A fluorescently-labeled reporter system used to monitor lysosome function.
Detection of changes in function could be used as a diagnostic for
neurodegenerative disorders, kidney disease and various infectious diseases.
- Provides a quick, sensitive, easy to use means to specifically monitor the
dynamic CMA activity in cells.
- Can be used in in vitro and in live cell applications.
CMA is a lysosomal mechanism responsible for the degradation of many
cytosolic proteins. CMA activity is highly selective and closely regulated
making it a suitable means to monitor functional autophagy in cells. Several
methods exist to measure CMA activity. However, these means are time consuming,
technically challenging, and do not work in live cells. A faster, easier
approach to quantify CMA activity could offer benefit to a number of conditions
involving changes in CMA function. Emory investigators have created a
fluorescently labeled molecule for monitoring CMA activity in live cells. This
molecule is a fusion construct made up of the KFERQ peptide motif (a protein ID
sequence recognized by chaperones during CMA) linked to a membrane-permeable
peptide and a fluorescent label. As such, this reporter molecule allows one to
determine the cell's CMA activity by observing and measuring the
intensity/dynamics of the fluorescent signal strength. Beyond this application,
the construct itself can be modified and can be extended to any situation where
the direct and specific measurement of CMA activity is needed, having relevance
to both in vivo and in vitro applications. This technology
thus creates a high throughput system that can be used for drug discovery, drug
screening, and disease diagnostic purposes.
Proof of concept has been established using the CMA reporter system.