FRET imaging of multiple focal planes to analyze the organization and conformation of Transferrin-Receptor in polarized cells

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1 FRET imaging of multiple focal planes to analyze the organization and conformation of Transferrin-Receptor in polarized cells Horst Wallrabe a, Ammasi Periasamy b and Margarida Barroso c a Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA b Center for Cardiovascular Sciences, Albany Medical College, Albany, NY ABSTRACT We developed a FRET-based assay to analyze multiple focal planes from z-staks collected using confocal imaging of polarized epithelial MDCK cells. To establish the imaging assay for multiple focal planes, we have used fixed cells, where changes in the organization and conformation of transferrin-receptor complexes between endosomes are expected to be minimized. Therefore, we indeed only see minor changes in E% throughout the endocytic pathway of polarized cells. In the future, we will apply this FRET-based assay to live polarized epithelial cells to investigate the factors involved in the regulation of the organization and conformation of membrane-bound receptors during endocytic trafficking. KEYWORDS FRET, transferrin, endosomes, apical, confocal, membrane trafficking 1. INTRODUCTION We have previously used our FRET assay to investigate clustered versus random distribution of receptor-ligand complexes during endocytosis and transcytosis in polarized epithelial MDCK cells 1-5. To draw conclusions about the organization and conformation of receptor-ligand complexes, we use energy transfer efficiency (E%) and its relationship to several parameters, such as acceptor fluorescence and donor:acceptor ratios (D/A) 3;5. In turn, E% can only be used with confidence, after the FRET signal has been corrected for spectral bleed-through (SBT) 6, which inevitably occurs as a result of the spectral overlap between donor and acceptor - necessary for FRET to occur in the first place. Other conditions for FRET are that donor and acceptor fluorophores have to be within a distance of Å and have a favorable dipole-dipole orientation between them. We have developed a highly sensitive algorithm based on single label reference specimens - called PFRET (processed FRET) - which corrects the contaminated FRET signal pixel-bypixel 3;5;6. Recently, the software has been upgraded to be capable of processing large amounts of data efficiently, as is the case with this data, where multiple focal plane images need to be analyzed with thousands of regions of interest (ROIs). Our main research objective is to investigate the organization and conformation of transferrin-receptor (TFR)-iron-bound transferrin (Tfn) complexes along the endocytic pathway in live polarized epithelial MDCK cells 3;5. Fully polarized MDCK cells can acquire a 10-15μm height and develop distinct endosomes with different ph levels and different protein populations serving different functions within the cell (Figure 1) 7-9. Both, apical and basolateral early endosomes (AEE and BEE) have an acidic environment, maintained by proton pumps. The peri-nuclear common endosome (CE) is also largely acidic, whereas the apical recycling endosome (ARE) provides a neutral ph space and acts as a one-way trafficking station leading to the apical plasma membrane (PM). Under normal physiological conditions, the majority of TFR-Tfn complexes enter the cell through the basolateral PM, release their Fe +++ cargo in the acidic BEE, and whereas some of the TFR-apo-Tfn complexes are then recycled to the basolateral PM, the majority however proceeds to the CE and is recycled back to the basolateral PM with a small percentage being misdirected to the apical PM via the ARE (Figure 1, black arrows) 7-9. Treatment with Brefeldin A (BFA) missorts a considerable proportion of TFR-Tfn complexes from the CE to the ARE and apical PM 10. Therefore, in the presence of BFA, Tfn can enter polarized cells Multiphoton Microscopy in the Biomedical Sciences IX, edited by Ammasi Periasamy, Peter T. C. So, Proc. of SPIE Vol. 7183, 71832F 2009 SPIE CCC code: /09/$18 doi: / Proc. of SPIE Vol F-1

2 from the apical PM to meet basolaterally co-internalized Tfn molecules throughout the polarized endocytic pathway, predominantly at the CE and ARE 4;5;10. Apical PM + BFA treatment ph: Basolateral PM Figure 1. Endocytic trafficking pathways in polarized MDCK cells. Treatment with BFA directs a higher proportion of TFR to the apical PM, compared with untreated conditions; this allows the internalization of significant quantities of labeled Tfn from the apical PM (dotted arrows) in addition to the main basolateral endocytosis (solid arrows). Both pathways enter the acidic BEE and AEE, proceed to the peri-nuclear CE, from where the majority is cycled to the basolateral PM. In the presence of BFA, a significant percentage is missorted to the neutral-ph ARE and subsequently to the apical PM. The endosomal ph gradient from early endosomes to ARE is displayed separately. TJ indicates the tight junctions, separating apical and basolateral PMs in polarized epithelial MDCK cells. FRET will be measured at different cell heights to determine the organization of TFR-Tfn complexes throughout the polarized endocytic pathways. Using the MDCK model systems, we have previously used a FRET assay based on E%'s independence from acceptor and negative dependence on D:A ratio, to show that different receptor-ligand complexes, including TFR and polymeric IgA-receptor, form clusters while being co-transported via the polarized endocytic pathways 4. We observed differences in E% levels between CE and ARE, which could be due to several factors, such as ph, iron release and the binding of sorting proteins. These differences may affect E% levels by altering the proximity of the fluorophores within the cluster due to changes in the organization and/or protein conformation of receptor-ligand complexes. Here, we have internalized differently fluorophore-labeled Tfn conjugates from opposite PM domains to measure FRET upon their clustering at different polarized endocytic compartments (Figure 1; FRET). Furthermore, we have tested the ability of the PFRET software to analyze multiple focal planes from z-stacks collected using confocal microscopy and established the FRET assay to analyze E% level changes throughout the endocytic pathway in polarized MDCK cells. Proc. of SPIE Vol F-2

3 B LI = -II I '-I 41:1 D -6pm 2L1 -ijo rn I 1 [I s = 2: 4': EU ufret ROIs 21:1 41J El:' Acceptor (iriterisitv.ipixels) Figure 2. FRET imaging of TFR-Tfn complexes at different focal planes in polarized MDCK cells. Left panels: ufret images; Middle panels, ufret images displaying selected ROIs; Right panels: E% vs. acceptor charts. Alexa Fluor 488-Tfn and Alexa Fluor 555-Tfn were co-internalized from opposite PMs into polarized MDCK cells in the presence of BFA. Starting from the apical region, 5 optical Z-sections were taken every 2μm steps under optimized FRET imaging conditions, using LSM Zeiss 510META. The ufret images shown here are those before spectral bleed-through correction plus the automatic ROI grid selection as described in Materials and Methods. The charts corresponding to each focal plane show broadly an independence of E% from acceptor intensity fluorescence levels, indicating a clustered distribution of TFR-Tfn complexes co-internalized from opposite PMs. Proc. of SPIE Vol F-3

4 2. MATERIALS AND METHODS Cellular uptake of TFR-Tfn complexes. Confluent MDCK-PTR cells, i.e. Martin Darby canine kidney cells that express the stably transfected human transferrin-receptor (TFR) at the plasma membrane (PM), are grown on membrane inserts for 3-4 days until they reach a polarized status 4;7;9. TFR behaves as a homo-dimeric type II transmembrane receptor; each TFR's monomer contains a small cytoplasmic domain, a single-pass transmembrane region and a large extracellular domain, which binds a Tfn ligand bearing two iron molecules. Therefore, a TFR homodimer binds two molecules of Tfn, at the basolateral PM (Figure 1) 11;12. Alexa Fluor 488 (Donor)-labeled and Alexa Fluor 555 (Acceptor)-labeled Tfn ligands are used to follow the intracellular trafficking of TFR in polarized MDCK-PTR cells (Figure 1) 3. The cells are pretreated with 10μm BFA for 30min, followed by the internalization of Alexa Fluor 488- and/or Alexa Fluor 555-Tfn from the opposite PM, i.e Alexa Fluor 488-Tfn (25μg/ml) from the apical PM and Alexa Fluor 555-Tfn (μg/ml) from the basolateral PM. Internalization proceeded for 30 min at 37 C in the presence of BFA. Cells are fixed with 4% paraformaldehyde, washed with PBS and imaged at room temperature. Confocal Imaging. A multi-track imaging approach was used on a Zeiss 510 confocal microscope with the following settings: 15% of Argon laser at 488nm and 12% of HeNe laser at 561nm with PMT levels at 6. The following emission filters were used: Donor emission BP0-5nm and Acceptor LP575nm. The z-stacks tool was used to collect focal plane images every 2μm steps, starting at apical PM at a zoom 2x. Imaging conditions were optimized achieving an acceptable uncorrected energy transfer signal in the double-label specimen (Donor excitation/acceptor emission - Dex/Aem) and minimal saturation in the donor channel (Donor excitation/donor emission Dex/Dem) and in the acceptor channel (Acceptor excitation/acceptor emission (Aex/Aem). As expected the back-bleedthrough channel (Acceptor excitation/donor emission - Aex/Dem), did not show any visible fluorescence. Single-label reference samples were imaged at identical conditions to determine the donor and acceptor bleedthrough levels using the PFRET SBT correction algorithm 3;5. Data Analysis. A proprietary version of the Processed FRET (PFRET) software was used to analyze the images as a plugin in ImageJ. As a first step, the level of background noise in each emission channel was established by manually selecting multiple random ROIs from the single-label donor sample at Aex/Aem and Aex/Dem, from the single label acceptor sample at Dex/Dem, Aex/Dem and Dex/Aem in areas of the specimen devoid of fluorophores. Background noise was low, different in each channel and subtracted specifically by channel with a module of the ImageJ-PFRET plugin. Next, the automatic 10x10 pixel ROI grid selection module was applied to a stack of background-subtracted uncorrected FRET images with a threshold of an average of 10 gray-level fluorescence values; ~10 ROIs were generated and analyzed as described below. 3. RESULTS AND DISCUSSION Confluent, polarized epithelial MDCK cells are a well-established model system in the biological sciences and ideal to investigate inter alia trafficking and signaling pathways in cells. After polarization, tight junctions are formed, distinct apical and basolateral PMs are present and several polarized endosomal compartments are fully developed 9 (Figure 1). There is considerable potential for imaging discrete focal planes in these ~ 10-15μm tall cells to track cellular events. Imaging multiple focal planes ('z-stacks ), each field of view containing several cells and data analysis with hundreds or thousands of ROIs presents many challenges, which can be overcome with high-end imaging systems and advanced data analysis software. The data presented here was generated on a LSM Zeiss Meta510 using the multi-track facility and data analysis with a proprietary version of the PFRET software. While we used initially fixed cells as proof-of-principle, the approach will primarily used in live cell imaging. To perform a detailed analysis of the FRET data, the PFRET plugin uses the background-subtracted images and the ROI coordinates generated by the grid selection to gather information about each ROI, including the number of non-saturated pixels as well as the average level per pixel of uncorrected FRET (ufret), energy transfer (pfret), percent of SBT correction, quenched donor (qd), unquenched/total donor (D = qd+pfret), acceptor (A), D/A ratio and E%; significantly, the PFRET plugin automatically removes saturated pixels from the analysis. Furthermore, this data was subjected to thresholds for the A & D:A ratios to eliminate potential outliers 3;5 ; the acceptor intensity was limited to a range of gray-level fluorescence values and D:A ratios to an average of ~1 (range ), which actually Proc. of SPIE Vol F-4

5 pn1fl1n111 includes the majority of the data. A Global Analysis is then produced with an average of all ROIs per image of the z- stack, providing a first quick glance of the data and an indication of the consistency and quality of the z-stack images (data not shown); by way of demonstrating the capabilities of the assay, here we show one representative z-stack (Figure 2). Figure 2 visualizes the different focal planes in 2μm steps starting at the apical region and proceeding towards the basal region. The ufret image is shown first (left panel) and then again displaying the location of numbered ROIs (middle panel). This analysis enables us to visually inspect a particular ROI that may show unusual properties, applying the ImageJ zoom tool to that particular ROI for examination. The identification of a number of ROIs (224) at the most apical focal planes (Figure 2A-B) is consistent with a significant percentage of TFR-Tfn complexes being missorted to the ARE, as expected as result from BFA treatment 4;10. The number of ROIs increases (361) as we enter the perinuclear CE region (Figure 2B-E), where the larger proportion of complexes is recycled to the basolateral PM. The most basolateral focal plane show lower number of ROIs (76) as expected due to the reduced trafficking of apical internalized Tfn molecules to the BEE (Figure 2E); more basolateral focal planes are not shown since they to not show significant levels of FRET and selected ROIs (data not shown). Inevitably, there is some overlap between ARE and CE (Figure 2A- B vs. C-D), which can actually be separated by visual inspection as described previously 4, the ARE being more central and the CE more peripheral. In Figure 2 (left panel), the E% vs. acceptor intensity levels charts show slightly negative slope values of the linear regression (s-values), as indicated by the trend lines. These results suggest that E% is largely independent of the acceptor most probably due to the clustering behavior of receptor-ligand complexes during endocytic trafficking InII C II- n 0 FF 0 IflIfl III FH 25-0 InnIn InIn In 25_m 0 I ApicI A BaoI IS FRET efficiency (E%) Figure 3. Distribution of E% by focal plane. Each panel represents a focal plane starting at the apical region and proceeding towards the basolateral area of the cells. Since the number of ROIs varies from focal plane to focal plane, absolute frequencies will vary also. In Figure 3, we summarize the frequency of the distribution of ROIs at different height levels in polarized cells. As expected, the majority of data points are at levels -2μm to -6μm, i.e. at the sub-apical to peri-nuclear regions, as explained above. Interestingly, a slight shift to the more basolateral focal planes having a larger proportion in the higher E%s is detected (Figure 3). This subtle difference is further confirmed by executing an ANOVA analysis of the five focal planes (p<0.001). Further experiments are necessary to demonstrate that this very slight E% shift is statistically significant in fixed cells. 4. CONCLUSIONS We have shown that the FRET assay investigating the trafficking of TRF-Tfn complexes in confluent, polarized epithelial MDCK cells can be successfully applied to multiple focal planes. A proprietary version of the PFRET software and the Zeiss 510 Meta multi- track module can generate and analyze a large amount of ROIs in a reasonably short time frame. In the future, we will test whether the slight statistical differences between E% levels at different polarized endocytic compartments are strengthened or weakened in live cells. In summary, we plan to apply this FRET-based assay to live polarized epithelial cells to investigate the factors involved in the regulation of the organization and conformation of membrane-bound receptors during endocytic trafficking. Proc. of SPIE Vol F-5

6 5. REFERENCES [1] Wallrabe, H., Stanley, M., Periasamy, A., Barroso, M. "One- and two-photon fluorescence resonance energy transfer microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes," J Biomed. Opt. 8, (2003). [2] Wallrabe, H., Elangovan, M., Burchard, A., Periasamy, A., Barroso, M. "Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes," Biophys.J 85, (2003). [3] Wallrabe, H., Chen, Y., Periasamy, A., Barroso, M. "Issues in confocal microscopy for quantitative FRET analysis," Microsc. Res. Tech. 69, (2006). [4] Wallrabe, H., Bonamy, G., Periasamy, A., Barroso, M. "Receptor Complexes Cotransported via Polarized Endocytic Pathways Form Clusters with Distinct Organizations," Mol. Cell Biol. 18, (2007). [5] Periasamy, A., Wallrabe, H., Chen, Y., Barroso, M. "Quantitation of Protein-Protein interactions: Confocal FRET Microscopy," Methods Cell Biol. 89, (2008). [6] Elangovan, M., Wallrabe, H., Chen, Y., Day, R.N., Barroso, M. and Periasamy, A. "Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy," Methods 29, (2003). [7] Brown, P.S., Wang, E., Aroeti, B., Chapin, S.J., Mostov, K.E. and Dunn, K.W. "Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling," Traffic 1, (2000). [8] Wang, E., Brown, P.S., Aroeti, B., Chapin, S.J., Mostov, K.E., and Dunn, K.W. "Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome," Traffic 1, (2000). [9] Barroso, M. and Sztul, E. "Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome," J Cell Biol. 124, (1994). [10] Wang, E., Pennington, J.G., Goldenring, J.R., Hunziker, W., Dunn, K.W. "Brefeldin A rapidly disrupts plasma membrane polarity by blocking polar sorting in common endosomes of MDCK cells," J Cell Sci. 114, (2001). [11] Cheng, Y., Zak, O., Aisen, P., Harrison, S.C., Walz, T. "Structure of the human transferrin receptor-transferrin complex," Cell 116, (2004). [12] Richardson, D.R. "Mysteries of the transferrin-transferrin receptor 1 interaction uncovered" Cell 116, (2004). Proc. of SPIE Vol F-6