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. Celleutplating og Transfeksjon <ol><li> I en cellekultur hette, plate ~ 300.000 til ~ 500.000 SN56 celler (en slags gave av Dr. Jane Rylett) på en 35 mm glass-bottom confocal tallerken og dekk med pre-transfeksjon media (Dulbeccos Modified Eagle Medium, DMEM, supplert med 10% FBS). </li><li> Inkuber celler over natten ved 37 ° C i en 5% CO 2 inkubator å spre seg. Merk: Cellene bør være ca 50% -70% konfluent før transfeksjon. </li><li> I en cellekultur hette, transfektere celler med plasmider...

<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>

Denne teknikken beskriver nøyaktig bilde-aktivering av membran proteiner merket med paGFP i TGN å visualisere påfølgende trafficking og cleavage. Mens paGFP ble skapt over et tiår siden 25, er dette det første eksempelet på nøyaktig fotoaktivering å følge smugling av begynnende proteiner til nedstrøms avdelinger. Tidligere studier med APP-konstruksjoner paGFP foto-aktivert en peri-atom region av cellen, som ble ansett for å være den 11 Golgi. Men så tydelig i våre mikrografer, lysoso...

Kjennetegnet av Alzheimers sykdom (AD) er tilstedeværelsen av senile plakk og nevrofibrillære floker (NFTs) i hjernen. Hovedbestanddelen av senile plakk er β-amyloid (AP). Ap er avledet fra dets forløper; amyloid forløper protein (APP) 1. Den amyloidogene spalting av APP begynner med fjerning av ectodomain fra APP av β-secretase to. Den gjenværende 99 rest-karboksyl-terminalt fragment (CTF) kan spaltes ved γ-sekretase å produsere Ap 3-7. Mens mange forsøk har dokumentert spalting av celleoverflaten APP etter intern fra celleoverflaten inn i endosomale / lysosomal system, har en rekke nyere studier antydet at intracellulær trafficking av APP er også viktig i å regulere sin behandling 8-11. Det har vært en rekke forsøk på å modulere nivåene av Ap med y-sekretaselignende inhibitorer og Ap Immunotherapies. Men nyere kliniske forsøk med disse behandlinger viste ingen fordel, og i noen tilfeller, forårsaket skade 12. En uutnyttet strategi for å modulere produksjon av Ap er å endre den sub-cellulære lokalisering av APP og γ-sekretase interaksjon. Golgi, plasma membran og endosomer / lysosomer har alle blitt foreslått som mulige steder for γ-spalting av APP. Forskning fra vårt laboratorium tyder på at APP og γ-secretase er bosatt proteiner av lysosomal membran 13. Videre har vi funnet at det lysosomale γ-sekretase har en sur optimal pH 13. I tillegg, alkalisering av den endosomale / lysosomale system med klorokin eller NH4CI, har vist seg å redusere produksjonen av Ap 14. y-sekretase hemming eller utstansing eller presenilin fører til APP-CTFs akkumulering i lysosomet 15-17. Videre forstyrre APP endocytose senker Ap produksjons 18-20. Til tross for viktigheten av Golgi som en sorteringsstasjon for begynnende proteiner og proteiner resirkulert fra endosomale / lysosomal system, har den intracellulære smugling av APP ikke blitt studert i detalj 21. Nyere arbeider har vist at APP kan resirkuleres til den trans-Golgi-nettet (TGN) via interaksjon med retromer komplekset. Nedregulering av retromer komplekse synker Ap produksjon 8,22-24. Imidlertid utslipp av APP fra Golgi har ikke blitt godt undersøkt. Mens etter endocytose av celleoverflateproteiner, slik som transferrin-reseptoren, er lett merket og etterfulgt, etter omsetning av intracellulære proteiner er mer utfordrende. Faktisk har noen proteiner hatt sine intracellulær trafficking avbildes. Ankomsten av fluorescerende protein koder, slik som fotoaktiverbar-GFP (paGFP), har gitt nye verktøy for å undersøke intracellulær trafficking. Fotoaktiverbar-GFP er en form for GFP som er nesten usynlig etter syntese, men utvikler sterk grønn (GFP) fluorescens etter å ha blitt aktivert ved 413 nm laserlys, og dette signalet er stabilt i flere dager 25,26. Konstruksjoner som bruker paGFP har blitt brukt for å demonstrere den intralysosomal omsetning av det lysosomale protein membranprotein 1 (LAMP1) 25, celleoverflaten levering av vesikulær stomatitt virus glykoprotein (VSVG, en markør for den sekretoriske vei) 27, og omsetningen av peroxisomes 28 og autophagosomes 29. For å visualisere sortering av APP fra TGN i levende celler, utformet vi plasmid uttrykker de siste 112 aminosyrer av APP koplet til fotoaktiverbar (paGFP) på C-terminal (referert til som βAPP-paGFP) 30. Vi har også utført disse eksperimentene med full lengde APP og oppnådde lignende resultater; den ΒAPP konstruksjonen er brukt her, fordi det gir klarere bilder. Vi så foto-activate &amp;# 946; APP-paGFP bare i TGN, som avgrenses av TGN markør Galactosyltransferase (Galt). Selv APP har blitt tagget med paGFP å visual rask aksonal transport av APP og klaring fra perinukleære regionen, er dette den første demonstrasjonen av APP trafficking fra en nøye definert rommet til en annen 11,31,32. Her viser vi nøyaktig bilde-aktivering i TGN og utslipp av APP til nedstrøms avdelinger og påfølgende spalting og klaring fra lysosomer 30. Den nøyaktige foto-aktivering i Golgi er allment gjeldende for andre proteinsystemer.

<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 (Ap) er den viktigste bestanddel av senile plakk som finnes i hjernen til Alzheimers sykdom pasienter. Ap er avledet fra sekvensiell spalting av amyloidforløperprotein (APP) med β og y-secretases. På tross av betydningen av Ap til AD patologi, er den subcellulære lokalisering av disse skillelinjer ikke er godt etablert. Arbeid i vårt laboratorium og andre implisere den endosomale / lysosomale system i APP prosessering etter internalisering fra celleoverflaten. Imidlertid er den intracellulære smugling av APP relativt studerte. Selv om celle-overflateproteiner er foranderlig for mange merkingsteknikker, er det ingen enkle metoder for å følge handel med membranproteiner fra Golgi. For å oppnå dette, har vi opprettet APP konstruksjoner som ble tagget med fotoaktiverbar GFP (paGFP) på C-terminalen. Etter syntese, har paGFP lav basal fluorescens, men det kan stimuleres med 413 nm lys to lage en sterk, stabil grønn fluorescens. Ved hjelp av Golgi markør galaktosyl transferase koblet til Cyan Fluorescent Protein (Galt-CFP) som et mål, er vi i stand til å nøyaktig photoactivate APP i trans-Golgi nettverk. Photo-aktivert APP-paGFP kan deretter bli fulgt som det traffics til nedstrøms avdelinger identifisert med fluorescently tagget kupé markørproteiner for tidlig endosomet (Rab5), avdøde endosomet (Rab9) og lysosomet (LAMP1). Videre, ved hjelp av inhibitorer for å APP prosessering, inkludert klorokin eller γ-sekretase inhibitor L685, 458, er vi i stand til å utføre puls-chase eksperimenter for å undersøke prosessering av APP i enkeltceller. Vi finner at en stor andel av APP beveger seg hurtig til lysosomet uten å virke på celleoverflaten, og er da fjernet fra lysosomet ved sekretaselignende spaltinger. Denne teknikken demonstrerer nytten av paGFP for å følge menneskehandel og behandling av intracellulære proteiner from Golgi til nedstrøms avdelinger.

Typiske resultater viser βAPP forlater TGN og ser ut til trafikk raskt til LAMP1 (Figur 1a og b). Under fotoaktiveringsperioden, kan vesikler sees avgang Golgi bestemt for lysosomer (Figur 1b). Uten inhibitor behandling, vil paGFP fluorescens være synlig i lysosomer mens det er fotoaktivering i TGN. Etter å ha stoppet fotoaktivering, er βAPP-paGFP raskt fjernet fra lysosomet (sammenlign figur 1a til 1b). Behandling med nocodazol fører til opphopnin...

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

Mens transport av celleoverflateproteiner som er forholdsvis lett studert, visualisering handel med intracellulære proteiner er mye vanskeligere. Her bruker vi konstruerer innlemme photoactivatable GFP og vise en metode for å nøyaktig følge amyloidforløperprotein fra Golgi-apparatet til nedstrøms avdelinger og følge sin klarering.

Imaging Intracellulær Trafficking of APP med 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 ...

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

Omprogrammering Menneskelige Somatiske celler i Utførte pluripotent stamceller (iPSCs) Bruke retrovirale Vector med 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

Funksjonell Imaging av brunt fett i Mus med 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

Lever Imaging av GFP-merket Proteiner i<em&gt; Drosophila</em&gt; Oocytter

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

Intramikros for Imaging subcellulære strukturer i levende mus Uttrykke Fluorescent Proteiner

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

Tomat / GFP-FLP / FRT Metode for Live Imaging av Mosaic Voksen<em&gt; Drosophila</em&gt; fotoreseptorcellene

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

Rapid Syntese og Screening av kjemisk Aktivert transkripsjonsfaktorer med GFP baserte Reportere

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

Fluorescens Imaging med One-nanometer Nøyaktighet (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

Real-time Imaging of Plant Cell Surface Dynamics med variabel vinkel epifluorescens Mikros

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