Dataset Information

Real-time measurements of cAMP production in live Dictyostelium cells.

ABSTRACT: Cyclic AMP has a crucial role during the entire developmental program of the social amoebae Dictyostelium, acting both as an intracellular second messenger and, when secreted, as a directional cue that is relayed to neighboring cells during chemotaxis. Although significant knowledge about cAMP production in chemotaxing cells has been derived from studies performed on cell populations, cAMP dynamics at the single cell level have not been investigated. To examine this, we used a FRET-based cAMP sensor that possesses high cAMP sensitivity and great temporal resolution. We show the transient profile of cAMP accumulation in live Dictyostelium cells and establish that chemoattractants control intracellular cAMP dynamics by regulating synthesis via the adenylyl cyclase ACA. aca(-) cells show no significant change in FRET response following chemoattractant addition. Furthermore, cells lacking ACB, the other adenylyl cyclase expressed in chemotaxing cells, behave similarly to wild-type cells. We also establish that the RegA is the major phosphodiesterase that degrades intracellular cAMP in chemotaxis-competent cells. Interestingly, we failed to measure intracellular cAMP compartmentalization in actively chemotaxing cells. We conclude that cytosolic cAMP, which is destined to activate PKA, is regulated by ACA and RegA and does not compartmentalize during chemotaxis.

SUBMITTER: Bagorda A

PROVIDER: S-EPMC2773191 | biostudies-other | 2009 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Real-time measurements of cAMP production in live Dictyostelium cells.

Bagorda Anna A Das Satarupa S Rericha Erin C EC Chen David D Davidson Jean J Parent Carole A CA

Journal of cell science 20091006 Pt 21

Cyclic AMP has a crucial role during the entire developmental program of the social amoebae Dictyostelium, acting both as an intracellular second messenger and, when secreted, as a directional cue that is relayed to neighboring cells during chemotaxis. Although significant knowledge about cAMP production in chemotaxing cells has been derived from studies performed on cell populations, cAMP dynamics at the single cell level have not been investigated. To examine this, we used a FRET-based cAMP se ...[more]

PMID: 19808889

Similar Datasets

Project description:Cell-surface glycans are attractive targets for molecule imaging due to their reflection of cellular processes associated with development and disease progression. In this paper, we describe the design, synthesis, and biological application of a new phosphine probe for real-time imaging of cell-surface glycans using bioluminescence. To accomplish this goal, we took advantage of the bioorthogonal chemical reporter technique. This strategy uses a two-step labeling procedure in which an unnatural sugar analogue containing a functional handle is (1) incorporated into sugar-bearing proteins via the cell's own biosynthetic machinery and then (2) detected with an exogenously added probe. We designed phosphine-luciferin reagent 1 to activate bioluminescence in response to Staudinger ligation with azide-labeled glycans. We chose to use a phosphine probe because, despite their slow reaction kinetics, they remain the best-performing reagents for tagging azidosugars in mice. Given the sensitivity and negligible background provided by bioluminescence imaging (BLI), we reasoned that 1 might be able to overcome some of the limitations encountered with fluorescent phosphine probes. In this work, we synthesized the first phosphine-luciferin probe for use in real-time BLI and demonstrated that azide-labeled cell-surface glycans can be imaged with 1 using concentrations as low as single digit nanomolar and times as little as 5 min, a feat that cannot be matched by any previous fluorescent phosphine probes. Even though we have only demonstrated its use in visualizing glycans, it can be envisioned that this probe could also be used for bioluminescence imaging of any azide-containing biomolecule, such as proteins and lipids, since azides have been previously incorporated into these molecules. The phosphine-luciferin probe is therefore poised for many applications in real-time imaging in cells and whole animals. These studies are currently in progress in our laboratory.

Dataset Information

Real-time measurements of cAMP production in live Dictyostelium cells.

Publications

Real-time measurements of cAMP production in live Dictyostelium cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets