Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Summary

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Abstract

Beta-amyloid (Aβ) is the major constituent of senile plaques found in the brains of Alzheimer’s disease patients. Aβ is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by β and γ-secretases. Despite the importance of Aβ to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.

While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the γ-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.

We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

Introduction

The hallmark of Alzheimer’s disease (AD) is the presence of senile plaques and neurofibrillary tangles (NFTs) in the brain. The major constituent of senile plaques is β-amyloid (Aβ). Aβ is derived from its precursor; amyloid precursor protein (APP) ¹. The amyloidogenic cleavage of APP begins with removal of the ectodomain from APP by β-secretase ². The remaining 99-residue carboxyl terminal fragment (CTF) can be cleaved by γ-secretase to produce Aβ ^3-7. While many experiments have documented the cleavage of cell surface APP after internalization from the cell surface into the endosomal/ lysosomal sys....

Protocol

1. Cell Plating and Transfection

1. In a cell culture hood, plate ~300,000 to ~500,000 SN56 cells (a kind gift of Dr. Jane Rylett) on a 35 mm glass-bottom confocal dish and cover with pre-transfection media (Dulbecco’s Modified Eagle Medium, DMEM, supplemented with 10% FBS).
2. Incubate cells overnight at 37 °C in a 5% CO₂ incubator to proliferate.
   Note: Cells should be approximately 50%-70% confluent before transfection.
3. In a cell culture hood, transfect cells with plasmids expressing a fluorescent protein tagged TGN marker (Galactosyl transferase coupled to Cyan Fluorescent Protein, GalT-CFP), a fluorescent prot....

Results

Typical results show βAPP leaving the TGN and appears to traffic rapidly to LAMP1 (Figure 1a and b). During the photo-activation period, vesicles can be seen departing the Golgi destined for lysosomes (Figure 1b). Without inhibitor treatment, paGFP fluorescence will be visible in lysosomes while there is photo-activation in the TGN. After stopping photoactivation, the βAPP-paGFP is rapidly cleared from the lysosome (compare Figure 1a to 1b

Discussion

This technique describes the accurate photo-activation of membrane proteins tagged with paGFP in the TGN to visualize subsequent trafficking and cleavage. While paGFP was created over a decade ago ²⁵, this is the first example of accurate photo-activation to follow the trafficking of nascent proteins to downstream compartments. Previous studies using APP-paGFP constructs photo-activated a peri-nuclear region of the cell, which was considered to be the Golgi ¹¹. However, as evident in our micrographs.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm glass bottom culture dishes	Matek	P-35G-1.5-20-C
Dulbecco's Phosphate Buffered Saline	Life Technologies	14190-144
Hanks Balanced Salt Solution	Life Technologies	14025-092
Lipofectamine 2000	Life Technologies	11668019
Dulbecco's Modified Eagle Medium	Life Technologies	11995-092
Penicilin/Streptomycin	Life Technologies	15140-122
dibutyrl cyclic AMP	Sigma	D0627
Heat in activated Fetal Bovine Serum	Life Technologies	10082147
Heated Microscopy stage insert P	PeCon GmbH
Tempcontrol 37–2 digital 2-channel	PeCon GmbH
Zeiss LSM-510 META laser- scanning microscope	Carl Zeiss		with laser diode, argon laser, and HeNe1 laser
Zeiss 63X 1.4 numerical aperture oil immersion lens	Carl Zeiss
L685, 458	EMD Millipore	565771	dissolved in DMSO
cholorquine	Sigma	C6628	dissolved in water
Nocodazole	Sigma	M1404	dissolved in DMSO
Dimethyl sulphoxide	Sigma	472301
Imaris	Bitplane		with colocalization package