Quantification of amyloid precursor protein and tau for the study of axonal traffic pathways.

We describe a procedure for quantifying the numbers of expressed fluorescent fusion proteins on vesicles transported in axons. The method can be used to estimate numbers of vesicle-anchored molecules moving in both anterograde and retrograde directions and is also applicable to cytosolic proteins. This is demonstrated using neurons (explanted retinal ganglion cells) transfected with yellow fluorescent protein-tagged amyloid precursor protein (APP–YFP) or cyan fluorescent protein–tau (CFP–tau). To calibrate the APP–YFP concentration on vesicles, standard solutions of recombinant YFP were imaged by confocal microscopy. For comparison, rotavirus-like particles containing a known number of 120 green fluorescent protein (GFP) molecules were imaged against standard solutions of GFP. On the basis of the calibration, the anterogradely and retrogradely moving APP vesicles contained 235 145 and 218 106 molecules, corresponding to mean fluxes of 2000 anterograde and 700 retrograde APP–YFP molecules per minute per axon. Using recombinant CFP standard solutions for calibration, exogenous CFP–tau concentrations depended on the levels of expression but were typically 3– 6 M. The value of this procedure is that it is of general use in cell biology, in which knowing the numbers of membrane-anchored molecules is desirable. For example, the amount of APP transported into axonal versus dendritic compartments is relevant to the physiological function of APP and pathological events in Alzheimer’s disease.