Optimisation of a protocol for the study of subcellular cAMP compartmentalisation in epithelial cells of Drosophila Melanogaster
Publication Date
July 18, 2023
Creator
Abstract
Rationale
The coordination and regulation of actin-based structures throughout the cell is tightly spatiotemporally regulated and linked to spatiotemporal activation of key players in actin polymerisation and de-polymerisation (Rac, RhoA and Cdc42). cAMP (cyclic adenosine monophosphate) and its effectors have been linked to the regulation of RhoGTPases Rac, Cdc42 and RhoA providing a convincing mechanism for their regulation, although direct evidence is not forthcoming due to limitations in currents methods of studying cAMP signalling dynamics within the cell. Most previous work has measured cAMP dynamics and concentrations within in vitro or within subcellular fractions, potentially conferring a misleading representation of cAMP dynamics. Here we present an optimised protocol for studying cAMP dynamics in vivo within live Drosophila melanogaster pupae, overcoming previous limitations in characterising cAMP dynamics in the study of actin membrane-based protrusions.
Methods
Using the Gal4-UAS system and the MARCM system, we expressed a genetically encoded cAMP FRET-based (Förster resonance energy transfer) biosensor within well-spaced epithelial cells and in epithelial cell clones in Drosophila melanogaster, using the tissue specific promoters Neuralized, in well-spaced epithelial cells, and Pannier, in epithelial cell clones, to restrict expression to the notum. Our aim was to employ this protocol in the measurement of cAMP gradients within actin membrane-based protrusions at high spatial resolution.
Results
We successfully created and optimised a confocal microscopy protocol for undertaking sensitised emission FRET measurements to assess localised cAMP gradients within intermediate-lateral actin protrusions in live Drosophila melanogaster pupae. This protocol overcomes current limitations in measuring cAMP signalling dynamics by providing high spatial resolution, allowing researchers to link precise cAMP domains to alterations in cellular morphology and interrogate the signalling events underpinning actin membrane-based protrusion dynamics. We observed statistically significant elevated relative basal cAMP concentrations within the tip of intermediate-lateral protrusions compared to the protrusion middle or base and cell cytosol within well-spaced epithelial neu-camps-epac1+cells, expressing a genetically encoded FRET-based cAMP biosensor. However, no statistically significant difference in relative cAMP concentration was detected within epithelial pnr-camps-epac1+ clones at any point within the cell cytosol or protrusions.
Conclusion
Our preliminary data highlights the utility of this approach to measure cAMP gradients at high spatial resolutions within three-dimensional, polarised cells in a live organism. We conclude that further exploratory work is required to the elucidate the full extent of the role for cAMP in actin protrusion regulation, while this represents a promising avenue for exploration.
Item Type
ethesis
Thesis Type
MRes
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
73862
UoN Repository URI
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MRes Final Version (Corrected).pdf
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Full-text
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Examined. Corrections
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