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Proceedings Paper

5D imaging approaches reveal the formation of distinct intracellular cAMP spatial gradients
Author(s): 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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Paper 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.

Paper Details

Date Published: 17 February 2017
PDF: 6 pages
Proc. SPIE 10070, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIV, 100700R (17 February 2017); doi: 10.1117/12.2253164
Show Author Affiliations
Thomas C. Rich, Univ. of South Alabama (United States)
Naga Annamdevula, Univ. of South Alabama (United States)
Kenny Trinh, Univ. of South Alabama (United States)
Andrea L. Britain, Univ. of South Alabama (United States)
Samuel A. Mayes, Univ. of South Alabama (United States)
John R. Griswold, Univ. of South Alabama (United States)
Joshua Deal, Univ. of South Alabama (United States)
Chase Hoffman, Univ. of South Alabama (United States)
Savannah West, Univ. of South Alabama (United States)
Silas J. Leavesley, Univ. of South Alabama (United States)


Published in SPIE Proceedings Vol. 10070:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIV
Thomas G. Brown; Carol J. Cogswell; Tony Wilson, Editor(s)

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