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Proceedings Paper

Fluorescence fluctuation analysis of BACE1-GFP fusion protein in cultured HEK293 cells
Author(s): Spencer Gardeen; Joseph L. Johnson; Ahmed A. Heikal
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Paper Abstract

Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer’s disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

Paper Details

Date Published: 3 October 2016
PDF: 15 pages
Proc. SPIE 9956, Ultrafast Nonlinear Imaging and Spectroscopy IV, 99560K (3 October 2016); doi: 10.1117/12.2237183
Show Author Affiliations
Spencer Gardeen, Univ. of Minnesota Duluth (United States)
Joseph L. Johnson, Univ. of Minnesota Duluth (United States)
Ahmed A. Heikal, Univ. of Minnesota Duluth (United States)

Published in SPIE Proceedings Vol. 9956:
Ultrafast Nonlinear Imaging and Spectroscopy IV
Zhiwen Liu, Editor(s)

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