Quantification of Amyloid Precursor Protein and Tau for the Study of Axonal Traffic Pathways

Abstract

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 Ϯ 145 and 218 Ϯ 106 molecules, corresponding to mean fluxes of ϳ2000 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.

Induced Dimerization of the Amyloid Precursor Protein Leads to Decreased Amyloid-β Protein Production

“…Both mass spectrometry and Tricine-urea gel electrophoresis showed the normal predicted profile of A␤ peptides without any alterations before or after dimerization from both APP F1 and APP F2 constructs. These findings are consistent with the apparent normal processing of APP GFP fusions used by a number of laboratories (38,(41)(42)(43)(44)(45). However, with this approach, our results unequivocally showed that forced dimerization of APP lowered rather than increased A␤ generation.…”

Protein-binding dynamics imaged in a living cell

“…Protein concentrations were determined from a calibration curve (Fig. 2D), where we measured the fluorescence intensities of purified CyTEM and YBLIP proteins diluted in HeLa cytoplasmic cell extract, using the confocal microscope setup employed for living cell measurements (14).…”

Quantification of the Brassinosteroid Insensitive1 Receptor in Planta

“…Quantitative confocal microscopy offers the advantage of revealing spatial information in a nondestructive manner (Sugiyama et al, 2005;Goldsbury et al, 2007;Verveer and Bastiaens, 2008;Wu et al, 2008). This is especially important when dealing with proteins, such as membrane receptors, that are continuously recycled.…”

“…Quantitative methods based on confocal laser scanning microscopy techniques have provided insight in the amount and subcellular localization of proteins (Verveer and Bastiaens, 2008). Confocal imaging is applicable for plant membrane, nuclear, and cytosolic proteins (Mathur, 2007;Berg et al, 2008) and is used in yeast (Wu and Pollard, 2005) and animal cells (Sugiyama et al, 2005;Goldsbury et al, 2007) to quantify fluorescently tagged proteins. In plants, fluorescence-based quantification techniques have been used to determine protein level in whole-tissue samples (Halfhill et al, 2005) and in extracts using ELISA (Richards et al, 2003) or intact cells using flow cytometry (Lu et al, 2007).…”