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 plasmids expressing a fluorescent protein tagged TGN marker (Galactosyl transferase coupled to Cyan Fluorescent Protein, GalT-CFP), a fluorescent protein tagged compartment marker (here, lysosome associated membrane protein 1, LAMP1, tagged with mRFP), and the protein of interest tagged with paGFP (&#946;APP-paGFP). (These constructs have previously been described 30)</li>
	<li>Incubate cells with transfection reagent and plasmid DNA for 24 hr at 37 &#176;C in a 5% CO2 incubator. Use a ratio of ~2 &#181;g of DNA to 5 &#181;l of transfection agent (e.g. Lipofectamine 2000) to best balance cytotoxicity and transfection efficiency. Actual concentrations may need to be optimized for different plasmids. In the experiments described here, use 1 &#181;g/confocal dish of &#946;APP-paGFP, 0.3 &#181;g/confocal dish of LAMP1-mRFP, and 0.2 &#181;g/confocal dish of GalT-CFP. 
	Note: The amount of plasmid used will depend on transfection efficiency.</li>
	<li>For neuronal cell lines: After step 1.4, in a cell culture hood, remove pre-transfection media and differentiate SN56 cells in 2 ml DMEM supplemented with 0.1% penicillin/streptomycin with 1 mM dibutyryl cyclic AMP (dbcAMP) for 24 hr.</li>
</ol>
2. Microscope Stage Preparation
<ol>
	<li>Pre-warm phosphate-buffered saline (PBS) and Hank&#8217;s Balanced Salt Solution (HBSS) to 37 &#176;C. Warm-up microscope stage before imaging to 37 &#176;C.</li>
	<li>Wash cells twice with PBS, to remove differentiation media and replace with 2 ml of HBSS at 37 &#176;C. Place cells on microscope stage warmed to 37 &#176;C. 
	Note: Cells are viable for approximately 1.5-2 hr in HBSS.</li>
	<li>Allow 5-10 min for the confocal plate temperature to equilibrate with the microscope stage.</li>
</ol>
3. Photo-activation of ROIs over the Golgi
<ol>
	<li>With the microscope eyepiece, find cells transfected with GalT-CFP, LAMP1-mRFP, and &#946;APP-paGFP.
	<ol>
		<li>Use an oil-immersion lens with high magnification (i.e. 63X or 100X). For visual conformation of fluorescence, use a filter set capable of visualizing FITC (for CFP and GFP fluorescence) and rhodamine (for mRFP fluorescence).</li>
	</ol>
	</li>
	<li>Set the confocal slice for each channel to 1 &#181;m. Take a low-resolution image.</li>
	<li>Crop to image only the cell of interest.</li>
	<li>Using the adjustment knob, manually set the focal plane to the middle of the cell. This is typically the part of the cell where the nucleus is the largest.</li>
	<li>Draw 3-5 circular Regions of Interests (ROIs) within the TGN (GalT-CFP fluorescence).
	<ol>
		<li>To draw ROIs (i.e. in the Zeiss LSM program), select the &#8216;Edit ROI&#8217; button in the command console.</li>
		<li>Select the circular ROI button.</li>
		<li>Click and drag over GalT-CFP positive regions of the cell. 
		Note: The photo-bleaching packages for many microscopes have this capability.</li>
	</ol>
	</li>
	<li>Take an image of the cell with the overlaid ROIs. Save a copy of the ROIs for later reference.
	<ol>
		<li>To save the ROIs to the image, select the &#8216;Macro&#8217; button.</li>
		<li>Press the &#8216;Load&#8217; button to start the &#8216;CopyRoisToOverlay&#8217; (file path: c:/AIM/Macros/AdvancedTimeSeries/Utility.lvb). 
		Note: A confocal microscope equipped with 475-525 nm (CFP) band pass (BP), a 500-530 nm BP (GFP), and a 560 nm long pass (LP) filter sets are required. An argon laser for 458 nm and 488 nm emission and a HeNe laser for 540 nm emission is also required.</li>
	</ol>
	</li>
	<li>Set-up the microscope for a bleach time course.
	<ol>
		<li>Set the laser diode (25 mW 405 nm laser) to maximum power. 
		Caution: Excessive photo-activation could lead to photo-toxicity or damage to cellular membranes. Use eye protection to avoid retinal damage.</li>
		<li>Set-up the microscope for 120 cycles of imaging. 
		Note: One imaging cycle consists of bleaching within the ROIs and imaging of the entire cell. This is a bleach time course.</li>
		<li>Set the microscope to bleach (photo-activate) only the ROIs for 20-30 iterations after every image. The time to image and bleach an image will vary. Set a time delay between images so each cycle is approximately 30 sec. 
		Note: The imaging portion of each cycle takes approximately 15-30 sec depending on image size. The bleaching for each ROI is about 50 msec. Bleaching of all 4 ROIs for 20 iterations will take 4 sec at the end of each bleach/image cycle.</li>
	</ol>
	</li>
	<li>Start the bleach time course.</li>
	<li>Turn off the bleaching after 15 min (30 cycles of imaging and bleaching), but continue capturing images of the cell. 
	Note: Time 0 to 15 min is the &#8216;pulse&#8217; period.</li>
	<li>Continue capturing images for 45 min (without bleaching), to follow the clearance of &#946;APP-paGFP. 
	Note: Time 15 to 45 min is the &#8216;chase&#8217; period.</li>
	<li>Using the analysis method described in step 6, co-localize the protein of interest (here, &#946;APP-paGFP) with the compartment of interest (here, LAMP1-mRFP) by making surfaces to each channel.</li>
</ol>
4. Determine the Downstream Compartment
<ol>
	<li>To determine if lysosomes (in these experiments) are the final compartment, add membrane-permeable protease inhibitors to cause proteins to accumulate in the lysosome.
	<ol>
		<li>For &#946;APP, use 0.5 &#181;M L685, 458 (specific &#947;-secretase inhibitor) overnight or 100 &#181;M chloroquine (deacidifies lysosomes) for 30 min before imaging.</li>
	</ol>
	</li>
	<li>Find cells and photoactivate as in step 3.1 &#8211; 3.8.</li>
	<li>Image the cell for an additional 45-min (without photo-activation) to determine where &#946;APP-paGFP accumulates in absence of cleavage.</li>
	<li>Analyze the delivery of protein to lysosomes (or other downstream compartment) and subsequent clearance according to the method described in step 6.</li>
</ol>
5. Ensure Accuracy of Photo-activation within the Golgi
<ol>
	<li>Warm-up HBSS to 37 &#176;C.</li>
	<li>Prepare a 2 ml solution, for every confocal plate to be imaged, of 66 &#181;M nocodazole (treatment) or DMSO (control) in HBSS.
	<ol>
		<li>For one confocal plate, pipette 2 ml of 37 &#176;C HBSS and add 7.96 &#181;l of nocodazole from a 16.60 mM nocodazole stock.</li>
		<li>As a control solution, pipette 2 ml of 37 &#176;C HBSS into a 2 ml tube and add 7.96 &#181;l of DMSO.</li>
	</ol>
	</li>
	<li>Incubate cells in HBSS with nocodazole or DMSO for 5 min before imaging.</li>
	<li>Find cells as in step 3.1.</li>
	<li>Before photoactivation, prepare the microscope to take a Z-stack after the photo-activation period.
	<ol>
		<li>Click on the &#8216;Z Stack&#8217; button. Start scanning using Fast XY.</li>
		<li>Adjust the focal plane with the microscope adjustment knob to the top of the cell. Press &#8216;Mark First&#8217; to set the first position in the stack.</li>
		<li>Adjust the focal plane with the microscope adjustment knob to the bottom (closest to the glass) of the cell. Press &#8216;Mark Last&#8217; to set the last position in the stack.</li>
		<li>In the Z stack panel, press the &#8216;Z sectioning&#8217; button. Set the &#8216;Interval&#8217; to 1 &#181;m. 
		Note: For a higher spatial resolution stack a smaller stack interval, but more images will need to be taken lengthening the imaging period for the stack.</li>
	</ol>
	</li>
	<li>Bleach the cells, as described in step 3.7-3.8. Photo-activate and image from 0 min to 15 min (30 frames).</li>
	<li>Stop imaging after 30 frames. Immediately save an image of the video. 
	Caution: Some microscopes may overwrite first video if it is not saved before starting a second imaging sequence.</li>
	<li>After saving the video, immediately acquire a Z-stack, using the parameters from step 5.5.</li>
	<li>Co-localize &#946;APP-paGFP with GalT-CFP (or other Golgi marker), as described in step 6. 
	Note: CFP photobleaches rapidly and may not be visible by the end of the imaging period. Colocalization may not be possible with GalT-CFP. If colocalization is required, use mRFP to tag GalT.</li>
</ol>
6. Vesicle Filtering and Colocalization
<ol>
	<li>Use an analysis program (e.g. Imaris) to make surfaces of the compartment (e.g. LAMP1-mRFP) and a protein of interest (e.g. &#946;APP-paGFP).</li>
	<li>Enter the &#8216;Surpass&#8217; section of the analysis program by clicking on the Surpass button on the top menu bar.</li>
	<li>Select the Surface wizard button at the top of the left most panel (5th button from the left).</li>
	<li>Select a channel to filter vesicles (i.e. &#946;APP-paGFP fluorescence, protein of interest).</li>
	<li>Perform background subtraction by submitting the diameter of the largest vesicle in the field to the wizard and press next. The analysis program automatically selects &#8212; based on a threshold derived from the largest vesicle in the field &#8212; an area in the channel of interest.
	<ol>
		<li>Manually adjust the threshold background to refine the selection if needed.</li>
	</ol>
	</li>
	<li>At the bottom of the Threshold selection screen, check the &#8216;Split touching objects&#8217; option to separate the combined Surfaces. 
	Note: If many vesicles are closely grouped together, the analysis program will group these objects under one surface.
	<ol>
		<li>Choose 10-15 representative vesicles and calculate the average vesicle diameter and enter the result into the wizard.</li>
	</ol>
	</li>
	<li>Refine the selected seed points by increasing or decreasing the threshold on &#8216;Quality&#8217; (intensity of the channel in the middle of each spot). 
	Note: Surfaces of the channel of interest will be created. Further refinement of the selected surfaces can be performed at this point. Threshold vesicles based on the number of pixels in each vesicle.</li>
	<li>Mask the original channel with this surface. To mask, select the 4th tab from the left in the surface creation wizard (pencil).</li>
	<li>Select &#8216;Mask All&#8217;. A new channel with the vesicles of interest will be created. 
	Note: During the mask process, an option to duplicate the original channel is offered. Select this option if the original data needs to be saved.</li>
	<li>Repeat steps 6.1 through 6.7 for the unfiltered channel (i.e. LAMP1 mRFP, compartment).</li>
	<li>At the top tool bar, select the colocalization tool.
	<ol>
		<li>In the top panel, as the histograms of the channels to be colocalized appear, select the recently created masked channels.</li>
		<li>Select all of available data. There are dark pixels that are sometimes present after analysis. Do not select these pixels in the histogram, they will skew the results.</li>
		<li>In the far right panel, select &#8216;Build Coloc Channel&#8217;. This creates a new channel containing only the colocalized pixels. The data will be in the same panel under the &#8216;Channel Statistics&#8217; button. The data can be exported as a .CSV file.</li>
		<li>Use the &#8216;Percent of Material colocalized&#8217; statistic. This option takes into account the intensity of the pixels and size of the object.</li>
	</ol>
	</li>
</ol>

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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>

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 Aufrufe.  Western University.  Während der Transport von Zelloberflächenproteinen relativ leicht studiert, Visualisieren des Handels mit intrazellulären Proteinen ist viel schwieriger. Hier verwenden wir Konstrukte Einbeziehung photoaktivierbaren GFP und zeigen eine Methode, um genau zu folgen das Amyloid-Vorläuferprotein aus dem Golgi-Apparat, um Downstream-Fächer und folgen Sie den Freiraum. 

Serie: Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

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 medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

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 labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

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

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

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

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

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

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 density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

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

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

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

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

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, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

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 Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

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

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

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 structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

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

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 cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

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

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.