We recently developed a new method for cytoplasmic protein localisation utilising a genetic tag, Apex, that is compatible with electron microscopy (EM) fused to a GFP-binding protein.
In 2016 we have applied these methods to study the effect of specific mutations in endocytic proteins associated with Parkinson’s disease and we have further developed these methods in a number of directions.
Firstly, we have optimised conditions to increase sensitivity of the method. This has allowed us to detect Apex-tagged proteins at the level of expression of endogenous proteins in genome- edited cells.
Secondly, we have further developed systems to allow EM detection of proteins in a model organism, the zebrafish Danio rerio.
Thirdly, we have developed complementary systems based on an Apex-mCherry nanobody.
Fourthly, we have developed a destabilised Apex-nanobody construct that causes degradation of the unbound nanobody and so increases the signal to noise ratio.
These applications are restricted to cytoplasmic proteins. We have therefore extended this approach to lumenal/ extracellular proteins by generating monovalent peroxidase-labelled fluorescent proteins. Uptake of the extracellular marker protein allowed correlation with novel fluorescent methods to determine mobility of the marker in cells and allowed detailed analysis of their properties in specific compartments.
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