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Method Article

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

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DOI:

10.3791/53153

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October 17th, 2015

 , 
1Department of Physiology and Pharmacology, Robarts Research Institute, Western University, 2Department of Clinical Neurological Sciences, Western University

In This Article

Summary

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

Abstract

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

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

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

Introduction

The hallmark of Alzheimer’s disease (AD) is the presence of senile plaques and neurofibrillary tangles (NFTs) in the brain. The major constituent of senile plaques is β-amyloid (Aβ). Aβ is derived from its precursor; amyloid precursor protein (APP) 1. The amyloidogenic cleavage of APP begins with removal of the ectodomain from APP by β-secretase 2. The remaining 99-residue carboxyl terminal fragment (CTF) can be cleaved by γ-secretase to produce Aβ 3-7. While many experiments have documented the cleavage of cell surface APP after internalization from the cell surface into the endosomal/ lysosomal 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 Aβ with γ-secretase inhibitors and Aβ immunotherapies. However, recent clinical trials with these therapies showed no benefit, and, in some cases, caused harm 12. An unexploited strategy to modulate Aβ production is to alter the sub-cellular localization of APP and γ-secretase interaction. The Golgi, plasma membrane, and endosomes/lysosomes have all been suggested as possible locales for γ-cleavage of APP. Research from our laboratory suggests that APP and the γ-secretase are resident proteins of the lysosomal membrane 13. Furthermore, we have found that the lysosomal γ-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 Aβ 14. γ-secretase inhibition or knockout or Presenilin leads to APP-CTFs accumulation in the lysosome 15-17. Moreover, disrupting APP endocytosis lowers Aβ 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 Aβ 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 βAPP-paGFP) 30. We have also performed these experiments with full length APP and achieved similar results; the ΒAPP construct is used here because it provides brighter images. We then photo-activate βAPP-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.

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Protocol

1. Cell Plating and Transfection

  1. In a cell culture hood, plate ~300,000 to ~500,000 SN56 cells (a kind gift of Dr. Jane Rylett) on a 35 mm glass-bottom confocal dish and cover with pre-transfection media (Dulbecco’s Modified Eagle Medium, DMEM, supplemented with 10% FBS).
  2. Incubate cells overnight at 37 °C in a 5% CO2 incubator to proliferate.
    Note: Cells should be approximately 50%-70% confluent before transfection.
  3. In a cell culture hood, transfect cells with plasmids expressing a fluorescent protein tagged TGN marker (Galactosyl transferase coupled to Cyan Fluorescent Protein, GalT-CFP), a fluorescent protein tagged compartment marker (here, lysosome associated membrane protein 1, LAMP1, tagged with mRFP), and the protein of interest tagged with paGFP (βAPP-paGFP). (These constructs have previously been described 30)
  4. Incubate cells with transfection reagent and plasmid DNA for 24 hr at 37 °C in a 5% CO2 incubator. Use a ratio of ~2 µg of DNA to 5 µl 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 µg/confocal dish of βAPP-paGFP, 0.3 µg/confocal dish of LAMP1-mRFP, and 0.2 µg/confocal dish of GalT-CFP.
    Note: The amount of plasmid used will depend on transfection efficiency.
  5. 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.

2. Microscope Stage Preparation

  1. Pre-warm phosphate-buffered saline (PBS) and Hank’s Balanced Salt Solution (HBSS) to 37 °C. Warm-up microscope stage before imaging to 37 °C.
  2. Wash cells twice with PBS, to remove differentiation media and replace with 2 ml of HBSS at 37 °C. Place cells on microscope stage warmed to 37 °C.
    Note: Cells are viable for approximately 1.5-2 hr in HBSS.
  3. Allow 5-10 min for the confocal plate temperature to equilibrate with the microscope stage.

3. Photo-activation of ROIs over the Golgi

  1. With the microscope eyepiece, find cells transfected with GalT-CFP, LAMP1-mRFP, and βAPP-paGFP.
    1. 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).
  2. Set the confocal slice for each channel to 1 µm. Take a low-resolution image.
  3. Crop to image only the cell of interest.
  4. 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.
  5. Draw 3-5 circular Regions of Interests (ROIs) within the TGN (GalT-CFP fluorescence).
    1. To draw ROIs (i.e. in the Zeiss LSM program), select the ‘Edit ROI’ button in the command console.
    2. Select the circular ROI button.
    3. Click and drag over GalT-CFP positive regions of the cell.
      Note: The photo-bleaching packages for many microscopes have this capability.
  6. Take an image of the cell with the overlaid ROIs. Save a copy of the ROIs for later reference.
    1. To save the ROIs to the image, select the ‘Macro’ button.
    2. Press the ‘Load’ button to start the ‘CopyRoisToOverlay’ (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.
  7. Set-up the microscope for a bleach time course.
    1. 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.
    2. 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.
    3. 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.
  8. Start the bleach time course.
  9. 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 ‘pulse’ period.
  10. Continue capturing images for 45 min (without bleaching), to follow the clearance of βAPP-paGFP.
    Note: Time 15 to 45 min is the ‘chase’ period.
  11. Using the analysis method described in step 6, co-localize the protein of interest (here, βAPP-paGFP) with the compartment of interest (here, LAMP1-mRFP) by making surfaces to each channel.

4. Determine the Downstream Compartment

  1. To determine if lysosomes (in these experiments) are the final compartment, add membrane-permeable protease inhibitors to cause proteins to accumulate in the lysosome.
    1. For βAPP, use 0.5 µM L685, 458 (specific γ-secretase inhibitor) overnight or 100 µM chloroquine (deacidifies lysosomes) for 30 min before imaging.
  2. Find cells and photoactivate as in step 3.1 – 3.8.
  3. Image the cell for an additional 45-min (without photo-activation) to determine where βAPP-paGFP accumulates in absence of cleavage.
  4. Analyze the delivery of protein to lysosomes (or other downstream compartment) and subsequent clearance according to the method described in step 6.

5. Ensure Accuracy of Photo-activation within the Golgi

  1. Warm-up HBSS to 37 °C.
  2. Prepare a 2 ml solution, for every confocal plate to be imaged, of 66 µM nocodazole (treatment) or DMSO (control) in HBSS.
    1. For one confocal plate, pipette 2 ml of 37 °C HBSS and add 7.96 µl of nocodazole from a 16.60 mM nocodazole stock.
    2. As a control solution, pipette 2 ml of 37 °C HBSS into a 2 ml tube and add 7.96 µl of DMSO.
  3. Incubate cells in HBSS with nocodazole or DMSO for 5 min before imaging.
  4. Find cells as in step 3.1.
  5. Before photoactivation, prepare the microscope to take a Z-stack after the photo-activation period.
    1. Click on the ‘Z Stack’ button. Start scanning using Fast XY.
    2. Adjust the focal plane with the microscope adjustment knob to the top of the cell. Press ‘Mark First’ to set the first position in the stack.
    3. Adjust the focal plane with the microscope adjustment knob to the bottom (closest to the glass) of the cell. Press ‘Mark Last’ to set the last position in the stack.
    4. In the Z stack panel, press the ‘Z sectioning’ button. Set the ‘Interval’ to 1 µm.
      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.
  6. Bleach the cells, as described in step 3.7-3.8. Photo-activate and image from 0 min to 15 min (30 frames).
  7. 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.
  8. After saving the video, immediately acquire a Z-stack, using the parameters from step 5.5.
  9. Co-localize βAPP-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.

6. Vesicle Filtering and Colocalization

  1. Use an analysis program (e.g. Imaris) to make surfaces of the compartment (e.g. LAMP1-mRFP) and a protein of interest (e.g. βAPP-paGFP).
  2. Enter the ‘Surpass’ section of the analysis program by clicking on the Surpass button on the top menu bar.
  3. Select the Surface wizard button at the top of the left most panel (5th button from the left).
  4. Select a channel to filter vesicles (i.e. βAPP-paGFP fluorescence, protein of interest).
  5. 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 — based on a threshold derived from the largest vesicle in the field — an area in the channel of interest.
    1. Manually adjust the threshold background to refine the selection if needed.
  6. At the bottom of the Threshold selection screen, check the ‘Split touching objects’ option to separate the combined Surfaces.
    Note: If many vesicles are closely grouped together, the analysis program will group these objects under one surface.
    1. Choose 10-15 representative vesicles and calculate the average vesicle diameter and enter the result into the wizard.
  7. Refine the selected seed points by increasing or decreasing the threshold on ‘Quality’ (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.
  8. Mask the original channel with this surface. To mask, select the 4th tab from the left in the surface creation wizard (pencil).
  9. Select ‘Mask All’. 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.
  10. Repeat steps 6.1 through 6.7 for the unfiltered channel (i.e. LAMP1 mRFP, compartment).
  11. At the top tool bar, select the colocalization tool.
    1. In the top panel, as the histograms of the channels to be colocalized appear, select the recently created masked channels.
    2. 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.
    3. In the far right panel, select ‘Build Coloc Channel’. This creates a new channel containing only the colocalized pixels. The data will be in the same panel under the ‘Channel Statistics’ button. The data can be exported as a .CSV file.
    4. Use the ‘Percent of Material colocalized’ statistic. This option takes into account the intensity of the pixels and size of the object.

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Results

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

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Discussion

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

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Disclosures

The authors have no competing financial interests or conflicts of interest.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
35 mm glass bottom culture dishes MatekP-35G-1.5-20-C
Dulbecco's Phosphate Buffered SalineLife Technologies14190-144
Hanks Balanced Salt SolutionLife Technologies14025-092
Lipofectamine 2000Life Technologies11668019
Dulbecco's Modified Eagle MediumLife Technologies11995-092
Penicilin/StreptomycinLife Technologies15140-122
dibutyrl cyclic AMPSigma D0627
Heat in activated Fetal Bovine SerumLife Technologies10082147
Heated Microscopy stage insert PPeCon GmbH
Tempcontrol 37–2 digital 2-channelPeCon GmbH
Zeiss LSM-510 META laser- scanning microscope Carl Zeisswith laser diode, argon laser, and HeNe1 laser
Zeiss 63X 1.4 numerical aperture oil immersion lensCarl Zeiss
L685, 458 EMD Millipore565771dissolved in DMSO
cholorquine SigmaC6628 dissolved in water
NocodazoleSigmaM1404dissolved in DMSO
Dimethyl sulphoxide Sigma472301
Imaris Bitplanewith colocalization package 

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APP TraffickingGolgi MarkerConfocal ImagingLysosome TrackingRab5 Rab9 LAMP1Gamma Secretase InhibitorPulse Chase ExperimentLive Cell MicroscopyIntracellular Protein Trafficking
Imaging the Intracellular Trafficking of APP with Photoactivatable GFP
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