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Bone Abstracts (2013) 1 PP232 | DOI: 10.1530/boneabs.1.PP232

ECTS2013 Poster Presentations Cell biology: osteoclasts and bone resorption (24 abstracts)

The use of photo-activatable fluorophores to study the turnover of the receptor activator of NFκB receptor in health and disease

David Mellis , Angela Duthie , Susan Clark & Julie Crockett


University of Aberdeen, Aberdeen, UK.


Familial expansile osteolysis (FEO) is characterised by focal areas of increased bone turnover driven by bone-resorbing osteoclasts. The syndrome is caused by a heterozygous tandem insertion duplication mutation within the signal peptide region of TNFRSF11a (encoding receptor activator of NFκB; RANK). Our recent research has demonstrated that heterotrimeric receptor formation may hold the key to the disease phenotype. We have shown previously that, whilst homozygous overexpression of FEO-RANK leads to accumulation of the protein within an ER-like compartment, heterozygous overexpression results in FEO-RANK expression at the plasma membrane likely as a result of interaction with wildtype-RANK. In this study we investigated whether turnover of the RANK receptor is affected by carriage of the mutation using live-cell confocal microscopy.

We generated several photoactivatable expression constructs containing either wildtype (WT) or mutant RANK (FEO) and tagged with either photoactivatable GFP or mCherry. The constructs either alone or in combination were over-expressed in HeLa cells and the fluorescence activated by exposure to ultraviolet (UV) light. Only proteins expressed at the time of UV exposure were activated and produced a fluorescent signal. The rate of receptor turnover was measured indirectly by monitoring the loss of fluorescent signal in live cells, where any newly synthesised proteins produced post-UV activation did not fluoresce. Using a live-cell imaging LSM710 confocal microscope, we analysed the rate at which the WT and FEO- RANK proteins were degraded over a period of 40 minutes. We consistently found that FEO-RANK (GFP or mCherry tagged) was degraded more slowly than WT-RANK (GFP or mCherry tagged) when expressed alone or in combination. This is an exciting observation since it may provide an explanation for the increased osteoclast activity in this syndrome and increase our understanding of the control of RANK signalling in osteoclasts.

Volume 1

European Calcified Tissue Society Congress 2013

Lisbon, Portugal
18 May 2013 - 22 May 2013

European Calcified Tissue Society 

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