- Email: [email protected]
Qualitative and quantitative analysis of endocytic recycling
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Qualitative and quantitative analysis of endocytic recycling James B. Reineke, Shuwei Xie, Naava Naslavsky, Steve Caplan1 Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA 1
Corresponding author: E-mail: [email protected]
CHAPTER OUTLINE Introduction ................................................................................................................ 2 1. Materials............................................................................................................... 4 1.1 Major Equipment .................................................................................... 4 1.2 Small Equipment .................................................................................... 4 1.3 Cells ...................................................................................................... 4 1.4 Reagents ................................................................................................ 5 1.5 Buffers................................................................................................... 5 2. Qualitative Assessment of Endocytic Recycling ........................................................ 5 2.1 Experimental Strategies ........................................................................... 5 2.1.1 Tf and EGF treatment ........................................................................... 6 2.1.2 Immunfluorescence.............................................................................. 6 2.1.3 Imaging................................................................................................ 9 2.1.4 Postimage processing ......................................................................... 10 2.1.5 Remarks ............................................................................................ 10 3. Quantitative Assessment of Endocytic Recycling .................................................... 11 3.1 Experimental Strategies ......................................................................... 11 3.1.1 Flow cytometry analysis of fluorescently labeled Tf .............................. 13 3.1.2 Remarks ............................................................................................ 14 4. Quantification of MHC I Antibody-Receptor Complex Recycling Using Immunofluorescence Microscopy and ImageJ ......................................................... 14 4.1 Remarks............................................................................................... 15 Acknowledgments ..................................................................................................... 16 References ............................................................................................................... 16
Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.04.002 © 2015 Elsevier Inc. All rights reserved.
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Abstract Endocytosis, which encompasses the internalization and sorting of plasma membrane (PM) lipids and proteins to distinct membrane-bound intracellular compartments, is a highly regulated and fundamental cellular process by which eukaryotic cells dynamically regulate their PM composition. Indeed, endocytosis is implicated in crucial cellular processes that include proliferation, migration, and cell division as well as maintenance of tissue homeostasis such as apical-basal polarity. Once PM constituents have been taken up into the cell, either via clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE), they typically have two fates: degradation through the late-endosomal/ lysosomal pathway or returning to the PM via endocytic recycling pathways. In this review, we will detail experimental procedures that allow for both qualitative and quantitative assessment of endocytic recycling of transmembrane proteins internalized by CDE and CIE, using the HeLa cervical cancer cell line as a model system.
INTRODUCTION To respond to extracellular cues, cells must dynamically regulate the localization of transmembrane receptors to the plasma membrane (PM). The myriad of endocytic pathways is one crucial way by which cells can control the cellular response to extracellular signal input. For example, the internalization of receptor tyrosine kinases is a means to attenuate their signaling pathways (Palfy, Remenyi, & Korcsmaros, 2012). Alternatively, internalization of adhesion receptors, such as b1-integrins at the cell rear, and their subsequent recycling to the cell front is required for polarized migration through the extracellular matrix (Caswell, Vadrevu, & Norman, 2009). Clathrin-dependent endocytosis (CDE) commences when receptors at the PM cluster into clathrin-coated pitsdoften triggered by ligand bindingdand are internalized into membrane-bound vesicles (Figure 1). The process of internalization is an active one and relies upon adaptor proteins that recognize amino acid motifs on the cytoplasmic tails of transmembrane receptor (Kirchhausen, Bonifacino, & Riezman, 1997). While a detailed explanation of internalization pathways is beyond the scope of this methods review, it is important to note that in addition to CDE, transmembrane proteins can also be internalized via membrane invaginations that are devoid of clathrin (clathrin-independent endocytosis, CIE; Figure 1) (Sandvig, Pust, Skotland, & van Deurs, 2011). Regardless of the initial mechanism used to internalize these receptors, most vesicles containing internalized proteins arrive at a common sorting station known as the early/sorting endosome (SE; Figure 1) (Huotari & Helenius, 2011; Jovic, Sharma, Rahajeng, & Caplan, 2010), although some receptors such as CD144 and CD98 bypass the SE (Eyster et al., 2009). The SE is a complex sorting structure and the mechanisms by which receptors are trafficked through this organelle have not been fully defined. However, cargos that sort to tubular-vesicular regions typically undergo recycling, whereas cargos that undergo post-translational modifications (such as ubiquitination) are often slated for degradation via the late-endosomal/ lysosomal (LE/Lys; Figure 1) pathway. Recycling receptors may either be recycled
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Introduction
Clathrin-dependent Endocytosis (CDE)
Clathrin-independent Endocytosis (CIE)
Endocytic Recycling Compartment (ERC) Early /Sorting Endosome (SE)
Lysosome (Lys)
N Late Endosome (LE)
FIGURE 1 Schematic diagram depicting the endocytic pathways. Receptors are internalized either through clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE) into vesicles that ultimately fuse with early/sorting endosomes (SE). Cargo may be transported to late endosomes (LE) and lysosomes (Lys) for degradation, or it may be recycled back to the plasma membrane directly from SE or indirectly via a perinuclear endocytic recycling compartment (ERC).
directly back to the PM via fast recycling or trafficked indirectly to the PM via a perinuclear/pericentrosomal endocytic recycling compartment (ERC; Figure 1). Due to the importance of endocytic trafficking, there are many proteins that regulate the intracellular trafficking of cargos. Rab proteins comprise a family of more than 60 small Ras-related GTP-binding proteins that control endocytic transport steps and localize to endocytic organelles (Pfeffer & Aivazian, 2004). In their GDP-bound form, Rabs are cytosolic and typically inactive, whereas active, GTPbound Rabs are usually associated with endocytic membranes. Active Rabs recruit effector proteins that regulate lipid content, membrane fusion/fission, and transport along microtubules or microfilaments (Pfeffer & Aivazian, 2004). Given the differential localization of each Rab protein (and their effectors), they serve as convenient markers of the endosomal system in immunofluorescence studies. Among other key regulators of endocytic transport are the C-terminal Eps15 homology domain (EHD) proteins, a family of four highly homologous membrane-associated ATPases. EHD1e4 and their binding partners regulate
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membrane tubulation and vesiculation and are required for the efficient transport of cargo proteins between endocytic compartments (Naslavsky & Caplan, 2011). EHD1 and EHD3 are both recruited to endocytic membranes through their interactions with molecule interacting with CasL-like1 (MICAL-L1). MICAL-L1 and the EHDs localize to a unique array of tubular recycling endosomes (TRE) that span a region from the cell periphery to the perinuclear ERC (Cai et al., 2013; Giridharan, Cai, Vitale, Naslavsky, & Caplan, 2013). It is hypothesized that the recruitment of EHD1 to TREs by MICAL-L1 is required for the efficient recycling of cargo back to the PM. Now that we have briefly introduced the endocytic pathway and some of the key regulatory proteins, we will focus on detailing methods that can be used to qualitatively and quantitatively measure endocytic recycling in human cells (HeLa cells). To this aim, we will show how commercially available fluorescently labeled ligands, such as transferrin (Tf) or epidermal growth factor (EGF), can be used as models to assess the functionality of the endocytic pathway for CDE cargos. In addition, we will describe how CIE can be monitored with antibodies directed against the extracellular domains of receptors such as MHC I. It is our goal to provide protocols that will be useful for researchers new to the realm of membrane trafficking.
1. MATERIALS 1.1 MAJOR EQUIPMENT Confocal or epifluorescence microscope Flow cytometer Mammalian cell culture incubator Laminar flow hood (Biosafety Level 2) 37 C incubator with CO2
1.2 SMALL EQUIPMENT Sterilized 12 mm circular glass coverslips of thickness #1.5 (12-545-81, Fisher Scientific) Double frosted microscope slides (12-552-5, Fisher Scientific) Forceps Cell culture dishes (Corning Life Sciences-Fisher Scientific) Parafilm 12-well dishes (for immunofluorescence) 15 mL Falcon tubes 1.5 mL microfuge tubes
1.3 CELLS Human cervical adenocarcinoma cells (HeLa, ATCC CCL-2)
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2. Qualitative assessment of endocytic recycling
1.4 REAGENTS DMEM Fetal bovine serum Glutamine Penicillin/streptomycin Bovine serum albumin (BSA, 5470, Sigma) Antibodies (Table 1) Formaldehyde 37% (F8775, Sigma) Saponin (47036, Sigma) DAPI (D1306, Invitrogen) Mounting media (FluoromountG, 0100-01 SouthernBiotech) Nail polish Microscope lens oil Cell Stripper nonenzymatic cell dissociation solution (25-056-CI, Corning) FACS tubes 12*75 mm polystyrene with clear cap (14-956-3D, Fisherbrand).
1.5 BUFFERS Phosphate-buffered saline (calcium and magnesium-free PBS): 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4 Stripping buffer: 0.5 M NaCl, 0.5% acetic acid, pH 3.0 Table 1 Antibodies and Ligands used for Endocytic Recycling Assays Antibody (or Labeled Ligand)
Company (Catalog Number)
Dilution
Transferrin-Alexa Fluor 488 Epidermal growth factor-Alexa Fluor 555 Rabbit anti-EEA1-marker of sorting endosome Rabbit anti-Rab11-marker of recycling endosome Rabbit anti-Rab7-marker of late endosome Goat antirabbit and goat antimouse Alexa Fluor conjugated antibodies W6/32 purified antihuman HLAA,B,C (anti-MHC I antibody)
Invitrogen (T13342) Invitrogen (E35350)
10 mg/mL 100 ng/mL
Cell Signaling Technology (3288)
1:100
Cell Signaling Technology (5589)
1:100
Cell Signaling Technology (9367)
1:100
Invitrogen
1:500
Leinco Technologies, Inc. (H199)
1:200
2. QUALITATIVE ASSESSMENT OF ENDOCYTIC RECYCLING 2.1 EXPERIMENTAL STRATEGIES Qualitative assessments of endocytic recycling have one significant advantage over most quantitative methods (including flow cytometry discussed later and
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biotinylation of cell-surface receptors): these microscopic analyses enable researchers to definitively identify the specific locations of internalized cargo as it traverses the endocytic pathways. Often combining quantitative and qualitative approaches provides the most optimal information. For example, if a select cell treatment of protein knockdown causes a delay in endocytic recycling (measured first quantitatively), many researchers might be interested in understanding at what stage of the endocytic pathway the receptor is delayed (or where it accumulates). Such studies require qualitative recycling assays that when combined with markers for the endocytic pathways can easily provide information on the subcellular localization of the internalized receptor. In this section, we will describe experimental strategies to monitor fluorescently labeled ligands such as Tf or EGF through the endocytic pathway using immunofluorescence microscopy. Both ligands induce ligand-dependent endocytosis when they bind to their cognate receptors at the PM and are convenient surrogate markers for their respective receptors. While both transferrin receptor (TfR) and EGFR are internalized via CDE, they follow distinct endocytic itineraries that are well documented in the literature. Under steady-state conditions, the majority of Tf/TfR complexes are recycled back to the PM via the slow recycling pathway (Figure 1). In contrast, EGF-binding leads to ubiquitination of EGFR, which causes EGFR to be sorted into the LE/Lys pathway and to undergo degradation (Figure 1). Thus, fluorescently labeled Tf and EGF can be used to monitor the recycling and degradation pathways, respectively (Maxfield & McGraw, 2004).
2.1.1 Tf and EGF treatment 1. HeLa cells should be passaged using standard techniques and plated 24 h prior to the experiment on 35-mm dishes containing round coverslips. Cells should be approximately 50e75% confluent. 2. The cells should be washed three times with sterile PBS or serum-free DMEM and then serum-starved in DMEM (containing 0.1% BSA) at 37 C for 30 min (Tf) or 120 min (EGF). 3. The Tf and/or EGF should be diluted in DMEM (containing 0.1% BSA; see Table 1). Enough solution should be prepared so that each coverslip can be inverted onto w50 mL of solution that has been spotted onto a sheet of parafilm inside a humidity chamber (see Figure 2). 4. Using forceps, transfer coverslips from 35 mm dish and gently lay them cell side down on top of the 50 mL ligand-containing solution. 5. Quickly place the humidity chamber in a 37 C incubator and follow the experimental flow chart shown in Figure 3 to monitor cargo through the various stages within the endocytic pathways.
2.1.2 Immunfluorescence 1. At each time point outlined in Figure 3, forceps should be used to quickly but carefully transfer each coverslip cell side up into a single well of a 12-well plate that already contains 1 mL of PBS.
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2. Qualitative assessment of endocytic recycling
Humidity chamber for immunofluorescence staining Bottom
Parafilm
Ab. in staining solution
Top (cover)
Glass coverslip
Moist filter paper attached to cover
FIGURE 2 Schematic illustration of a humidity chamber. The moist filter paper attached to the cover of the plate on which the glass coverslips are incubated with the antibody solution ensures that the coverslip and cells do not dry out.
2. Cells should then be fixed in 3.7% formaldehyde in PBS for 10 min at room temperature (can be done in the same 12-well plate). 3. The coverslips should be washed three times with PBS, gently removing the fluid by aspiration, and pipetting in 1e2 mL PBS. 4. If only visualizing cargo (and not staining for endocytic markers), proceed to Step 12. 5. Block and permeabilize cells in PBS containing 3% BSA and 0.2% w/v saponin (Abdil solution) for 30 min at room temperature. 6. Dilute antibodies in Abdil solution. To save on reagents, place cells onto a drop of solution inside a humidity chamber. 7. Place cells cell side down on top of the drop containing the primary antibody solution for 1 h at room temperature or overnight at 4 C in a humidity chamber. 8. Carefully transfer coverslips back to a 12-well dish and wash three times with PBS. 9. Dilute the appropriate Alexa Fluor-conjugated secondary antibody in Abdil solution. If visualizing Tf-488 and endocytic organelles, use anti-rabbit or anti-mouse 568. If visualizing EGF-555, use anti-rabbit or antimouse 488.
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Workflow and expected results for Tf-568 and EGF- 488 pulse-chase Pulse cells for 7 minutes with labeled ligand(s) Wash cells three with PBS ee times ti
Pulse only: Fix to visualize both cargos localized to SE with Tf localized to the tubulo-vesicular compartment and EGF localized to the vacuolar compartment.
Pulse + chase: Incubate cells in complete media containing unlabeled ligand (DMEM+FBS) and chase for 20 minutes to follow trafficking into the ERC (Tf) or late endosomes (EGF), or either 45 minutes (Tf) or 120 minutes (EGF) to visualize recycling or degradation, respectively.
Expected results 7 min. pulse only
20 min. pulse-chase 120 or 45 min. pulse-chase 120’
EGF-488
45’
Tf-568
Merge (inset)
FIGURE 3 Schematic workflow for qualitative monitoring of internalization and recycling of transferrin (Tf) and epidermal growth factor (EGF) and their respective receptors. Anticipated results are depicted in the micrographs showing Tf and EGF internalized for 7 min, and then “chased” for 20 min (both cargos), 45 min (Tf), or 120 min (EGF).
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2. Qualitative assessment of endocytic recycling
10. Place the coverslips onto drops containing the secondary antibody cell side down and incubate for 1 h at room temperature. 11. Wash cells three times with PBS in a 12-well dish. 12. If desired, counterstain with DAPI to label the nuclei (follow the manufacturer’s recommendations). 13. The cells should be rinsed once with ddH2O and the coverslips mounted cell side down onto a drop of FluormountG (w10e20 mL) that is placed on microscope slides. Allow coverslips to air dry for w1 h at room temperature. 14. Seal the coverslips with clear nail polish and visualize by confocal or epifluorescence microscopy. Once the nail polish has dried, gently clean the coverslip with a damp Kimwipe.
2.1.3 Imaging 1. Essentially, any epifluorescence or confocal microscope equipped with a camera can be used. We will describe the imaging process with an inverted Zeiss LSM5 Pascal confocal microscope. 2. Place a small drop of oil on the coverslip, and use a wide-excitation mercury bulb and appropriate filters. Use the eyepiece to visualize the cells. Internalized Tf and EGF will appear as small puncta within the cells. 3. To image the internalized cargo, switch to the confocal mode and use the appropriate laser and filter combinations to scan. For example, a 543 nm laser will activate Alexa Fluor 568, and orange-red light and photons can be collected with a long-pass 560 nm filter (collects all light above 560 nm) or a 560e620 nm band-pass filter. Fast scans with the lowest laser intensity possible should be used initially to prevent photobleaching during the focusing and calibration stage. Typically, Alexa Fluor dyes are quite resistant to photobleaching under most imaging conditions. 4. Set the gain so that the pixels are slightly oversaturated. Since a small amount of photobleaching will occur when obtaining averaged and higher resolution final images, the oversaturation ensures that the signal will be of sufficient intensity. 5. The gain level and all other parameters must be recorded and kept at identical settings throughout the imaging process to ensure that comparisons are valid. Note: many microscopy systems keep computerized records of the settings and microscopy software programs typically have a “reuse” button to set the parameters back to the same values if changed. 6. If the signal is dim, which may occur with short pulses of a labeled ligand, one option is to open the pinhole on a confocal microscope to collect fluorescence from a greater-sized slice. Typical pinhole settings are at one Airy unit, which usually correlates with 0.4e0.7 mm. If pinhole values are altered, be sure to apply this setting to all subsequent images. Other options include increasing the laser power, increasing the gain, or decreasing the number of pixels in the image.
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7. If a sample is co-labeled for endosomes, it is convenient to find the plane of focus for the endosomal marker rather than the weaker internalized ligand (to minimize photobleaching). 8. Because endosomes are localized at different localizations within the cell (SE are in the periphery and close to the PM while LE and the ERC are found deeper in the cell near the nucleus) it may be necessary to perform z-sections. We typically use 0.5e1.0 mm size steps. 9. Blue fluorescence light (if the cells are labeled with DAPI), green, and red fluorescence are best acquired sequentially. This reduces the possibility of cross-activation and/or bleed-through artifacts, and the only disadvantage for fixed cells is a slightly longer image acquisition process. Images are typically obtained with pixel dimensions of 1024 1024, but for weaker signals, 512 512 may be helpful. 10. Save images and export them as 16-bit tiffs. If z-sections are used, export individual sections or use 3D processing to make maximal projection XY images.
2.1.4 Postimage processing 1. Import 16-bit tiff file into ImageJ, Photoshop CS or alternate program. 2. It is vital that the investigator takes appropriate caution when manipulating images in Photoshop. Very minimal image manipulation should be done. It is best practice to collect the optimal image during acquisition. If adjustments are necessary (brightness/contrast), the linear “levels” function in Photoshop may be used, with adjustments made for an entire micrograph. 3. If the investigator intends to compare different time points or treatments, it is essential that both image acquisition and image processing be maintained identically for all image samples. 4. For publication, most journals require images of at least 300 dpi with pixel dimensions of 512 512 or 1024 1025. Pixel dimensions can be decreased in size, but not increased beyond the size obtained during acquisition (for example, images obtained with 512 512 pixels cannot be changed to 1024 1024 pixels during processing).
2.1.5 Remarks The above-mentioned protocols may be used to demonstrate the remarkable degree of regulation within the endocytic pathways. While TfR and EGFR are both internalized via CDE, their endocytic fates are vastly different under normal conditions. However, perturbations of key endocytic regulatory proteins disrupt the trafficking of the receptors. For instance, defects in the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway slow EGFR degradation and result in prolonged growth factor signaling, which can lead to oncogenesis (Mosesson, Mills, & Yarden, 2008). In addition, siRNA-mediated depletion of EHD1 or EHD3 delays trafficking of cargos by blocking movement out of the ERC or SE, respectively (Naslavsky & Caplan, 2011). Given that large genomewide screens have implicated
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kinases/phosphatases, lipid biosynthetic pathways, and many other proteins in endocytic regulation, we expect that many investigators will find the need to study endocytic trafficking (Collinet et al., 2010; Liberali, Snijder, & Pelkmans, 2014). While the above-mentioned protocols are useful starting points, we wish to point out several caveats that the investigator should be aware of: 1. It is likely that researchers may want to carry out the above-mentioned experiments after treatment with inhibitors or siRNAs to disrupt a certain protein or pathway. Given that siRNA transfection is a relatively long process (48e72 h), the levels of PM receptors may change during this time period and may need to be accounted for. For instance, depletion of certain endocytic proteins can actually decrease PM levels of TfR or EGFR (presumably by affecting the steady-state trafficking of the receptor). 2. The protocols outlined earlier are for fixed cells, thus they only give a snapshot of what occurs inside the cell. Live cell imaging can provide a more accurate assessment of the kinetics of transport from one endocytic organelle to the next. However, this often involves overexpression of fluorescently labeled proteins, which can affect trafficking. 3. Given that endocytic recycling is a fundamental process for all cells, most of the defects caused by siRNA depletion or chemical inhibitors are not complete. For example, EHD1-depletion slows recycling but does not block recycling. Similarly, EHD3-depletion blocks transport of cargo from the SE to the ERC; however, cargo may utilize the fast recycling pathway as a compensatory mechanism. Thus, receptor recycling is only partially inhibited. 4. In this section, we used fluorescently labeled ligands, which cause internalization of the receptor/ligand complex via CDE. However, as will be described later, antibodies against the extracellular domain of receptors can be used to follow the trafficking of receptors (see the MHC class I example in the next section). However, it is not always clear if the antibody mimics the endogenous ligand thereby causing ligand-dependent endocytosis or if the antibody simply labels the receptor and allows for visualization of the constitutive endocytic pathway.
3. QUANTITATIVE ASSESSMENT OF ENDOCYTIC RECYCLING 3.1 EXPERIMENTAL STRATEGIES In this section we will discuss two fluorescence-based experimental strategies to quantitatively assess endocytic recycling. It should be noted that there are a variety of additional methods that may be used to quantify recycling, often based on the labeling of cell-surface receptors by biotinylation or even cell-surface iodination. In this chapter, we have decided to focus on fluorescence-based methods as being potentially the most popular and easiest techniques to apply. The first experimental strategy that we discuss measures the disappearance of labeled ligands such as Tf from within the cell (Figure 4; part 1), whereas the second measures the
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Workflow for measuring TfR or MHC I recycling (1) Pulse cells for 15 minutes with Tf-488 Wash cells three times with PBS Fix to measure the internalized Tf-488 (Tube A)
In Incubate cells in complete DMEM and chase for 30 minutes Fix to measure easu the amount of remaining Tf-488 in the cell after a 30 minute chase (Tube B)
(2) Pulse cells for 30 m minutes with anti-MHC I antibody
= Tf-488 = MHC I/antibody complex
Acid strip for 1 minute
= goat anti-mouse 568 antibody
Wash cells three times with PBS Fix and stain with goat anti-mouse 568 in the presence of saponin to measure the amount of internalized MHC I (Tube A)
Fix and d stain stain with goat anti-mouse 568 in the absence of saponin to measure the amount of internalized MHC I (Tube B)
Incubate cells in In DMEM for 3 hours
complete
Wash cells three e time times with PBS Fix and stain with h goat goa anti-mouse 568 in the absence of saponin to measure the amount of MHC I that has recycled back to plasma membrane (Tube C)
FIGURE 4 Schematic workflow for quantitatively measuring recycling of Tf and its receptor or MHC I by flow cytometry.
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reappearance of internalized antibodyereceptor complexes (such as major histocompatibility complex class I (MHC I) and CD59) at the PM (Figure 4; part 2). Flow cytometry is the method of choice that we describe to measure the disappearance (recycling) of internalized Tf in HeLa cells, and we detail the use of confocal microscopy coupled with post-imaging quantitation via free software programs such as ImageJ to quantify the reappearance of antibodyereceptor complexes on the surface of the PM.
3.1.1 Flow cytometry analysis of fluorescently labeled Tf 1. Passage and plate HeLa cells on a 100-mm dish 24 h prior to the experiment, as described earlier. To have sufficient cells for treatment, they should be approximately 60e80% confluent. 2. Serum-starve cells as described in the previous section. 3. Incubate the cells in 4 mL of Cell Stripper (Mediatech Inc.) for 1 min on ice. Pipet up and down to detach cells from the bottom of the dish. Collect the cells in 15 mL Falcon tubes and pellet them by centrifugation at 1300 rpm at 4 C for 5 min. 4. Carefully aspirate the supernatant without disrupting the cell pellet and wash the cells three times in 1 mL of cold PBS, pelleting the cells by centrifugation as described earlier after each wash. 5. After the third PBS wash, resuspend cells in 1 mL of PBS and divide the cells into three aliquots (w330 mL) in 1.5 mL Eppendorf tubes. Tube A will serve as the Tf pulse treatment (see later), Tube B will serve as the Tf chase treatment, and Tube C will serve as the preuptake control, which accounts for autofluorescence artifacts and background noise. 6. For Tube C, add 330 mL of 7.4% PBSeformaldehyde (final concentration 3.7%) and fix the cells for 10 min at room temperature. 7. Dilute Tf-488 in DMEM (containing 0.1% BSA; see Table 1) to a concentration of 25 mg/mL. Enough solution should be prepared so that cells from each treatment can be resuspended in 150e300 mL of solution. 8. Resuspend the cells in Tubes A and B in 150 mL of Tf-488 solution. Rotate the tubes in a CO2-regulated incubator at 37 C for 15 min (pulse). 9. Wash cells three times with 1 mL of cold PBS, pelleting cells by centrifugation at 1300 rpm at 4 C for 5 min after each wash. 10. For Tube A, resuspend the pellet in 3.7% paraformaldehyde in PBS and fix for 10 min at room temperature. 11. For Tube B, resuspend the pellet in 1 mL of complete DMEM (containing fetal bovine serum) and rotate at 37 C for 45 min (chase). Wash and fix cells as described in Steps 9 and 10. 12. Transfer fixed cells from Tubes A, B, and C into flow cytometry-compatible tubes and measure 488 fluorescence by flow cytometry. 13. Assess the median fluorescence intensity (MFI, arbitrary units) of different samples by flow cytometry. Use Tube C to “gate” (set the fluorescence
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threshold) for Tubes A and B. The MFI of Tube A equals the total amount of internalized Tf while Tube B represents the amount of Tf remaining in the cells after the chase (nonrecycled). The recycling rate is calculated by the following equation (Eqn (1)):
ðMFI of chase sampleðTube BÞÞ 100% Recycling rate ¼ 1 ðMFI of pulse only sampleðTube AÞÞ
(1)
3.1.2 Remarks As with all experiments, recycling assays need to be repeated at least three times, and the recycling rates measured in each experiment may be presented in a bar graph with standard deviation and appropriate statistical significance tests.
4. QUANTIFICATION OF MHC I ANTIBODY-RECEPTOR COMPLEX RECYCLING USING IMMUNOFLUORESCENCE MICROSCOPY AND IMAGEJ 1. HeLa cells should be passaged and plated on 35 mm plates containing three coverslips to measure the recycling rate of MHC I. Cells should be approximately 50e75% confluent; higher confluency helps maintain cell viability during the acid stripping of noninternalized antibodyereceptor complexes (see later). 2. W6/32 anti-MHC I antibody should be diluted in complete DMEM. Each coverslip should be inverted onto 50 mL of antibody-containing solution in a humidity chamber (see previous section) for 30 min at 37 C (“pulse”). 3. Transfer the coverslips to a 12-well plate and remove noninternalized MHC I-antibody by incubating the coverslips in 1 mL of acid-stripping buffer (prewarmed to 37 C) for 1 min. Gently agitate the plate to achieve consistent stripping. 4. Wash coverslips three times with 1 mL prewarmed PBS. 5. Fix two of the coverslips, denoted A and B, respectively, in 3.7% formaldehydePBS for 10 min at room temperature (“pulse-only”). Incubate the rest of the coverslips in 2 mL prewarmed complete DMEM media in a 12-well plate for 3 h (chase) at 37 C. Fix this coverslip (C). Alexa-568 goat antimouse antibody staining is performed as described in section “Immunofluorescence,” Steps 9e14. Note that for coverslip A, staining is performed in the Abdil solution containing 0.2% w/v saponin, while the staining of B and C is done without this permeabilizing reagent. Coverslips AeC measure the following: A. the total internalized MHC I after pulse; B. the basal surface level of MHC I remaining after the pulse and stripping; and C. the level of MHC I that is recycled back to PM after the chase.
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4. Quantification of MHC i antibody-receptor complex recycling
6. Image acquisition is performed as described in the “Imaging” section. However, to maximize the objectivity and repeatability of this method, additional attention should be paid to parameter settings and the selection of cell fields for imaging. a. Cell fields need to be selected randomly, and the fields imaged should have cells that are evenly spread out without growing on top of each other. b. Avoid selecting fields from the same area of the coverslip. c. Before acquiring images, adjust the value of the detector gain so that there is no oversaturation. Pixel oversaturation appears red in the Range Indicator. d. All parameters including the gain level and cropping/magnification of images should remain unchanged during the image acquisition process. It is also important that no image be manipulated by image editing programs. e. Import 16-bit tiff files into ImageJ (http://rsbweb.nih.gov/ij/links.html). For each image, circle the outline of a cell with the Freehand selections tool. The MFI (arbitrary unit) is assessed by the Measure Tool. Collect the area and mean value for each selected cell; the area and mean fluorescence allow calculation of the total fluorescence (Eqn (2)). The investigator should be cautious about cell selection in the following manner: (1) select cells that are intact, flat, and preferably spread out so that the cell edges are easily recognizable; and (2) to better visualize the cells, DAPI staining of the cell nuclei is recommended. However, for a dual/multicolor image, make sure to split the channels in ImageJ and measure only the channel displaying MHC I staining. 7. Measure at least 60 cells from different fields of each sample and repeat the experiment at least three times. 8. For each independent experiment, calculate the total fluorescence using Eqn (2). Note that the measurement of Area is in pixels. Calculate the average of total fluorescence for each sample. The rate of MHC I recycling is calculated by the following equation (Eqn (3)) and the significance of the different tubes is described in Step 5: Total fluorescence ¼ Area Mean Recycling rate ¼
(2)
Total fluorescence of C Total fluorescence of B 100% (3) Total fluorescence of A
As with all experiments, such recycling assays need to be repeated at least three times, and the recycling rates measured in each experiment may be presented in a bar graph with standard deviation.
4.1 REMARKS These protocols are designed for the measurement of endocytic recycling of different receptors. However, internalized receptors are not only sorted to the recycling pathway. While Tf primarily recycles to the PM, MHC I is internalized via CIE. In
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the latter case, with each round of endocytosis, approximately half of the internalized MHC I molecules are recycled, with the rest sorted to the lysosomal pathway for degradation (Naslavsky, Weigert, & Donaldson, 2003). A decreased recycling rate results from either enhanced intracellular retentions or increased degradation. For example, when the lipid modifier diacylglycerol kinase a is depleted from cells, the MHC I recycling rate is dramatically decreased (Xie, Naslavsky, & Caplan, 2014). The shunting of receptors from recycling to degradation pathways, or vice versa, is not uncommon. Depletion of Rabenosyn-5 results in missorting and extensive degradation of TfR (Navaroli et al., 2012). In addition, the nontoxic domain 4 of the bacterial toxin intermedilysin (rILYd4) induces rapid CD59 degradation in lysosomes, whereas in the absence of the toxin CD59 is primarily recycled (Cai et al., 2014). Such cases show the importance of combining recycling and degradation assays to monitor and understand the subcellular itineraries of internalized proteins.
ACKNOWLEDGMENTS This work was supported by the National Institutes of Health Grants R01GM087455, R01GM074876, and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant number 5P30GM106397.
REFERENCES Cai, B., Giridharan, S. S., Zhang, J., Saxena, S., Bahl, K., Schmidt, J. A., et al. (2013). Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes. Journal of Biological Chemistry, 288(42), 30172e30180. Cai, B., Xie, S., Liu, F., Simone, L. C., Caplan, S., Qin, X., et al. (2014). Rapid degradation of the complement regulator, CD59, by a novel inhibitor. Journal of Biological Chemistry, 289(17), 12109e12125. Caswell, P. T., Vadrevu, S., & Norman, J. C. (2009). Integrins: masters and slaves of endocytic transport. Nature Reviews Molecular Cell Biology, 10(12), 843e853. Collinet, C., Stoter, M., Bradshaw, C. R., Samusik, N., Rink, J. C., Kenski, D., et al. (2010). Systems survey of endocytosis by multiparametric image analysis. Nature, 464(7286), 243e249. Eyster, C. A., Higginson, J. D., Huebner, R., Porat-Shliom, N., Weigert, R., Wu, W. W., et al. (2009). Discovery of new cargo proteins that enter cells through clathrin-independent endocytosis. Traffic, 10(5), 590e599. Giridharan, S. S., Cai, B., Vitale, N., Naslavsky, N., & Caplan, S. (2013). Cooperation of MICAL-L1, syndapin2, and phosphatidic acid in tubular recycling endosome biogenesis. Molecular Biology of the Cell, 24(11), 1776e1790. S1771e1715. Huotari, J., & Helenius, A. (2011). Endosome maturation. EMBO Journal, 30(17), 3481e3500. Jovic, M., Sharma, M., Rahajeng, J., & Caplan, S. (2010). The early endosome: a busy sorting station for proteins at the crossroads. Histology and Histopathology, 25(1), 99e112.
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Kirchhausen, T., Bonifacino, J. S., & Riezman, H. (1997). Linking cargo to vesicle formation: receptor tail interactions with coat proteins. Current Opinion in Cell Biology, 9(4), 488e495. Liberali, P., Snijder, B., & Pelkmans, L. (2014). A hierarchical map of regulatory genetic interactions in membrane trafficking. Cell, 157(6), 1473e1487. Maxfield, F. R., & McGraw, T. E. (2004). Endocytic recycling. Nature Reviews in Molecular Cell Biology, 5(2), 121e132. Mosesson, Y., Mills, G. B., & Yarden, Y. (2008). Derailed endocytosis: an emerging feature of cancer. Nature Reviews Cancer, 8(11), 835e850. Naslavsky, N., & Caplan, S. (2011). EHD proteins: key conductors of endocytic transport. Trends in Cell Biology, 21(2), 122e131. Naslavsky, N., Weigert, R., & Donaldson, J. G. (2003). Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides. Molecular Biology of the Cell, 14(2), 417e431. Navaroli, D. M., Bellve, K. D., Stanley, C., Lifshitz, L. M., Cardia, J., Lambright, D., et al. (2012). Rabenosyn-5 defines the fate of the transferrin receptor following clathrinmediated endocytosis. Proceedings of the National Academy of Sciences of the United States of America, 109(8), E471eE480. Palfy, M., Remenyi, A., & Korcsmaros, T. (2012). Endosomal crosstalk: meeting points for signaling pathways. Trends in Cell Biology, 22(9), 447e456. Pfeffer, S., & Aivazian, D. (2004). Targeting Rab GTPases to distinct membrane compartments. Nature Reviews in Molecular Cell Biology, 5(11), 886e896. Sandvig, K., Pust, S., Skotland, T., & van Deurs, B. (2011). Clathrin-independent endocytosis: mechanisms and function. Current Opinion in Cell Biology, 23(4), 413e420. Xie, S., Naslavsky, N., & Caplan, S. (2014). Diacylglycerol kinase alpha regulates tubular recycling endosome biogenesis and major histocompatibility complex class I recycling. Journal of Biological Chemistry, 289(46), 31914e31926.
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Qualitative and quantitative analysis of endocytic recycling James B. Reineke, Shuwei Xie, Naava Naslavsky, Steve Caplan1 Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA 1
Corresponding author: E-mail: [email protected]
CHAPTER OUTLINE Introduction ................................................................................................................ 2 1. Materials............................................................................................................... 4 1.1 Major Equipment .................................................................................... 4 1.2 Small Equipment .................................................................................... 4 1.3 Cells ...................................................................................................... 4 1.4 Reagents ................................................................................................ 5 1.5 Buffers................................................................................................... 5 2. Qualitative Assessment of Endocytic Recycling ........................................................ 5 2.1 Experimental Strategies ........................................................................... 5 2.1.1 Tf and EGF treatment ........................................................................... 6 2.1.2 Immunfluorescence.............................................................................. 6 2.1.3 Imaging................................................................................................ 9 2.1.4 Postimage processing ......................................................................... 10 2.1.5 Remarks ............................................................................................ 10 3. Quantitative Assessment of Endocytic Recycling .................................................... 11 3.1 Experimental Strategies ......................................................................... 11 3.1.1 Flow cytometry analysis of fluorescently labeled Tf .............................. 13 3.1.2 Remarks ............................................................................................ 14 4. Quantification of MHC I Antibody-Receptor Complex Recycling Using Immunofluorescence Microscopy and ImageJ ......................................................... 14 4.1 Remarks............................................................................................... 15 Acknowledgments ..................................................................................................... 16 References ............................................................................................................... 16
Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.04.002 © 2015 Elsevier Inc. All rights reserved.
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Abstract Endocytosis, which encompasses the internalization and sorting of plasma membrane (PM) lipids and proteins to distinct membrane-bound intracellular compartments, is a highly regulated and fundamental cellular process by which eukaryotic cells dynamically regulate their PM composition. Indeed, endocytosis is implicated in crucial cellular processes that include proliferation, migration, and cell division as well as maintenance of tissue homeostasis such as apical-basal polarity. Once PM constituents have been taken up into the cell, either via clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE), they typically have two fates: degradation through the late-endosomal/ lysosomal pathway or returning to the PM via endocytic recycling pathways. In this review, we will detail experimental procedures that allow for both qualitative and quantitative assessment of endocytic recycling of transmembrane proteins internalized by CDE and CIE, using the HeLa cervical cancer cell line as a model system.
INTRODUCTION To respond to extracellular cues, cells must dynamically regulate the localization of transmembrane receptors to the plasma membrane (PM). The myriad of endocytic pathways is one crucial way by which cells can control the cellular response to extracellular signal input. For example, the internalization of receptor tyrosine kinases is a means to attenuate their signaling pathways (Palfy, Remenyi, & Korcsmaros, 2012). Alternatively, internalization of adhesion receptors, such as b1-integrins at the cell rear, and their subsequent recycling to the cell front is required for polarized migration through the extracellular matrix (Caswell, Vadrevu, & Norman, 2009). Clathrin-dependent endocytosis (CDE) commences when receptors at the PM cluster into clathrin-coated pitsdoften triggered by ligand bindingdand are internalized into membrane-bound vesicles (Figure 1). The process of internalization is an active one and relies upon adaptor proteins that recognize amino acid motifs on the cytoplasmic tails of transmembrane receptor (Kirchhausen, Bonifacino, & Riezman, 1997). While a detailed explanation of internalization pathways is beyond the scope of this methods review, it is important to note that in addition to CDE, transmembrane proteins can also be internalized via membrane invaginations that are devoid of clathrin (clathrin-independent endocytosis, CIE; Figure 1) (Sandvig, Pust, Skotland, & van Deurs, 2011). Regardless of the initial mechanism used to internalize these receptors, most vesicles containing internalized proteins arrive at a common sorting station known as the early/sorting endosome (SE; Figure 1) (Huotari & Helenius, 2011; Jovic, Sharma, Rahajeng, & Caplan, 2010), although some receptors such as CD144 and CD98 bypass the SE (Eyster et al., 2009). The SE is a complex sorting structure and the mechanisms by which receptors are trafficked through this organelle have not been fully defined. However, cargos that sort to tubular-vesicular regions typically undergo recycling, whereas cargos that undergo post-translational modifications (such as ubiquitination) are often slated for degradation via the late-endosomal/ lysosomal (LE/Lys; Figure 1) pathway. Recycling receptors may either be recycled
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Clathrin-dependent Endocytosis (CDE)
Clathrin-independent Endocytosis (CIE)
Endocytic Recycling Compartment (ERC) Early /Sorting Endosome (SE)
Lysosome (Lys)
N Late Endosome (LE)
FIGURE 1 Schematic diagram depicting the endocytic pathways. Receptors are internalized either through clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE) into vesicles that ultimately fuse with early/sorting endosomes (SE). Cargo may be transported to late endosomes (LE) and lysosomes (Lys) for degradation, or it may be recycled back to the plasma membrane directly from SE or indirectly via a perinuclear endocytic recycling compartment (ERC).
directly back to the PM via fast recycling or trafficked indirectly to the PM via a perinuclear/pericentrosomal endocytic recycling compartment (ERC; Figure 1). Due to the importance of endocytic trafficking, there are many proteins that regulate the intracellular trafficking of cargos. Rab proteins comprise a family of more than 60 small Ras-related GTP-binding proteins that control endocytic transport steps and localize to endocytic organelles (Pfeffer & Aivazian, 2004). In their GDP-bound form, Rabs are cytosolic and typically inactive, whereas active, GTPbound Rabs are usually associated with endocytic membranes. Active Rabs recruit effector proteins that regulate lipid content, membrane fusion/fission, and transport along microtubules or microfilaments (Pfeffer & Aivazian, 2004). Given the differential localization of each Rab protein (and their effectors), they serve as convenient markers of the endosomal system in immunofluorescence studies. Among other key regulators of endocytic transport are the C-terminal Eps15 homology domain (EHD) proteins, a family of four highly homologous membrane-associated ATPases. EHD1e4 and their binding partners regulate
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membrane tubulation and vesiculation and are required for the efficient transport of cargo proteins between endocytic compartments (Naslavsky & Caplan, 2011). EHD1 and EHD3 are both recruited to endocytic membranes through their interactions with molecule interacting with CasL-like1 (MICAL-L1). MICAL-L1 and the EHDs localize to a unique array of tubular recycling endosomes (TRE) that span a region from the cell periphery to the perinuclear ERC (Cai et al., 2013; Giridharan, Cai, Vitale, Naslavsky, & Caplan, 2013). It is hypothesized that the recruitment of EHD1 to TREs by MICAL-L1 is required for the efficient recycling of cargo back to the PM. Now that we have briefly introduced the endocytic pathway and some of the key regulatory proteins, we will focus on detailing methods that can be used to qualitatively and quantitatively measure endocytic recycling in human cells (HeLa cells). To this aim, we will show how commercially available fluorescently labeled ligands, such as transferrin (Tf) or epidermal growth factor (EGF), can be used as models to assess the functionality of the endocytic pathway for CDE cargos. In addition, we will describe how CIE can be monitored with antibodies directed against the extracellular domains of receptors such as MHC I. It is our goal to provide protocols that will be useful for researchers new to the realm of membrane trafficking.
1. MATERIALS 1.1 MAJOR EQUIPMENT Confocal or epifluorescence microscope Flow cytometer Mammalian cell culture incubator Laminar flow hood (Biosafety Level 2) 37 C incubator with CO2
1.2 SMALL EQUIPMENT Sterilized 12 mm circular glass coverslips of thickness #1.5 (12-545-81, Fisher Scientific) Double frosted microscope slides (12-552-5, Fisher Scientific) Forceps Cell culture dishes (Corning Life Sciences-Fisher Scientific) Parafilm 12-well dishes (for immunofluorescence) 15 mL Falcon tubes 1.5 mL microfuge tubes
1.3 CELLS Human cervical adenocarcinoma cells (HeLa, ATCC CCL-2)
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1.4 REAGENTS DMEM Fetal bovine serum Glutamine Penicillin/streptomycin Bovine serum albumin (BSA, 5470, Sigma) Antibodies (Table 1) Formaldehyde 37% (F8775, Sigma) Saponin (47036, Sigma) DAPI (D1306, Invitrogen) Mounting media (FluoromountG, 0100-01 SouthernBiotech) Nail polish Microscope lens oil Cell Stripper nonenzymatic cell dissociation solution (25-056-CI, Corning) FACS tubes 12*75 mm polystyrene with clear cap (14-956-3D, Fisherbrand).
1.5 BUFFERS Phosphate-buffered saline (calcium and magnesium-free PBS): 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4 Stripping buffer: 0.5 M NaCl, 0.5% acetic acid, pH 3.0 Table 1 Antibodies and Ligands used for Endocytic Recycling Assays Antibody (or Labeled Ligand)
Company (Catalog Number)
Dilution
Transferrin-Alexa Fluor 488 Epidermal growth factor-Alexa Fluor 555 Rabbit anti-EEA1-marker of sorting endosome Rabbit anti-Rab11-marker of recycling endosome Rabbit anti-Rab7-marker of late endosome Goat antirabbit and goat antimouse Alexa Fluor conjugated antibodies W6/32 purified antihuman HLAA,B,C (anti-MHC I antibody)
Invitrogen (T13342) Invitrogen (E35350)
10 mg/mL 100 ng/mL
Cell Signaling Technology (3288)
1:100
Cell Signaling Technology (5589)
1:100
Cell Signaling Technology (9367)
1:100
Invitrogen
1:500
Leinco Technologies, Inc. (H199)
1:200
2. QUALITATIVE ASSESSMENT OF ENDOCYTIC RECYCLING 2.1 EXPERIMENTAL STRATEGIES Qualitative assessments of endocytic recycling have one significant advantage over most quantitative methods (including flow cytometry discussed later and
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biotinylation of cell-surface receptors): these microscopic analyses enable researchers to definitively identify the specific locations of internalized cargo as it traverses the endocytic pathways. Often combining quantitative and qualitative approaches provides the most optimal information. For example, if a select cell treatment of protein knockdown causes a delay in endocytic recycling (measured first quantitatively), many researchers might be interested in understanding at what stage of the endocytic pathway the receptor is delayed (or where it accumulates). Such studies require qualitative recycling assays that when combined with markers for the endocytic pathways can easily provide information on the subcellular localization of the internalized receptor. In this section, we will describe experimental strategies to monitor fluorescently labeled ligands such as Tf or EGF through the endocytic pathway using immunofluorescence microscopy. Both ligands induce ligand-dependent endocytosis when they bind to their cognate receptors at the PM and are convenient surrogate markers for their respective receptors. While both transferrin receptor (TfR) and EGFR are internalized via CDE, they follow distinct endocytic itineraries that are well documented in the literature. Under steady-state conditions, the majority of Tf/TfR complexes are recycled back to the PM via the slow recycling pathway (Figure 1). In contrast, EGF-binding leads to ubiquitination of EGFR, which causes EGFR to be sorted into the LE/Lys pathway and to undergo degradation (Figure 1). Thus, fluorescently labeled Tf and EGF can be used to monitor the recycling and degradation pathways, respectively (Maxfield & McGraw, 2004).
2.1.1 Tf and EGF treatment 1. HeLa cells should be passaged using standard techniques and plated 24 h prior to the experiment on 35-mm dishes containing round coverslips. Cells should be approximately 50e75% confluent. 2. The cells should be washed three times with sterile PBS or serum-free DMEM and then serum-starved in DMEM (containing 0.1% BSA) at 37 C for 30 min (Tf) or 120 min (EGF). 3. The Tf and/or EGF should be diluted in DMEM (containing 0.1% BSA; see Table 1). Enough solution should be prepared so that each coverslip can be inverted onto w50 mL of solution that has been spotted onto a sheet of parafilm inside a humidity chamber (see Figure 2). 4. Using forceps, transfer coverslips from 35 mm dish and gently lay them cell side down on top of the 50 mL ligand-containing solution. 5. Quickly place the humidity chamber in a 37 C incubator and follow the experimental flow chart shown in Figure 3 to monitor cargo through the various stages within the endocytic pathways.
2.1.2 Immunfluorescence 1. At each time point outlined in Figure 3, forceps should be used to quickly but carefully transfer each coverslip cell side up into a single well of a 12-well plate that already contains 1 mL of PBS.
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Humidity chamber for immunofluorescence staining Bottom
Parafilm
Ab. in staining solution
Top (cover)
Glass coverslip
Moist filter paper attached to cover
FIGURE 2 Schematic illustration of a humidity chamber. The moist filter paper attached to the cover of the plate on which the glass coverslips are incubated with the antibody solution ensures that the coverslip and cells do not dry out.
2. Cells should then be fixed in 3.7% formaldehyde in PBS for 10 min at room temperature (can be done in the same 12-well plate). 3. The coverslips should be washed three times with PBS, gently removing the fluid by aspiration, and pipetting in 1e2 mL PBS. 4. If only visualizing cargo (and not staining for endocytic markers), proceed to Step 12. 5. Block and permeabilize cells in PBS containing 3% BSA and 0.2% w/v saponin (Abdil solution) for 30 min at room temperature. 6. Dilute antibodies in Abdil solution. To save on reagents, place cells onto a drop of solution inside a humidity chamber. 7. Place cells cell side down on top of the drop containing the primary antibody solution for 1 h at room temperature or overnight at 4 C in a humidity chamber. 8. Carefully transfer coverslips back to a 12-well dish and wash three times with PBS. 9. Dilute the appropriate Alexa Fluor-conjugated secondary antibody in Abdil solution. If visualizing Tf-488 and endocytic organelles, use anti-rabbit or anti-mouse 568. If visualizing EGF-555, use anti-rabbit or antimouse 488.
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Workflow and expected results for Tf-568 and EGF- 488 pulse-chase Pulse cells for 7 minutes with labeled ligand(s) Wash cells three with PBS ee times ti
Pulse only: Fix to visualize both cargos localized to SE with Tf localized to the tubulo-vesicular compartment and EGF localized to the vacuolar compartment.
Pulse + chase: Incubate cells in complete media containing unlabeled ligand (DMEM+FBS) and chase for 20 minutes to follow trafficking into the ERC (Tf) or late endosomes (EGF), or either 45 minutes (Tf) or 120 minutes (EGF) to visualize recycling or degradation, respectively.
Expected results 7 min. pulse only
20 min. pulse-chase 120 or 45 min. pulse-chase 120’
EGF-488
45’
Tf-568
Merge (inset)
FIGURE 3 Schematic workflow for qualitative monitoring of internalization and recycling of transferrin (Tf) and epidermal growth factor (EGF) and their respective receptors. Anticipated results are depicted in the micrographs showing Tf and EGF internalized for 7 min, and then “chased” for 20 min (both cargos), 45 min (Tf), or 120 min (EGF).
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10. Place the coverslips onto drops containing the secondary antibody cell side down and incubate for 1 h at room temperature. 11. Wash cells three times with PBS in a 12-well dish. 12. If desired, counterstain with DAPI to label the nuclei (follow the manufacturer’s recommendations). 13. The cells should be rinsed once with ddH2O and the coverslips mounted cell side down onto a drop of FluormountG (w10e20 mL) that is placed on microscope slides. Allow coverslips to air dry for w1 h at room temperature. 14. Seal the coverslips with clear nail polish and visualize by confocal or epifluorescence microscopy. Once the nail polish has dried, gently clean the coverslip with a damp Kimwipe.
2.1.3 Imaging 1. Essentially, any epifluorescence or confocal microscope equipped with a camera can be used. We will describe the imaging process with an inverted Zeiss LSM5 Pascal confocal microscope. 2. Place a small drop of oil on the coverslip, and use a wide-excitation mercury bulb and appropriate filters. Use the eyepiece to visualize the cells. Internalized Tf and EGF will appear as small puncta within the cells. 3. To image the internalized cargo, switch to the confocal mode and use the appropriate laser and filter combinations to scan. For example, a 543 nm laser will activate Alexa Fluor 568, and orange-red light and photons can be collected with a long-pass 560 nm filter (collects all light above 560 nm) or a 560e620 nm band-pass filter. Fast scans with the lowest laser intensity possible should be used initially to prevent photobleaching during the focusing and calibration stage. Typically, Alexa Fluor dyes are quite resistant to photobleaching under most imaging conditions. 4. Set the gain so that the pixels are slightly oversaturated. Since a small amount of photobleaching will occur when obtaining averaged and higher resolution final images, the oversaturation ensures that the signal will be of sufficient intensity. 5. The gain level and all other parameters must be recorded and kept at identical settings throughout the imaging process to ensure that comparisons are valid. Note: many microscopy systems keep computerized records of the settings and microscopy software programs typically have a “reuse” button to set the parameters back to the same values if changed. 6. If the signal is dim, which may occur with short pulses of a labeled ligand, one option is to open the pinhole on a confocal microscope to collect fluorescence from a greater-sized slice. Typical pinhole settings are at one Airy unit, which usually correlates with 0.4e0.7 mm. If pinhole values are altered, be sure to apply this setting to all subsequent images. Other options include increasing the laser power, increasing the gain, or decreasing the number of pixels in the image.
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7. If a sample is co-labeled for endosomes, it is convenient to find the plane of focus for the endosomal marker rather than the weaker internalized ligand (to minimize photobleaching). 8. Because endosomes are localized at different localizations within the cell (SE are in the periphery and close to the PM while LE and the ERC are found deeper in the cell near the nucleus) it may be necessary to perform z-sections. We typically use 0.5e1.0 mm size steps. 9. Blue fluorescence light (if the cells are labeled with DAPI), green, and red fluorescence are best acquired sequentially. This reduces the possibility of cross-activation and/or bleed-through artifacts, and the only disadvantage for fixed cells is a slightly longer image acquisition process. Images are typically obtained with pixel dimensions of 1024 1024, but for weaker signals, 512 512 may be helpful. 10. Save images and export them as 16-bit tiffs. If z-sections are used, export individual sections or use 3D processing to make maximal projection XY images.
2.1.4 Postimage processing 1. Import 16-bit tiff file into ImageJ, Photoshop CS or alternate program. 2. It is vital that the investigator takes appropriate caution when manipulating images in Photoshop. Very minimal image manipulation should be done. It is best practice to collect the optimal image during acquisition. If adjustments are necessary (brightness/contrast), the linear “levels” function in Photoshop may be used, with adjustments made for an entire micrograph. 3. If the investigator intends to compare different time points or treatments, it is essential that both image acquisition and image processing be maintained identically for all image samples. 4. For publication, most journals require images of at least 300 dpi with pixel dimensions of 512 512 or 1024 1025. Pixel dimensions can be decreased in size, but not increased beyond the size obtained during acquisition (for example, images obtained with 512 512 pixels cannot be changed to 1024 1024 pixels during processing).
2.1.5 Remarks The above-mentioned protocols may be used to demonstrate the remarkable degree of regulation within the endocytic pathways. While TfR and EGFR are both internalized via CDE, their endocytic fates are vastly different under normal conditions. However, perturbations of key endocytic regulatory proteins disrupt the trafficking of the receptors. For instance, defects in the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway slow EGFR degradation and result in prolonged growth factor signaling, which can lead to oncogenesis (Mosesson, Mills, & Yarden, 2008). In addition, siRNA-mediated depletion of EHD1 or EHD3 delays trafficking of cargos by blocking movement out of the ERC or SE, respectively (Naslavsky & Caplan, 2011). Given that large genomewide screens have implicated
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kinases/phosphatases, lipid biosynthetic pathways, and many other proteins in endocytic regulation, we expect that many investigators will find the need to study endocytic trafficking (Collinet et al., 2010; Liberali, Snijder, & Pelkmans, 2014). While the above-mentioned protocols are useful starting points, we wish to point out several caveats that the investigator should be aware of: 1. It is likely that researchers may want to carry out the above-mentioned experiments after treatment with inhibitors or siRNAs to disrupt a certain protein or pathway. Given that siRNA transfection is a relatively long process (48e72 h), the levels of PM receptors may change during this time period and may need to be accounted for. For instance, depletion of certain endocytic proteins can actually decrease PM levels of TfR or EGFR (presumably by affecting the steady-state trafficking of the receptor). 2. The protocols outlined earlier are for fixed cells, thus they only give a snapshot of what occurs inside the cell. Live cell imaging can provide a more accurate assessment of the kinetics of transport from one endocytic organelle to the next. However, this often involves overexpression of fluorescently labeled proteins, which can affect trafficking. 3. Given that endocytic recycling is a fundamental process for all cells, most of the defects caused by siRNA depletion or chemical inhibitors are not complete. For example, EHD1-depletion slows recycling but does not block recycling. Similarly, EHD3-depletion blocks transport of cargo from the SE to the ERC; however, cargo may utilize the fast recycling pathway as a compensatory mechanism. Thus, receptor recycling is only partially inhibited. 4. In this section, we used fluorescently labeled ligands, which cause internalization of the receptor/ligand complex via CDE. However, as will be described later, antibodies against the extracellular domain of receptors can be used to follow the trafficking of receptors (see the MHC class I example in the next section). However, it is not always clear if the antibody mimics the endogenous ligand thereby causing ligand-dependent endocytosis or if the antibody simply labels the receptor and allows for visualization of the constitutive endocytic pathway.
3. QUANTITATIVE ASSESSMENT OF ENDOCYTIC RECYCLING 3.1 EXPERIMENTAL STRATEGIES In this section we will discuss two fluorescence-based experimental strategies to quantitatively assess endocytic recycling. It should be noted that there are a variety of additional methods that may be used to quantify recycling, often based on the labeling of cell-surface receptors by biotinylation or even cell-surface iodination. In this chapter, we have decided to focus on fluorescence-based methods as being potentially the most popular and easiest techniques to apply. The first experimental strategy that we discuss measures the disappearance of labeled ligands such as Tf from within the cell (Figure 4; part 1), whereas the second measures the
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Workflow for measuring TfR or MHC I recycling (1) Pulse cells for 15 minutes with Tf-488 Wash cells three times with PBS Fix to measure the internalized Tf-488 (Tube A)
In Incubate cells in complete DMEM and chase for 30 minutes Fix to measure easu the amount of remaining Tf-488 in the cell after a 30 minute chase (Tube B)
(2) Pulse cells for 30 m minutes with anti-MHC I antibody
= Tf-488 = MHC I/antibody complex
Acid strip for 1 minute
= goat anti-mouse 568 antibody
Wash cells three times with PBS Fix and stain with goat anti-mouse 568 in the presence of saponin to measure the amount of internalized MHC I (Tube A)
Fix and d stain stain with goat anti-mouse 568 in the absence of saponin to measure the amount of internalized MHC I (Tube B)
Incubate cells in In DMEM for 3 hours
complete
Wash cells three e time times with PBS Fix and stain with h goat goa anti-mouse 568 in the absence of saponin to measure the amount of MHC I that has recycled back to plasma membrane (Tube C)
FIGURE 4 Schematic workflow for quantitatively measuring recycling of Tf and its receptor or MHC I by flow cytometry.
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reappearance of internalized antibodyereceptor complexes (such as major histocompatibility complex class I (MHC I) and CD59) at the PM (Figure 4; part 2). Flow cytometry is the method of choice that we describe to measure the disappearance (recycling) of internalized Tf in HeLa cells, and we detail the use of confocal microscopy coupled with post-imaging quantitation via free software programs such as ImageJ to quantify the reappearance of antibodyereceptor complexes on the surface of the PM.
3.1.1 Flow cytometry analysis of fluorescently labeled Tf 1. Passage and plate HeLa cells on a 100-mm dish 24 h prior to the experiment, as described earlier. To have sufficient cells for treatment, they should be approximately 60e80% confluent. 2. Serum-starve cells as described in the previous section. 3. Incubate the cells in 4 mL of Cell Stripper (Mediatech Inc.) for 1 min on ice. Pipet up and down to detach cells from the bottom of the dish. Collect the cells in 15 mL Falcon tubes and pellet them by centrifugation at 1300 rpm at 4 C for 5 min. 4. Carefully aspirate the supernatant without disrupting the cell pellet and wash the cells three times in 1 mL of cold PBS, pelleting the cells by centrifugation as described earlier after each wash. 5. After the third PBS wash, resuspend cells in 1 mL of PBS and divide the cells into three aliquots (w330 mL) in 1.5 mL Eppendorf tubes. Tube A will serve as the Tf pulse treatment (see later), Tube B will serve as the Tf chase treatment, and Tube C will serve as the preuptake control, which accounts for autofluorescence artifacts and background noise. 6. For Tube C, add 330 mL of 7.4% PBSeformaldehyde (final concentration 3.7%) and fix the cells for 10 min at room temperature. 7. Dilute Tf-488 in DMEM (containing 0.1% BSA; see Table 1) to a concentration of 25 mg/mL. Enough solution should be prepared so that cells from each treatment can be resuspended in 150e300 mL of solution. 8. Resuspend the cells in Tubes A and B in 150 mL of Tf-488 solution. Rotate the tubes in a CO2-regulated incubator at 37 C for 15 min (pulse). 9. Wash cells three times with 1 mL of cold PBS, pelleting cells by centrifugation at 1300 rpm at 4 C for 5 min after each wash. 10. For Tube A, resuspend the pellet in 3.7% paraformaldehyde in PBS and fix for 10 min at room temperature. 11. For Tube B, resuspend the pellet in 1 mL of complete DMEM (containing fetal bovine serum) and rotate at 37 C for 45 min (chase). Wash and fix cells as described in Steps 9 and 10. 12. Transfer fixed cells from Tubes A, B, and C into flow cytometry-compatible tubes and measure 488 fluorescence by flow cytometry. 13. Assess the median fluorescence intensity (MFI, arbitrary units) of different samples by flow cytometry. Use Tube C to “gate” (set the fluorescence
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threshold) for Tubes A and B. The MFI of Tube A equals the total amount of internalized Tf while Tube B represents the amount of Tf remaining in the cells after the chase (nonrecycled). The recycling rate is calculated by the following equation (Eqn (1)):
ðMFI of chase sampleðTube BÞÞ 100% Recycling rate ¼ 1 ðMFI of pulse only sampleðTube AÞÞ
(1)
3.1.2 Remarks As with all experiments, recycling assays need to be repeated at least three times, and the recycling rates measured in each experiment may be presented in a bar graph with standard deviation and appropriate statistical significance tests.
4. QUANTIFICATION OF MHC I ANTIBODY-RECEPTOR COMPLEX RECYCLING USING IMMUNOFLUORESCENCE MICROSCOPY AND IMAGEJ 1. HeLa cells should be passaged and plated on 35 mm plates containing three coverslips to measure the recycling rate of MHC I. Cells should be approximately 50e75% confluent; higher confluency helps maintain cell viability during the acid stripping of noninternalized antibodyereceptor complexes (see later). 2. W6/32 anti-MHC I antibody should be diluted in complete DMEM. Each coverslip should be inverted onto 50 mL of antibody-containing solution in a humidity chamber (see previous section) for 30 min at 37 C (“pulse”). 3. Transfer the coverslips to a 12-well plate and remove noninternalized MHC I-antibody by incubating the coverslips in 1 mL of acid-stripping buffer (prewarmed to 37 C) for 1 min. Gently agitate the plate to achieve consistent stripping. 4. Wash coverslips three times with 1 mL prewarmed PBS. 5. Fix two of the coverslips, denoted A and B, respectively, in 3.7% formaldehydePBS for 10 min at room temperature (“pulse-only”). Incubate the rest of the coverslips in 2 mL prewarmed complete DMEM media in a 12-well plate for 3 h (chase) at 37 C. Fix this coverslip (C). Alexa-568 goat antimouse antibody staining is performed as described in section “Immunofluorescence,” Steps 9e14. Note that for coverslip A, staining is performed in the Abdil solution containing 0.2% w/v saponin, while the staining of B and C is done without this permeabilizing reagent. Coverslips AeC measure the following: A. the total internalized MHC I after pulse; B. the basal surface level of MHC I remaining after the pulse and stripping; and C. the level of MHC I that is recycled back to PM after the chase.
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4. Quantification of MHC i antibody-receptor complex recycling
6. Image acquisition is performed as described in the “Imaging” section. However, to maximize the objectivity and repeatability of this method, additional attention should be paid to parameter settings and the selection of cell fields for imaging. a. Cell fields need to be selected randomly, and the fields imaged should have cells that are evenly spread out without growing on top of each other. b. Avoid selecting fields from the same area of the coverslip. c. Before acquiring images, adjust the value of the detector gain so that there is no oversaturation. Pixel oversaturation appears red in the Range Indicator. d. All parameters including the gain level and cropping/magnification of images should remain unchanged during the image acquisition process. It is also important that no image be manipulated by image editing programs. e. Import 16-bit tiff files into ImageJ (http://rsbweb.nih.gov/ij/links.html). For each image, circle the outline of a cell with the Freehand selections tool. The MFI (arbitrary unit) is assessed by the Measure Tool. Collect the area and mean value for each selected cell; the area and mean fluorescence allow calculation of the total fluorescence (Eqn (2)). The investigator should be cautious about cell selection in the following manner: (1) select cells that are intact, flat, and preferably spread out so that the cell edges are easily recognizable; and (2) to better visualize the cells, DAPI staining of the cell nuclei is recommended. However, for a dual/multicolor image, make sure to split the channels in ImageJ and measure only the channel displaying MHC I staining. 7. Measure at least 60 cells from different fields of each sample and repeat the experiment at least three times. 8. For each independent experiment, calculate the total fluorescence using Eqn (2). Note that the measurement of Area is in pixels. Calculate the average of total fluorescence for each sample. The rate of MHC I recycling is calculated by the following equation (Eqn (3)) and the significance of the different tubes is described in Step 5: Total fluorescence ¼ Area Mean Recycling rate ¼
(2)
Total fluorescence of C Total fluorescence of B 100% (3) Total fluorescence of A
As with all experiments, such recycling assays need to be repeated at least three times, and the recycling rates measured in each experiment may be presented in a bar graph with standard deviation.
4.1 REMARKS These protocols are designed for the measurement of endocytic recycling of different receptors. However, internalized receptors are not only sorted to the recycling pathway. While Tf primarily recycles to the PM, MHC I is internalized via CIE. In
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the latter case, with each round of endocytosis, approximately half of the internalized MHC I molecules are recycled, with the rest sorted to the lysosomal pathway for degradation (Naslavsky, Weigert, & Donaldson, 2003). A decreased recycling rate results from either enhanced intracellular retentions or increased degradation. For example, when the lipid modifier diacylglycerol kinase a is depleted from cells, the MHC I recycling rate is dramatically decreased (Xie, Naslavsky, & Caplan, 2014). The shunting of receptors from recycling to degradation pathways, or vice versa, is not uncommon. Depletion of Rabenosyn-5 results in missorting and extensive degradation of TfR (Navaroli et al., 2012). In addition, the nontoxic domain 4 of the bacterial toxin intermedilysin (rILYd4) induces rapid CD59 degradation in lysosomes, whereas in the absence of the toxin CD59 is primarily recycled (Cai et al., 2014). Such cases show the importance of combining recycling and degradation assays to monitor and understand the subcellular itineraries of internalized proteins.
ACKNOWLEDGMENTS This work was supported by the National Institutes of Health Grants R01GM087455, R01GM074876, and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant number 5P30GM106397.
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