This work was funded by a grant from the Canadian Institute for Health Research MOP 82890 to SHP. The authors wish to thank G.H. Patterson and J. Lippincott Schwartz for the paGFP construct. SN56 cells were a gift of Dr. Jane Rylett.

1. Cell Plating and Transfection
<ol>
	<li>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&#8217;s Modified Eagle Medium, DMEM, supplemented with 10% FBS).</li>
	<li>Incubate cells overnight at 37 &#176;C in a 5% CO2 incubator to proliferate. 
	Note: Cells should be approximately 50%-70% confluent before transfection.</li>
	<li>In a cell culture hood, transfect cells with ...

<ol>
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A., Lopez-Coviella, I., Slack, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+dynamin-dependent+endocytosis+increases+shedding+of+the+amyloid+precursor+protein+ectodomain+and+reduces+generation+of+amyloid+beta+protein.">Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid beta protein.</a> BMC Cell Biol. 6, 30 (2005).</li><li>Traub, L. M., Kornfeld, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trans-Golgi+network:+a+late+secretory+sorting+station.">The trans-Golgi network: a late secretory sorting station.</a> Curr. Opin. Cell Biol. 9 (4), 527-533 (1997).</li><li>Vieira, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval+of+the+Alzheimer's+amyloid+precursor+protein+from+the+endosome+to+the+TGN+is+S655+phosphorylation+state-dependent+and+retromer-mediated.">Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated.</a> Mol Neurodegener. 5 (1), 40 (2010).</li><li>Sullivan, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retromer+disruption+promotes+amyloidogenic+APP+processing.">Retromer disruption promotes amyloidogenic APP processing.</a> Neurobiol. Dis. 43 (2), 338-3345 (2011).</li><li>Fjorback, A. 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The authors have no competing financial interests or conflicts of interest.

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...

The hallmark of Alzheimer&#8217;s disease (AD) is the presence of senile plaques and neurofibrillary tangles (NFTs) in the brain. The major constituent of senile plaques is &#946;-amyloid (A&#946;). A&#946; is derived from its precursor; amyloid precursor protein (APP) 1. The amyloidogenic cleavage of APP begins with removal of the ectodomain from APP by &#946;-secretase 2. The remaining 99-residue carboxyl terminal fragment (CTF) can be cleaved by &#947;-secretase to produce A&#946; 3-7. While many experiments have documented the cleavage of cell surface APP after internalization from the cell surface into the endosomal/ lysosomal system, a number of recent studies have suggested that intracellular trafficking of APP is also important in regulating its processing 8-11.
There have been a number of attempts at modulating the levels of A&#946; with &#947;-secretase inhibitors and A&#946; immunotherapies. However, recent clinical trials with these therapies showed no benefit, and, in some cases, caused harm 12. An unexploited strategy to modulate A&#946; production is to alter the sub-cellular localization of APP and &#947;-secretase interaction. The Golgi, plasma membrane, and endosomes/lysosomes have all been suggested as possible locales for &#947;-cleavage of APP. Research from our laboratory suggests that APP and the &#947;-secretase are resident proteins of the lysosomal membrane 13. Furthermore, we have found that the lysosomal &#947;-secretase has an acidic optimal pH 13. In addition, alkalization of the endosomal/lysosomal system with chloroquine or NH4Cl, has been shown to decrease the production of A&#946; 14. &#947;-secretase inhibition or knockout or Presenilin leads to APP-CTFs accumulation in the lysosome 15-17. Moreover, disrupting APP endocytosis lowers A&#946; production 18-20.
Despite the importance of the Golgi as a sorting station for nascent proteins and proteins recycled from the endosomal/lysosomal system, the intracellular trafficking of APP has not been studied in detail 21. Recent work has shown that APP can be recycled to the trans-Golgi network (TGN) via interaction with the retromer complex. Down regulation of the retromer complex decreases A&#946; production 8,22-24. However, the egress of APP from the Golgi has not been well studied.
While following the endocytosis of cell-surface proteins, such as the transferrin receptor, are easily labeled and followed, following the trafficking of intracellular proteins is more challenging. In fact, few proteins have had their intracellular trafficking imaged. The advent of fluorescent protein tags, such as photo-activatable-GFP (paGFP), has provided new tools to examine intracellular trafficking. Photo-activatable-GFP is a form of GFP that is nearly invisible after synthesis, but develops strong green (GFP) fluorescence after being activated by 413 nm laser light and this signal is stable for days 25,26. Constructs using paGFP have been used to demonstrate the intralysosomal trafficking of the Lysosomal Protein membrane protein 1 (LAMP1) 25, the cell surface delivery of the Vesicular Stomatitis Virus Glycoprotein (VSVG, a marker of the secretory pathway) 27, and the turnover of peroxisomes 28 and autophagosomes 29.
In order to visualize the sorting of APP from the TGN in live cells, we designed plasmid expressing the last 112 amino acids of APP coupled to photo-activatable (paGFP) on the C-terminal (referred to as &#946;APP-paGFP) 30. We have also performed these experiments with full length APP and achieved similar results; the &#914;APP construct is used here because it provides brighter images. We then photo-activate &#946;APP-paGFP only in the TGN, as demarcated by the TGN marker Galactosyltransferase (GalT). Although APP has been tagged with paGFP to visualize rapid axonal transport of APP and clearance from the perinuclear region, this is the first demonstration of APP trafficking from one carefully defined compartment to another 11,31,32. Here, we demonstrate accurate photo-activation within the TGN and the egress of APP into downstream compartments and subsequent cleavage and clearance from lysosomes 30. The accurate photo-activation within the Golgi is widely applicable to other protein systems.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>35-mm glass bottom culture dishes&#160;</td>
			<td>Matek</td>
			<td>P-35G-1.5-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Life Technologies</td>
			<td>11995-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>dibutyrl cyclic AMP</td>
			<td>Sigma&#160;</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat in activated Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Microscopy stage insert P</td>
			<td>PeCon GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tempcontrol 37&#8211;2 digital 2-channel</td>
			<td>PeCon GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM-510 META laser- scanning microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>with laser diode, argon laser, and HeNe1 laser</td>
		</tr>
		<tr>
			<td>Zeiss 63&#215; 1.4 numerical aperture oil immersion lens&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L685, 458&#160;</td>
			<td>EMD Millipore</td>
			<td>565771</td>
			<td>dissolved in DMSO</td>
		</tr>
		<tr>
			<td>cholorquine&#160;</td>
			<td>Sigma</td>
			<td>C6628&#160;</td>
			<td>dissolved in water</td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma</td>
			<td>M1404</td>
			<td>dissolved in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide&#160;</td>
			<td>Sigma</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris&#160;</td>
			<td>Bitplane</td>
			<td></td>
			<td>with colocalization package&#160;</td>
		</tr>
	</tbody>
</table>

imaging the intracellular trafficking of app with photoactivatable gfp

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; 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 &#947;-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.

Typical results show &#946;APP 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 &#946;APP-paGFP is rapidly cleared from the lysosome (compare Figure 1a to <strong...

Watch this Scientific Journal Video about Imaging the Intracellular Trafficking of APP with Photoactivatable GFP at JoVE.com

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

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.

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

imaging-the-intracellular-trafficking-of-app-with-photoactivatable-gfp

Research

JoVE Journal

Biology

11.7K visualizações.  Western University.  Embora o transporte de proteínas de superfície celular está relativamente facilmente estudado, visualizando o tráfico de proteínas intracelulares é muito mais difícil. Aqui, usamos construções que incorporam GFP fotoactivável e demonstrar um método para seguir com precisão a proteína precursora de amilóide do aparelho de Golgi para compartimentos a jusante e siga a sua depuração. 