Name: imaging the intracellular trafficking of app with photoactivatable gfp
Uploaded: 2015-10-17T14:00:00
Description: Watch this Scientific Journal Video about Imaging the Intracellular Trafficking of APP with Photoactivatable GFP at JoVE.com

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>
	<li>Masters, C. L., 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=Amyloid+plaque+core+protein+in+Alzheimer+disease+and+Down+syndrome.">Amyloid plaque core protein in Alzheimer disease and Down syndrome.</a> Proc. Natl. Acad. Sci. U.S.A. 82 (12), 4245-4249 (1985).</li><li>Vassar, R., 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=Beta-secretase+cleavage+of+Alzheimer's+amyloid+precursor+protein+by+the+transmembrane+aspartic+protease+BACE.">Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.</a> Science. 286 (5440), 735-741 (1999).</li><li>De Strooper, B., 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=Deficiency+of+presenilin-1+inhibits+the+normal+cleavage+of+amyloid+precursor+protein.">Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.</a> Nature. 391 (6665), 387-390 (1998).</li><li>Xia, W., 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=Presenilin+complexes+with+the+C-terminal+fragments+of+amyloid+precursor+protein+at+the+sites+of+amyloid+beta-protein+generation.">Presenilin complexes with the C-terminal fragments of amyloid precursor protein at the sites of amyloid beta-protein generation.</a> Proc. Natl. Acad. Sci. U.S.A. 97 (16), 9299-9304 (2000).</li><li>Li, Y. M., 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=Presenilin+1+is+linked+with+gamma-secretase+activity+in+the+detergent+solubilized+state.">Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state.</a> Proc. Natl. Acad. Sci. U.S.A. 97 (11), 6138-6143 (2000).</li><li>Kim, W., Hecht, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+determinants+of+enhanced+amyloidogenicity+of+Alzheimer+A{beta}42+peptide+relative+to+A{beta}40.">Sequence determinants of enhanced amyloidogenicity of Alzheimer A{beta}42 peptide relative to A{beta}40.</a> J. Biol. Chem. 280 (41), 35069-35076 (2005).</li><li>Pawar, A. 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=Prediction+of+'aggregation-prone'+and+"aggregation-susceptible.">Prediction of 'aggregation-prone' and "aggregation-susceptible.</a> J. Mol. Biol. 350 (2), 379-392 (2005).</li><li>Wen, L., 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=VPS35+haploinsufficiency+increases+Alzheimer's+disease+neuropathology.">VPS35 haploinsufficiency increases Alzheimer's disease neuropathology.</a> J. Cell Biol. 195 (5), 765-779 (2011).</li><li>Rogaeva, E., 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=The+neuronal+sortilin-related+receptor+SORL1+is+genetically+associated+with+Alzheimer+disease.">The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease.</a> Nat. Genet. 39 (2), 168-177 (2007).</li><li>Andersen, O. M., 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=Neuronal+sorting+protein-related+receptor+sorLA/LR11+regulates+processing+of+the+amyloid+precursor+protein.">Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (38), 13461-13466 (2005).</li><li>Schmidt, V., 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=SorLA/LR11+regulates+processing+of+amyloid+precursor+protein+via+interaction+with+adaptors+GGA+and+PACS-1.">SorLA/LR11 regulates processing of amyloid precursor protein via interaction with adaptors GGA and PACS-1.</a> J. Biol. Chem. 282 (45), 32956-32964 (2007).</li><li>Moreth, J., Mavoungou, C., Schindowski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+anti-amyloid+immunotherapy+in+Alzheimer's+disease:+What+are+the+most+promising+targets.">Passive anti-amyloid immunotherapy in Alzheimer's disease: What are the most promising targets.</a> Immun Ageing. 10 (1), 18 (2013).</li><li>Pasternak, S. H., 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=Presenilin-1,+nicastrin,+amyloid+precursor+protein,+and+gamma-secretase+activity+are+co-localized+in+the+lysosomal+membrane.">Presenilin-1, nicastrin, amyloid precursor protein, and gamma-secretase activity are co-localized in the lysosomal membrane.</a> J. Biol. Chem. 278 (29), 26687-26694 (2003).</li><li>Schrader-Fischer, G., Paganetti, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+alkalizing+agents+on+the+processing+of+the+beta-amyloid+precursor+protein.">Effect of alkalizing agents on the processing of the beta-amyloid precursor protein.</a> Brain Res. 716 (1-2), 91-100 (1996).</li><li>Golde, T., Estus, S., Younkin, L., Selkoe, D., Younkin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+the+amyloid+protein+precursor+to+potentially+amyloidogenic+derivatives.">Processing of the amyloid protein precursor to potentially amyloidogenic derivatives.</a> Science. 255 (5045), 728-730 (1992).</li><li>Higaki, J., Quon, D., Zhong, Z., Cordell, B. <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+beta-amyloid+formation+identifies+proteolytic+precursors+and+subcellular+site+of+catabolism.">Inhibition of beta-amyloid formation identifies proteolytic precursors and subcellular site of catabolism.</a> Neuron. 14 (3), 651-659 (1995).</li><li>Chen, F., 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=Carboxyl-terminal+Fragments+of+Alzheimer+beta+-Amyloid+Precursor+Protein+Accumulate+in+Restricted+and+Unpredicted+Intracellular+Compartments+in+Presenilin+1-deficient+Cells.">Carboxyl-terminal Fragments of Alzheimer beta -Amyloid Precursor Protein Accumulate in Restricted and Unpredicted Intracellular Compartments in Presenilin 1-deficient Cells.</a> J. Biol. Chem. 275 (47), 36794-36802 (2000).</li><li>Perez, R. G., 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=Mutagenesis+identifies+new+signals+for+beta-amyloid+precursor+protein+endocytosis,+turnover,+and+the+generation+of+secreted+fragments,+including+Abeta42.">Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42.</a> J. Biol. Chem. 274 (27), 18851-18856 (1999).</li><li>Cirrito, J. R., 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=Endocytosis+Is+Required+for+Synaptic+Activity-Dependent+Release+of+Amyloid-&#946;+In+Vivo.">Endocytosis Is Required for Synaptic Activity-Dependent Release of Amyloid-&#946; In Vivo.</a> Neuron. 58 (1), 42-51 (2008).</li><li>Carey, R. M., Balcz, B. 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. W., 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+binds+the+FANSHY+sorting+motif+in+SorLA+to+regulate+amyloid+precursor+protein+sorting+and+processing.">Retromer binds the FANSHY sorting motif in SorLA to regulate amyloid precursor protein sorting and processing.</a> J. Neurosci. 32 (4), 1467-1480 (2012).</li><li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Photoactivatable+GFP+for+Selective+Photolabeling+of+Proteins+and+Cells.">A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells.</a> Science. 297 (5588), 1873-1877 (2002).</li><li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+photolabeling+of+proteins+using+photoactivatable+GFP.">Selective photolabeling of proteins using photoactivatable GFP.</a> Methods. 32 (4), 445-450 (2004).</li><li>Hirschberg, K., 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=Kinetic+analysis+of+secretory+protein+traffic+and+characterization+of+golgi+to+plasma+membrane+transport+intermediates+in+living+cells.">Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells.</a> J. Cell Biol. 143 (6), 1485-1503 (1998).</li><li>Kim, P. K., Mullen, R. T., Schumann, U., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+maintenance+of+mammalian+peroxisomes+involves+a+de+novo+PEX16-dependent+pathway+from+the+ER.">The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER.</a> J. Cell Biol. 173 (4), 521-532 (2006).</li><li>Hailey, D. W., 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=Mitochondria+supply+membranes+for+autophagosome+biogenesis+during+starvation.">Mitochondria supply membranes for autophagosome biogenesis during starvation.</a> Cell. 141 (4), 656-667 (2010).</li><li>Tam, J. H. K., Seah, C., Pasternak, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Amyloid+Precursor+Protein+is+rapidly+transported+from+the+Golgi+apparatus+to+the+lysosome+and+where+it+is+processed+into+beta-amyloid.">The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid.</a> Mol. Brain. 7 (1), 54 (2014).</li><li>Scott, D. A., Das, U., Tang, Y., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+logic+underlying+the+axonal+transport+of+cytosolic+proteins.">Mechanistic logic underlying the axonal transport of cytosolic proteins.</a> Neuron. 70 (3), 441-454 (2011).</li><li>Herl, L., 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=Mutations+in+amyloid+precursor+protein+affect+its+interactions+with+presenilin/gamma-secretase.">Mutations in amyloid precursor protein affect its interactions with presenilin/gamma-secretase.</a> Mol. Cell. Neurosci. 41 (2), 166-174 (2009).</li><li>Lorenzen, A., 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=Rapid+and+Direct+Transport+of+Cell+Surface+APP+to+the+Lysosome+defines+a+novel+selective+pathway.">Rapid and Direct Transport of Cell Surface APP to the Lysosome defines a novel selective pathway.</a> Mol. Brain. 3 (1), 11 (2010).</li><li>Thinakaran, G., Teplow, D. B., Siman, R., Greenberg, B., Sisodia, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+the+'Swedish'+amyloid+precursor+protein+variant+in+neuro2a+(N2a)+cells.+Evidence+that+cleavage+at+the+beta-secretase";site+occurs+in+the+golgi+apparatus.">Metabolism of the 'Swedish' amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the beta-secretase";site occurs in the golgi apparatus.</a> J. Biol. Chem. 271 (16), 9390-9397 (1996).</li><li>Thinakaran, G., Koo, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+Precursor+Protein+Trafficking,+Processing,+and+Function.">Amyloid Precursor Protein Trafficking, Processing, and Function.</a> J. Biol. Chem. 283 (44), 29615-29619 (2008).</li><li>Schneider, M., Barozzi, S., Testa, I., Faretta, M., Diaspro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Photon+Activation+and+Excitation+Properties+of+PA-GFP+in+the+720-920-nm+Region.">Two-Photon Activation and Excitation Properties of PA-GFP in the 720-920-nm Region.</a> Biophys. J. 89 (2), 1346-1352 (2005).</li><li>Sevier, C. S., Weisz, O. A., Davis, M., Machamer, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+export+of+the+vesicular+stomatitis+virus+G+protein+from+the+endoplasmic+reticulum+requires+a+signal+in+the+cytoplasmic+tail+that+includes+both+tyrosine-based+and+di-acidic.">Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic.</a> 11 (1), 13-22 (2000).</li><li>Muresan, V., Varvel, N. H., Lamb, B. T., Muresan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cleavage+products+of+amyloid-beta+precursor+protein+are+sorted+to+distinct+carrier+vesicles+that+are+independently+transported+within+neurites.">The cleavage products of amyloid-beta precursor protein are sorted to distinct carrier vesicles that are independently transported within neurites.</a> J. Neurosci. 29, 3565-3578 (2009).</li></ol>

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 ...

Research

JoVE Journal

Biology

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence Imaging with One-nanometer Accuracy FIONA

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...