RESUMO
Cyclic AMP is a ubiquitous second messenger that orchestrates a variety of cellular functions over different timescales. The mechanisms underlying specificity within this signaling pathway are still not well understood. Several lines of evidence suggest the existence of spatial cAMP gradients within cells, and that compartmentalization underlies specificity within the cAMP signaling pathway. However, to date, no studies have visualized cAMP gradients in three spatial dimensions (3D: x, y, z).This is in part due to the limitations of FRET-based cAMP sensors, specifically the low signal-to-noise ratio intrinsic to all intracellular FRET probes. Here, we overcome this limitation, at least in part, by implementing spectral imaging approaches to estimate FRET efficiency when multiple fluorescent labels are used and when signals are measured from weakly expressed fluorescent proteins in the presence of background autofluorescence and stray light. Analysis of spectral image stacks in two spatial dimensions (2D) from single confocal slices indicates little or no cAMP gradients formed within pulmonary microvascular endothelial cells (PMVECs) under baseline conditions or following 10 min treatment with the adenylyl cyclase activator forskolin. However, analysis of spectral image stacks in 3D demonstrates marked cAMP gradients from the apical to basolateral face of PMVECs. Results demonstrate that spectral imaging approaches can be used to assess cAMP gradients-and in general gradients in fluorescence and FRET-within intact cells. Results also demonstrate that 2D imaging studies of localized fluorescence signals and, in particular, cAMP signals, whether using epifluorescence or confocal microscopy, may lead to erroneous conclusions about the existence and/or magnitude of gradients in either FRET or the underlying cAMP signals. Thus, with the exception of cellular structures that can be considered in one spatial dimension, such as neuronal processes, 3D measurements are required to assess mechanisms underlying compartmentalization and specificity within intracellular signaling pathways.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Masculino , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Razão Sinal-RuídoRESUMO
Incidental appendectomies (IAs) are often performed in laparotomies to prevent future complications caused by the buildup of scar tissue. Although neoplastic findings are rare, all appendectomy specimens should be sent for histopathological analysis. We present the case of a 38-year-old man found to have an appendiceal neuroendocrine tumor (NET) after receiving an IA secondary to a traumatic rectal perforation requiring exploratory laparotomy. Well-differentiated NETs isolated to the appendix have an excellent prognosis. Appendectomies are considered curative for NETs smaller than 2 cm that have not metastasized beyond the appendix. Appendiceal NETs are capable of secreting vasoactive substances and, therefore, causing carcinoid syndrome. However, the progression to carcinoid syndrome generally coincides with metastasis to the liver, indicating a poor prognosis. While histopathological analysis of appendectomy specimens rarely yields atypical findings, this analysis is crucial to ensure that the proper treatment is selected based on tumor progression in an appendectomy specimen staining positive for somatotropin and chromogranin.
RESUMO
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-to-noise 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. This work was supported by NIH P01HL066299, R01HL058506, S10RR027535, AHA 16PRE27130004 and the Abraham Mitchell Cancer Research Fund.