and acceptor - Turquoise and Venus fluorescent proteins. We observed cAMP gradients in response to 0.1 or 1 μM isoproterenol, 0.1 or 1 μM PGE₁, or 50 μM forskolin. Forskolin- and isoproterenol-induced cAMP gradients formed from the apical (high cAMP) to basolateral (low cAMP) face of cells. In contrast, PGE1-induced cAMP gradients originated from both the basolateral and apical faces of cells. Data suggest that 2D (x,y) studies of cAMP compartmentalization may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D (x,y,z) studies are required to assess mechanisms of signaling specificity. Results demonstrate that 5D imaging technologies are powerful tools for measuring biochemical processes in discrete subcellular domains.

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Thomas C. Rich, Naga Annamdevula, Kenny Trinh, Andrea L. Britain, Samuel A. Mayes, John R. Griswold, Joshua Deal, Chase Hoffman, Savannah West, and Silas J. Leavesley "5D imaging approaches reveal the formation of distinct intracellular cAMP spatial gradients", Proc. SPIE 10070, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIV, 100700R (17 February 2017); https://doi.org/10.1117/12.2253164

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KEYWORDS

Fluorescence resonance energy transfer

Hyperspectral imaging

3D metrology

3D image processing

Signal to noise ratio

Venus

Visualization

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