All-optical mapping of cAMP transport reveals rules of sub-cellular localization

Abstract

Cyclic adenosine monophosphate (cAMP) is a second messenger that mediates diverse intracellular signals. Studies of cAMP transport in cells have produced wildly different results, from reports of nearly free diffusion to reports that cAMP remains localized in nanometer-scale domains. We developed an all-optical toolkit, termed cAMP-SITES, to locally perturb and map cAMP transport. In MDCK cells and in cultured neurons, cAMP had a diffusion coefficient of ~120 μm2/s, similar to the diffusion coefficients of other small molecules in cytoplasm. In neuronal dendrites, a balance between diffusion and degradation led to cAMP domains with a length scale of ~30 μm. Geometrical confinement by membranes led to subcellular variations in cAMP concentration, but we found no evidence of nanoscale domains or of distinct membrane-local and cytoplasmic pools. We introduce theoretical relations between cell geometry and small-molecule reaction-diffusion dynamics and transport to explain our observations.

Phosphatases control the duration and range of cAMP/PKA microdomains

“…Interestingly, in our experiments we did not observe any cAMP gradients independently of the cAMP hydrolyzing ability of each cell line. Our data are in line with recent reports suggesting that PDE activity alone is not sufficient for the generation of intracellular cAMP gradients( 28 , 29 ).…”

All-optical mapping of Ca2+transport and homeostasis in dendrites

“…Using an analogous experiment with a blue-light activated adenylyl cyclase and a red-shifted cAMP reporter, we previously measured a dendritic length constant of cAMP transport φ cAMP ~30 μm, and a dendritic diffusion coefficient of D cAMP ~ 120 μm 2 /s. 18 Allbritton and coworkers measured diffusion of inositol 1,4,5-trisphosphate (IP 3 ) in free cytosol and determined D IP3 ~ 283 μm 2 /s. They estimated a range of action of φ IP3 ~ 24 μm, though both numbers would be expected to be smaller in dendrites due to the presence of occlusions and traps.…”

“…Alloptical genetically encoded tools for targeted perturbation and measurement are a powerful approach to mapping spatiotemporal responses of cellular signals. For example, we have developed all-optical systems for perturbation and measurement of voltage, [15][16][17] of cAMP, 18 and of the embryonic morphogen Nodal. 19 Here we co-expressed a blue-excited calcium-selective channelrhodopsin CapChR2 20 with a farred fluorescent Ca 2+ indicator protein FR-GECO1c 21 in cultured rat hippocampal neurons.…”