Visualizing GABA A receptor trafficking dynamics with fluorogenic protein labeling

Abstract

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAARs), exhibit highly dynamic trafficking and cell surface mobility. Regulated trafficking to and from the surface is a critical determinant of GABAAR neurotransmission. Receptors delivered by exocytosis diffuse laterally in the plasma membrane, with tethering and reduced movement at synapses occurring through receptor interactions with the sub-synaptic scaffold. After diffusion away from synapses, receptors are internalized by clathrin-dependent endocytosis at extrasynaptic sites and can be either recycled back to the cell membrane or degraded in lysosomes. To study the dynamics of these key trafficking events in neurons, we have developed novel optical methods based around receptors containing a dual-tagged γ2 subunit (γ2pHFAP) in combination with fluorogen dyes. Specifically, the GABAAR γ2 subunit is tagged with a pH-sensitive green fluorescent protein and a fluorogen-activating peptide (FAP). The FAP allows receptor labeling with fluorogen dyes that are optically silent until bound to the FAP. Combining FAP/fluorescent imaging with organelle labeling allows novel and accurate measurement of receptor turnover and accumulation into intracellular compartments under basal conditions and scenarios ranging from in vitro seizure models to drug exposure paradigms. Here we provide a protocol to track and quantify receptors in transit from the neuronal surface to endosomes and lysosomes. This protocol is readily applicable to cell lines and primary cells, allowing rapid quantitative measurements of receptor surface levels and post-endocytic trafficking decisions.

INTRODUCTION:

Binding of the neurotransmitter GABA to GABA type A receptors (GABAARs), heteropentameric ligand-gated chloride (Cl־) ion channels, is central for inhibiting and sculpting electrical activity in the central nervous system. Decreased GABAAR activity through changes in receptor surface levels or localization can result in anxiety, agitation, seizures, chronic pain and sleep disturbance. Synaptic GABAAR are largely composed of two α, two β and a γ2 subunit. The γ2 subunit is essential for viability (Gunther et al., 1995) and is required for accumulation and maintenance of most synaptic GABAAR clusters and the post-synaptic scaffold gephyrin (Essrich, Lorez, Benson, Fritschy, & Luscher, 1998; Schweizer et al., 2003). Like other neurotransmitter receptors, GABAARs exhibit highly dynamic and regulated cell surface trafficking that controls the strength of GABAergic neurotransmission (reviewed in (J. M. Lorenz-Guertin & Jacob, 2018; Luscher, Fuchs, & Kilpatrick, 2011). Thus, real-time visualization of receptor sub-cellular localization and trafficking measurements provides vital information on regulation of receptor signaling. Although the addition of traditional genetically encoded fluorophore tags allows localization of proteins and their movement within living cells, remaining challenges include resolving surface from internal protein populations and the reduced ability to detect and measure dynamic events due to the signal of pre-existing proteins of interest. Other approaches used for trafficking studies such as surface biotinylation approaches, labeling of live cells with antibodies to extracellular epitopes, and quantum dot-based methods also have specific caveats and limitations. For example, covalent modification of free amines during cell surface biotinylation has the potential to affect cellular activity and trafficking can be altered by surface antigen clustering or capping in antibody-based studies. Quantum dot nanomaterials can initiate nonspecific macropinocytosis and uptake (although this can be reduced with polyethylene glycol coating of the nanoparticles (Chang, Yu, Colvin, & Drezek, 2005)), and may produce cell toxicity and alter trafficking through bioaccumulation in internal compartments (L. W. Zhang & Monteiro-Riviere, 2009).

To advance GABAAR imaging capabilities, we have developed methods using fluorogen dyes in concert with a γ2 subunit tagged with both a pH-sensitive green fluorescent protein (superecliptic pHluorin, (Sankaranarayanan, De Angelis, Rothman, & Ryan, 2000)) and a fluorogen-activating peptide (FAP, (Szent-Gyorgyi et al., 2008)). This dual-tagged γ2 subunit (γ2pHFAP) enables steady-state surface receptor level measurements from the pH-sensitive green fluorescent protein in concert with membrane-localization specific high-affinity labeling techniques using a fluorogen-based reporter system (J. M. Lorenz-Guertin et al., 2017). The fluorogen-based reporter system consists of a genetically encoded FAP tag and exogenously applied fluorogen dyes. Both the FAP tag and fluorogen dyes are non-fluorescent but become exceedingly fluorescent upon binding together. FAPs are derived from antibody single chain variable fragments (scFvs) which have been characterized to selectively bind malachite green (MG) and thiazole orange (TO) synthetic fluorogen dyes with high specificity and affinity (Szent-Gyorgyi et al., 2008). Modification of these fluorogen synthetic dyes has generated dyes with distinct characteristics including cell permeability, pH-sensitivity, and various fluorescence properties (Fisher et al., 2010; Grover et al., 2012; Perkins et al., 2018; Szent-Gyorgyi et al., 2013; M. Zhang et al., 2015). This optical reporter system, in combination with labeling of early endosomes or lysosomes, provides opportunities to visualize and quantify targeting of surface receptors into sub-cellular compartments using both fixed and live imaging protocols and confocal microscopy experiments.

The FAP-dye system provides numerous advantage for imaging-based approaches: (i) within seconds of direct dye addition to the culture dish, target FAPs are saturated producing a robust signal; (ii) a single genetically encoded FAP can be targeted with a number of distinct dyes; and (iii) there is high specificity of the dyes binding to their target FAP, allowing use of multiple FAPs and dyes. Importantly, the high affinity of fluorogen-FAP binding produces a stable fluorescent module where surface receptors, and receptors undergoing internalization and recycling can be accurately measured (Pratt, He, Wang, Barth, & Bruchez, 2015; Szent-Gyorgyi et al., 2013; Yan et al., 2015). The protocols described here are only a brief foray into the utility of this system which has widespread applications as described later in the background commentary section.

Basic Protocol 1 describes the method of preparation of substrate, transfection of neurons and plating of neurons to be able to perform Basic Protocol 2 and 3. Protocol 2 details identification of internalized receptors in early endosomal compartments. Protocol 3 details a live-imaging confocal microscopy method used for quantitation of multiple stages of receptor trafficking including receptor surface, synaptic, and lysosomal levels. These techniques can be used to investigate receptor trafficking under basal conditions, following in vitro pathological models and during drug treatment paradigms (J. M. Lorenz-Guertin, Bambino, & Jacob, 2018; J. M. Lorenz-Guertin et al., 2017).

STRATEGIC PLANNING (optional)

A central consideration for using the outlined method is insertion of the pHluorin (pHGFP) and FAP tags in the extracellular portions of the protein of interest when it is delivered to the plasma membrane. For example, GABAAR subunit N-terminal domains reside in the vesicular lumen during trafficking and become extracellular after receptor insertion into the plasma membrane. This enables specific labeling and measurement of cell surface receptor endocytosis from the cell surface and subsequent transit through organelles including endosomes and lysosomes. The N-terminal tag location also allows for studies of receptor insertion or recycling, as previously done with GABAAR subunits tagged with pHluorin and the minimal α-bungarotoxin (Bgt) binding site peptide (BBS) (Brady, Moon, & Jacob, 2014; Jacob et al., 2009; Saliba, Gu, Yan, & Moss, 2009; Saliba, Kretschmannova, & Moss, 2012; Saliba, Michels, Jacob, Pangalos, & Moss, 2007). It is also important to show that addition of a genetic tag (pHluorin, FAP) and fluorogen dye are functionally silent. We have previously shown this for the γ2 subunit containing GABAAR reporter (γ2pHFAP) used here (J. M. Lorenz-Guertin et al., 2017). In addition, standard controls should confirm the tagged receptor is appropriately localized in neurons and expressed at similar levels to well-characterized untagged constructs, thus reducing concerns of tags inducing structural based changes in trafficking measurements. The most expedient and robust way of assessing these is usually through studies in a recombinant system such as HEK-293 cells, prior to use in neurons.

Statement of Ethical Approval

Applying this protocol in neurons requires the use of animal material. Thus, use of the described method requires approval for tissue collection by the Institutional Animal Care and Use Committee (IACUC), in accordance with national and local regulations.

BASIC PROTOCOL 1

BASIC PROTOCOL TITLE : Preparation of cortical neuronal cultures for imaging assays

Basic Protocol 1 describes the method of preparation of substrate, transfection of neurons and plating of neurons to be able to perform Basic Protocol 2 and 3.

Caution: Paraformaldehyde is a hazardous chemical waste, and appropriate protective gloves and eye protection should be worn. Paraformaldehyde waste should be collected and disposed of according to the institution’s regulations.

Materials:

Glass bottom tissue culture dish, MatTek Corporation, P35G-1.5–14-C - Mattek Dishes

Coverslips (round cover glass), #1 thickness, 12 mm, Warner Instruments, 640–0702

Poly-D lysine, Sigma, P6407

Biological safety cabinet/ tissue culture hood

Incubator for cultured cells

CO2 tank for tissue culture incubator & regulator

Water bath, 37C

MilliQ or similar cell culture grade water

Primary cortical neurons, tissue isolated acutely from E18 rats

Bright-line phase hemacytometer or cell counting device, Fisher, 02–671-6

Neurobasal media: Neuronal media (+10ml B27 Supplement, 5ml L-glutamine, 0.25 ml pen/strep, 6.6 ml 45% D-Glucose), Invitrogen/Life Technologies, 21103049

P3 Primary Cell 4D kit, Lonza, V4XP-3024

4D-NucleofectorTM Core and X Units, Lonza 4D-NucleofectorTM Core and X Units, AAF-1002X and AAF-1002B

Protocol steps — Step annotations:

Use sterile technique and reagents throughout Part 1, with all steps performed in tissue culture hood.

Prepare glass substrate for growing neurons.

For imaging studies compatible with immunofluorescence methods to identify intracellular compartments, use poly-D-lysine coated glass coverslips (0.1 mg/ml in H2O). For live imaging studies employing intracellular compartment live labels, prepare glass bottomed 3.5 cm dishes (for microscopy, No. 1.5 is the preferred coverslip thickness for optimized image quality using high numerical aperture objectives as described here).

1. Pipette a droplet of poly-D-lysine (0.1 mg/ml) onto glass substrate.

   For each of 4 coverslips (12 mm diameter) in a 3.5 cm dish use 70 μl poly-D lysine. For glass bottomed 3.5 cm tissue culture dish, pipette 200 μl poly-D-lysine onto embedded 14 mm glass.2) Leave the prepared dishes in the biological safety cabinet/tissue culture hood overnight.

   To minimize poly-D-lysine evaporation and keep surfaces sterile in the tissue culture hood, close the hood window sash completely and turn off the blower overnight. Note: UV lights are not used during the overnight incubation.

2. The next morning after turning the hood on and returning the tissue culture window sash to working height, wash the dishes by pipetting 2 ml of H2O (milliQ or similar grade water) per dish, remove by aspiration, repeat 3x.

3. Following removal of the last H2O wash, add 2 ml of neuronal media to each 3.5 cm dish and leave dishes in 37°C tissue-culture incubator for media to equilibrate until ready to plate the neurons in step 7.

Culturing and transfection of primary neuron cultures.

• 4.Prepare acutely dissociated cortical neurons from embryonic rats at day 18 (E18) (modified from (Hall, 2006; Kaech & Banker, 2006)). Alternatively rat embryonic neurons can be purchased from various online vendors.

  Note: this same substrate preparation and transfection protocol works well for growing hippocampal neurons.

• 5.Nucleofect freshly dissociated neurons on the day of culturing with 1–4 μg of maxiprep construct DNA using the Lonza P3 Primary Cell 4D kit, in this case γ2pHFAP GABAAR subunit.

  Note: typically, 3 μg of DNA is transfected into 1–2 million neurons, with a viability of 50 % and a transfection efficiency of 50 %. Recommendations are to follow the manufacturer’s protocol for cell type specific reagent kits. For culturing of rat hippocampal or cortical neurons, high viability protocols are recommended rather than high efficiency protocols.

  For example, starting with 2 million neurons: (2X10⁶ neurons X 0.5 viability factor) provides approximately 1 million neurons; 500,000 of which are nucleofected (Jacob et al., 2005). Varied amounts of construct DNA can be transfected initially, when optimizing expression levels, followed later by appropriate characterization of construct localization and function with the transfected DNA amount kept constant between independent cultures.

• 6.Following nucleofection of cortical neurons, plate neurons based on intended uses.

  For immunofluorescence studies on fixed neurons, plate approximately 2.0 × 10⁵ cortical neurons into 3.5 cm tissue culture dish containing 4 poly D-lysine coated coverslips.

  For live imaging studies using intracellular compartment labels, plate 40,000 neurons on the 14 mm glass area of a glass bottomed tissue culture dish.

• 7.Replace the media 18–22 hrs after preparation of neuronal cultures with fresh neuronal media, then allow neurons to develop in the incubator until 14–17 days in vitro (DIV) or desired developmental stage.

BASIC PROTOCOL 2: Surface receptor internalization and trafficking to early endosomes

Basic Protocol 2 enables the user to label surface receptors and follow the internalization of these receptors into early endosomal compartments. Surface resident receptors are labeled using a 2 min 100 nM MG-BTau dye pulse-labeling protocol in HBS (Hepes Buffered Saline) at room temperature, then dye is removed by rapid wash steps, followed by either a 30 minute incubation at 37°C to allow for receptor trafficking into cellular compartments or 10°C to block internalization. Next, neurons are fixed using a 4% paraformaldehyde sucrose solution and an immuno-fluorescence method using an antibody against EEA-1 (Early endosome antigen 1) is used to label endosomes. Finally, confocal microscopy and image analysis is used to quantify the intensity and area of MG-BTau labeled receptors colocalized with EEA1-positive intracellular vesicles.

Materials:

Incubator for cultured cells

Fluorogen dye, MG-BTau, Marcel Bruchez Lab.

For dye preparation and quantification see https://bruchez-lab.mbic.cmu.edu/resources/dyes/ Desiccated dye stock is dissolved in 500 uL-1000 uL of 1% Acetic acid (glacial) in ethanol for quantification of concentration, store at 4°C in dark. Diluted working stocks are made fresh on the experimental day, however they can be kept at 4°C for a maximum of a week.

Hepes Buffered Saline (HBS), 135 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 10 mM HEPES, 2.5 mM CaCl2, 11 mM glucose, pH 7.4,

Peltier device for rapid temperature regulation: Torrey Pines Scientific Inc, Echotherm (various models available)

Mounting medium, Dako, cS703

Paraformaldehyde sucrose fixing solution, 4 % PFA, 4% sucrose in Phosphate Buffered Saline, DPBS without calcium and magnesium, Invitrogen/Life Technologies, 14190235

Permeabilization solution (antibody block solution with 0.2% TX100) (see Reagents and Solutions)

Mouse anti-EEA1, BD Biosciences, 610457

Secondary antibody, goat anti-mouse-IgG conjugated to Alexa 405

Fluor 405 (1:1000; A31553, Invitrogen)

Antibody blocking solution(see Reagents and Solutions)

Coverslip tweezers

Microscope slides, VWR, 48312–003

Nikon Ti-E A1 Confocal microscope, 60x oil objective, Nikon

NIS Elements software, Nikon

Surface Receptor Labelling

1. Warm HBS to room temperature (RT) to be used for preparation of MG-BTau dye and subsequent wash steps. For each 3.5 cm dish used, approximately 10 ml of HBS at RT will be needed for the wash steps.

2. In preparation for step 7, set a Peltier device (Torrey Pines Scientific Inc, Echotherm) to 10°C.

3. Prepare the required volume of 100 nM MG-BTau dye solution in HBS. The stock solution of MG-Btau dye is 1 mM and is stored at 4 °C. The dye is used at a 100 nM final concentration. Per 3.5 cm dish, 1 ml of 100 nM MG-BTau dye in HBS is needed.

   1. For volumes of 10 ml or greater, the dye can be diluted at 1:1000: Add 1 ul of 1mM dye per 10 ml HBS.
   2. However, if smaller volumes are needed, a sequential dilution is recommended. For example: 1) first prepare dye at 1 uM (a 1:1000 dilution, 1 ul of 1mM dye in 1 ml HBS) and then 2) prepare 100 nM dye by adding 10 ul of 1uM dye to 1 ml HBS.

4. Remove neuronal culture 3.5 cm dishes from incubator and place on bench. Replace neuronal media with HBS, rapidly processing one dish at a time as follows: aspirate media, replacing with 2ml of HBS at RT.

5. Aspirate off HBS and replace with 1ml of of 100 nM MG-BTau dye solution and incubate for two minutes at room temperature (RT).

6. Perform three rapid wash steps with HBS to remove all MG-BTau dye solution so subsequently inserted receptors are not labeled

   1. Aspirate off MG-BTau dye solution and pipette on 2ml of HBS at RT for wash step 1.
   2. Repeat 2ml HBS wash x2, completing a total of 3 wash steps with 6 ml HBS.

7. The culture dishes containing HBS should now be divided into two treatment groups.

   Transfer

   1. group one into an incubator at 37°C to allow for receptor trafficking into cellular compartments
   2. group two onto a Peltier device set to 10°C to block internalization.

8. At chosen endocytosis time point (30 min suggested), take dishes from 37°C incubator or peltier device set to 10°C, aspirate HBS and fix in 1ml of 4 % paraformaldehyde/4 % sucrose for 20 min.

9. Remove fix and place in PFA waste container and wash dishes twice with 2 ml room temperature DPBS without calcium and magnesium.

10. Incubate coverslips at room temperature for 10 min in immunofluorescence block solution (5% horse serum, 0.5 % BSA in DPBS without calcium and magnesium) containing 0.2 % Triton X-100 to permeabilize the neurons and enable immunostaining of intracellular early endosomal compartments with EEA1, or other vesicular compartments of interest.

    In the sample data provided here, we examined if MG-BTau labeled surface receptors could be identified in early endosomes pathways following internalization by immunostaining with the early endosome marker EEA1 (Figure 1A).

11. Remove solution and incubate coverslips in immunofluorescence block solution without Triton X-100 for 20–30 minutes.

12. Perform primary antibody incubations of coverslips in block for several hours at room temperature or overnight at 4°C.

    Note: incubation of coverslips with primaries at 4°C overnight routinely produces improved immunofluorescence results.

13. Remove antibody solution by aspiration, replacing with 2ml of DPBS without calcium and magnesium. Incubate for 5 min, then repeat this wash step two more times.

14. Incubate coverslips with secondary antibodies in block solution for 1 hour at room temperature.

15. Wash coverslips 3 times with DPBS without calcium and magnesium (5 min for each wash step).

16. Mount coverslips, handling each coverslip individually: remove excess liquid from back of coverslip with lab tissue, then invert coverslip onto 4 μl of mounting medium on a glass slide.

17. Allow slides to dry at room temperature covered for 30 min then store at 4 °C until ready to perform microscopy.

Figure 1. MG-BTau dye and γ2^pHFAP GABAA receptor endocytosis assay with early endosomal compartment colocalization.

(A) DIV 12 γ2^pHFAP expressing neurons were pulse labeled with 100 nM MG-BTau dye for 2 min, then incubated in HBS at 37°C or 10°C for 30 min prior to fixation. MG-BTau (blue) labeled neurons were permeabilized and stained with early endosome marker EEA1 antibody (red). pHGFP (green) is visible throughout the cell after fixation. Boxed area is enlarged to the right with EEA1 (red), MG-BTau (blue), and Merge panels. Yellow circles show colocalized EEA1 and MG-BTau signal. (B) The intensity and area of MG-BTau labeled receptors colocalized with EEA1-positive intracellular vesicles is increased when endocytosis is not inhibited by 10°C incubation. (*p≤0.05, **p < 0.01, Student’s t-test; n = 25 neurons from 3 cultures for each condition; error bars represent ± s.e.m). Scale bars = 5 μm. Figure reproduced with permission from Journal of Cell Science (J. M. Lorenz-Guertin et al., 2017).

Image Acquisition and Analysis

• 18.
  Recommended image acquisition and analysis parameters:

  1. 60X oil NA 1.49 immersion objective
  2. Lasers: Argon gas 488 nm, 561 diode, 640 diode
  3. Emission band pass filters: 500–550, 575–625 and 650 long pass filter (to avoid any spectral bleed-through between channels)
• 19.Acquire single Z section images of a neuronal cell body with several dendritic processes in focus.
• 20.Quantify MG-Btau signal colocalized with EEA1 immunostaining along 20 μm of 3 to 4 proximal dendrites per neuron.

  Data can be analyzed using NIS Elements software (Nikon, NY) or similar software. The researcher should be blinded to treatment group during image acquisition and data analysis. A region of interest (ROI) is drawn around the cell body in the image. Next thresholds for individual channels (EEA1 and MG-BTau) are set using binary masks to selectively identify brightly fluorescent objects above background. The colocalized signal is identified by overlap of areas shared between the EEA1 and MG-BTau binaries. Finally, the total pixel intensity of MG-BTau labeled receptors colocalized with EEA1 is quantified within the cell body ROI.

• For each independent channel use the same threshold for analysis of all endocytosis data.

Analyze data from 10–12 neurons for each time point, repeating the experiment with several independent neuronal cultures (typically 3–5 cultures). In the sample data provided here, neurons maintained at 37°C demonstrated greater mean intensity and area of MG-BTau colocalized in EEA1-positve vesicles compared to those kept at 10°C where internalization was inhibited (Figure 1B). Thus, MG-BTau labeling FAP-tagged receptor subunits can be used to track, define and quantify internalized GABAAR pools.

BASIC PROTOCOL 3: Measurement of receptor steady state surface level, synaptic level and lysosomal targeting

Protocol 3 is a live-imaging confocal microscopy method used for quantitation of receptor surface and lysosomal levels. Neuronal surface resident receptors are labeled with MG-BTau dye and lysosomes are labeled with Lysotracker. Confocal microscopy and image analysis are used to measure multiple parameters: 1) steady state surface receptor level via the pHGFP signal; 2) remaining surface receptor following defined time periods of internalization via the MG-BTau surface signal; and 3) receptor populations in lysosomes via colocalization of MG-BTau with the live lysosomal label LysoTracker. Example data is provided analyzing changes in receptor trafficking following an in vitro seizure paradigm (J. M. Lorenz-Guertin et al., 2017). This method has also been employed to measure enhanced receptor lysosomal targeting due to prolonged treatment with benzodiazepines (Joshua M. Lorenz-Guertin, Bambino, Das, Weintraub, & Jacob, 2019), an important sedative-hypnotic clinical drug class that are GABAAR positive modulators.

Caution: LysoTracker dyes are supplied in DMSO, a polar organic reagent, and nitrile gloves should be used while working with DMSO based solutions.

Materials:

MG-BTau, Bruchez/Waggoner Labs

Hepes Buffered Saline (HBS)(see Reagents and Solutions)

Nikon Ti-E A1 Confocal microscope, 60x oil objective, Nikon

NIS Elements software, Nikon

Biological safety cabinet/ tissue culture hood

(−)-bicuculline methiodide, Tocris, 2503

LysoTracker Blue DND-22, Life Technologies, L7525

Neuronal cultures

Protocol steps — Step annotations:

Labelling of Neuronal Cultures

All steps until step 7 are performed in the tissue culture hood.

1. Perform treatments, at 14–16 DIV on neuronal cultures transfected as outline in protocol 1 with γ2pHFA, that are growing in mattek dishes (Glass bottom tissue culture dish, MatTek, see protocol 1).

2. Prepare fresh LysoTracker solution from stock solution for Step 7. LysoTracker is supplied at 1mM in DMSO, a polar organic reagent, and nitrile gloves should be used while working with DMSO based solutions. Dilute LysoTracker at 1:20 in HBS to generate a 50 μM stock, For each 3.5 cm TC dish (typical volume of media is 1.5–2 ml), a volume of 1.5–2 μl of 50 μM Lysotracker stock will be needed.

3. Take neuron dish from incubator, place into tissue culture hood, then remove media (keep conditioned media and reserve in incubator at 37°C for step 5), replacing with 2ml of HBS at RT.

   1. For live imaging time series experiments, only one dish is used at a time.

4. Aspirate HBS off of neuron dish and replace with 1ml of of 100 nM MG-BTau dye in HBS and incubate for two minutes at room temperature. Pulse label γ2pHFAP transfected neurons with MG-BTau dye.

5. Perform three rapid wash steps with HBS to remove all MG-BTau dye so subsequently inserted receptors are not labeled:

   1. Aspirate off MG-BTau dye solution and pipette on 2ml of HBS at RT for wash step 1.
   2. Repeat 2ml HBS wash x2, completing a total of 3 wash steps with 6 ml HBS.

6. Aspirate last HBS wash and replace with conditioned media retained during step 2, return dish to 37°C incubator for 1 hour to allow for receptor endocytosis and trafficking to lysosomal compartments.

   Once proficient in the protocol, after approximately either 15 or 35 minutes into the 1 hour 37°C incubation, you can begin the process for the next mattek dish, thus allowing more dishes to be processed within a shorter period of time. If not, the total experimental time (not including analysis) for two-three neurons in a single dish within a treatment group is approximately 1 hour and 20 minutes.

7. At 30 minutes into the 1-hour incubation period add LysoTracker Blue DND-22 dye, final concentration 50 nM, to identify lysosomes.

   Note: At the same time as lysotracker dye is added, the lysosomal inhibitor, Leupeptin can also be included (200 μM Amresco) (Joshua M. Lorenz-Guertin et al., 2019) to reduce receptor degradation in lysosomes and improved detection of lysosomally targeted receptors.

8. After 1 hour take dish from 37°C incubator, remove conditioned medium by aspiration, replacing with 2ml of 4°C HBS. Rapidly repeat HBS wash step four additional times.

   This wash step can be performed on the bench, rather than in the tissue culture hood.

Live Confocal Imaging

• 9.
  Acquire images on a confocal microscope, using the following parameters

  1. Neurons are imaged (blind to experimental condition) using a 60X oil NA 1.49 immersion objective at a zoom of 2.
  2. Lasers: 405 diode (for Lysotracker blue, excitation/emission maxima ~373/422 nm); Argon gas 488 nm (pHluorin signal), and 640 diode (MG-BTau dye signal).
  3. Filters: Samples for confocal imaging should be sequentially scanned with individual lasers and an appropriate emission band pass filter (500–550 and 575–625 nm) or long pass filter (650LP) to avoid any spectral bleed-through between channels.
• 10.Acquire single Z section images of two-three neurons per culture dish within 10 min of washing, using the same image acquisition settings. The cell body and several dendritic processes need to be clearly in focus to provide quantitative data.

  Data should be acquired from 15 neurons for each treatment group, repeating the experiment with several independent neuronal cultures (typically 3–5 cultures).

• 11.
  Data analysis uses NIS Elements software (Nikon, NY), with colocalization determined as previously described (Tretter et al., 2008).

  1. For image analysis, independent ROIs are drawn to capture the soma, three 10 μm sections of dendrite and the whole cell.
  2. Binary thresholds and colocalization measurements are performed to identify lysosomes (Lysotracker), surface synaptic GABA_AR clusters (pHGFP) and MG-BTau labeled GABA_AR on the surface and in intracellular compartments.
  3. For each individual channel, thresholds should be kept constant across a data set.
  4. Total surface pHGFP expression is determined by taking the entire cell surface signal following background subtraction.

REAGENTS AND SOLUTIONS:

1X Hepes Buffered Saline (HBS):135 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 10 mM HEPES, 2.5 mM CaCl2, 11 mM glucose, pH 7.4. This is typically made from a 10X stock without glucose, or CaCl2. 1X HBS made complete with glucose and CaCl2 is adjusted to pH 7.4 and kept for a few days at 4°C. To prepare 500 ml of 1 X HBS, dilute 50 ml of 10X HBS with 450 ml Milli-Q water, then add 1.25 ml of 1 M CaCl2 (sterile filtered at kept at 4°C) and 99 g of D-glucose, to pH to 7.4 takes approximately 2.5–3 ml 1 N NaOH.

Paraformaldehyde sucrose fixing solution: 4 % PFA, 4% sucrose in Phosphate Buffered Saline. Caution: Formaldehyde is toxic. Please read the MSDS before working with this chemical. Gloves and safety glasses should be worn and solutions made inside a fume hood. A large volume is made (400 ml) and then aliquoted to smaller volumes (5–10 ml aliquots), frozen and kept at −80°C for over a year.

Antibody blocking solution: 5 ml horse serum (Horse Serum, Invitrogen/Life Technologies, 26050–088), 0.25g BSA (BSA - Albumin bovine fraction V powder, Sigma, A7906), 45 mL DPBS without calcium or magnesium (DPBS without calcium and magnesium, Invitrogen/Life Technologies, 14190235). Typically, a 50 ml volume is made and stored at 4°C for a month.

Permeabilization solution: antibody blocking solution with 0.2% TX100 (TX-100, Sigma, T8787). On the experimental day, the needed volume for the experiment (1–2 ml per 3.5 cm dish) is made using the above antibody blocking solution with TX100 added.

COMMENTARY

BACKGROUND INFORMATION:

In 2008, Szent-Gyorgi et al introduced a new dual-reporter system based on protein reporters that produce fluorescence from dark molecules (fluorogens). These fluorogen activating peptides (FAPs), are based on antibody single chain variable fragments (scFvs) optimized to selectively bind malachite green (MG) and thiazole orange (TO), derived synthetic dyes with high specificity and affinity (Szent-Gyorgyi et al., 2008). MG and TO dyes are fluorogens, molecules that are dark in solution until bound by their respective FAP. Through chemical modifications, versions of these dyes are available with distinct properties including cell permeability, pH-sensitivity, and various fluorescence parameters (Fisher et al., 2014; Grover et al., 2012; Saunders et al., 2012; M. Zhang et al., 2015). FAPs and dyes bind via non-covalent interactions with up to sub-nanomolar affinity, producing an increase in dye fluorescence up to thousand-fold. This massively enhanced fluorescent signal results from FAP constraint of the rotation of a single bond within the chromophore (Saliba et al., 2009; Saliba et al., 2012).

We previously developed a labeling method to follow GABA_AR trafficking based on incorporation of an α-bungarotoxin binding site which selectively binds cell-excluded fluorescent bungarotoxin, allowing for specific monitoring of receptor insertion and internalization (Brady et al., 2014). As Alexa dye coupled bungarotoxins are inherently fluorescent, these methods require extensive washing after labeling to remove background signal from dye not coupled to the receptor target. In addition, subsequently functional studies identified that 5 μM bungarotoxin could function as an antagonist at α2β2γ2 GABAARs, somewhat complicating the use of these reagents (Hannan, Mortensen, & Smart, 2015), although lower concentrations did not reduce GABA currents, and trafficking assays typically use more than 10 fold lower concentrations (Brady & Jacob, 2015). However, to improve GABA_AR imaging methods and leverage several advantages of the FAP-based system, we incorporated the dl5** FAP into the N-terminus of a GABAAR subunit, also tagged with the pH sensitive GFP variant pHluorin. This allows for quantitation of steady state surface receptor levels via the pHluorin signal, while enabling additional pulse-labeling of receptors via FAP based methods. The sulfonated analog of the malachite green (MG) fluorogen used in this protocol, MG-BTau, is cell impermeable, thus allowing for selectively labeling of cell surface receptors. The FAP, dl5**, based on immunoglobulin (IgG) variable light (VL) domains, binds MG-BTau with subnanomolar affinity, generating a fluorescent signal that can be quantified within seconds of dye addition.

The general advantages of the genetic FAP tag - fluorogen dye system include: (1) selective, saturating and extremely rapid fluorescence generated when the dyes bind to the FAP expressing protein, allowing direct dye addition to a culture dish in physiological buffer or standard culture mediums; (2) additional unbound dye is nonfluorescent, removing the need for dye washout; (3) a number of distinct dyes can be used for the same genetically encoded FAP; (4) at approximately 26 kDa, FAPs can be easily genetically engineered into constructs (or inserted into endogenous genes via CRISPR) and (5) use of cell-permeant vs impermeant dyes allows for visualization of FAP fusion proteins on the cell surface, within trafficking vesicles, or at other intracellular locations. In particular, the high affinity fluorogen-FAP binding is extremely valuable for measuring receptors undergoing internalization and recycling (Pratt et al., 2015; Szent-Gyorgyi et al., 2013; Yan et al., 2015). Thus this FAP-fluorogen dual system has immense versatility, ranging from assessment of exquisite high resolution trafficking events in neurons via live imaging to large scale pharmacological screening of drug effects on receptor trafficking (Fisher et al., 2010; Fisher et al., 2014; Grover et al., 2012; Pratt et al., 2015; Snyder et al., 2015; Wu, Tapia, Jarvik, Waggoner, & Sklar, 2014) or siRNA library screening to identify novel endogenous regulators of receptor levels (Larsen et al., 2019). Additional applications based on this technology include fluorescence-synapse labeling to provide connectivity analysis in brain tissue (Kuljis et al., 2019), sorting of cells based on surface expression of FAP fusion proteins, in vivo imaging of tumors (Wang et al., 2017), chemoptogenetic damage of mitochondria via targeted FAP (Mito-FAP) delivery of a photosensitizer MG-2I dye (Qian et al., 2019), and visualization of the fusion pore openings when neuropeptides are delivered by dense core vesicles to synapses (Bulgari et al., 2019).

CRITICAL PARAMETERS:

FAP and pHluorin tagged protein.

It is important to find an appropriate location for insertion of the FAP and pHluorin tags that does not interfere with protein function. An extracellular site is needed; generally N terminal insertions in larger domains are well handled, shortly following the signal peptide sequence. Preliminary studies should include validation and evaluation of transfected tagged protein expression level in neurons.

Resources.

Consider the availability of resources including temperature controlling equipment and microscope accessibility for time-series imaging. In many cases, alternative approaches can be tailored to the available equipment. For example, if a Peltier cooling device is not available to hold neurons at 10°C and block endocytosis, a T30 at 37°C vs T0 time point can be compared.

Data acquisition and analysis.

Critical parameters include the health of neurons and good competency with confocal microscopy and image analysis. Before conducting these protocols one should determine the typical cell surface turnover rate for the protein of interest. Sufficient time should be invested prior to finalizing experimental details to allow the researcher to determine appropriate imaging conditions considering the specific protein of interest’s expression level and turnover. Photobleaching of fluorescent signals should be minimized through optimization and reducing the frequency of image acquisition during time-series acquisition. Like any microscopy-based method, different treatment groups should be acquired using the same settings. Furthermore, blinding of all experiments prior to imaging and analysis is important to generate robust unbiased data.

STATISTICAL ANALYSIS:

If two conditions are compared, standard student’s t-tests are applicable. If multiple timepoints are compared, then a one-way ANOVA is appropriate. If the experimental design includes multiple genotypes and treatments, then a two-way ANOVA is needed.

UNDERSTANDING RESULTS:

These protocols describe how GABAA receptors containing the γ2pHFAP subunit, combined with MG dye application can be used to examine multiple stages of receptor trafficking and targeting including receptor surface, synaptic, early-endosome and lysosomal levels. Imaging based assays rely on the healthy development of neurons and synaptic circuitry in vitro. This requires the careful preparation of a substrate promoting neuronal growth, an optimal plating density of neurons, and efficient transfection methods as described in Protocol 1. Protocol 2, describes how MG-BTau labeled surface receptors can be quantified in early endosomes pathways following internalization by combining an endocytosis assay with immunostaining of the early endosome marker EEA1 (Figure 1A). In the sample data provided, neurons maintained at 37°C demonstrate greater mean intensity and area of MG-BTau colocalized at EEA1 vesicles compared to those kept at 10°C where internalization was inhibited (Figure 1B). This shows how MG-BTau labeling FAP-tagged receptor subunits can be used to track, define and quantify internalized GABAAR pools.

Finally, Protocol 3, describes how the γ2pHFAP GABAA receptor construct in combination with MG dyes can be used to measure surface, synaptic and lysosomal receptor levels, showing how this approach can be used to reveal how an in-vitro seizure paradigm alters receptor trafficking. At DIV 12–14 γ2pHFAP neurons were pulse-labeled with MG-BTau dye and then returned to conditioned media +/− the GABAA receptor antagonist bicuculline at 37°C for 1 h. 50 nM LysoTracker was added 30 min prior to the end of treatment to identify association of receptors with lysosomes. Representative images indicate MG-BTau labels synaptic GABAAR clusters on the surface of dendrites as seen by colocalization of MG-BTau (blue) and pHGFP (green) (Figure 2A, B). MG-BTau also reveals internalized receptors within the cell body in lysosomes (Figure 2C, Lysotracker in red). These data demonstrate that the binding of MG-BTau to γ2pHFAP GABAARs and its resulting fluorescence is stable even in very low pH environments such as lysosomes, consistent with previous findings using different FAP-tagged receptors colocalized with LysoTracker in cell culture (Grover et al., 2012). In the sample data provided, image analysis uncovered no significant difference in total surface expression of γ2pHFAP between DMSO control and bicuculline treated cells when measuring pHGFP signal (Figure 2D). There was a trend towards a decrease in steady state synaptic levels of γ2pHFAP in bicuculline treated neurons (75 ± 13% of control), determined by pHGFP cluster fluorescence, but this was not significant. In contrast, (Figure 2E) shows bicuculline treatment reduced total and synaptic MG-BTau signal by 41 ± 10% and 67 ± 8%, respectively, indicating the population of pulse-labeled γ2pHFAP receptors had decreased dramatically. In support of enhanced receptor turnover, bicuculline treatment increased association of labeled receptors with lysosomes by 107 ± 41% compared to control. These findings suggest that bicuculline-induced seizure activity leads to augmented GABAAR synaptic turnover, lysosomal targeting, and a compensatory increase in new, non-recycled GABAAR insertion to mitigate this response. These results clearly demonstrate how the γ2pHFAP-dye system allows measurement of several trafficking events, enabling users to address complex biological questions in neurons.

Figure 2. Enhanced GABA_AR turnover rates and targeting to lysosomes following a bicuculline-induced in-vitro seizure paradigm.

(A) γ2^pHFAP neurons were pulse-labeled with 100 nM MG-BTau for 2 min then returned to 37°C conditioned media +/− 50 μM bicuculline for 1 h. LysoTracker (50 nM) was added directly to the media after 30 min to label lysosomes. pHGFP fluorescence is shown in green, LysoTracker in red, and MG-BTau in blue in the Merge panels. Smaller boxed areas in Merge panel identify surface synaptic receptors (enlarged in B). Larger boxed area identifies internalized receptors present in endosomes and lysosomes in cell body of neuron (enlarged in C). (B) Surface synaptic receptors on dendrites are seen with colocalization of MG-BTau and pHGFP signals. (C) Enlargements of cell body area show colocalization of internalized MG-BTau labeled GABA_ARs and lysosomes. (D) Quantification of pHGFP signal showed synaptic and total surface levels were not changed following bicuculline treatment. (E) In contrast, quantification of MG-BTau signal revealed reductions in total and synaptic receptor levels after the bicuculline seizure paradigm. Bicuculline treatment also enhanced the ratio of MG-BTau labeled receptors associated with lysosomes over total MG-BTau signal. (*p < 0.05, ****p < 0.0001, Student’s t-test; synaptic measurements performed on three 10 μm regions located on dendrites; n = 13 neurons per treatment from three independent cultures; error bars represent ± s.e.m.). Scale bars: 20 μm in A and 2 μm in B,C. Figure reproduced with permission from Journal of Cell Science (J. M. Lorenz-Guertin et al., 2017).

TIME CONSIDERATIONS:

Glass coverslip preparation with poly-D-lysine occurs overnight at room temperature. Nucleofection and plating of neurons takes approximately one hour. The length of imaging assays may vary depending on the cell surface turnover rate of the particular protein of interest, but typical timepoints for trafficking of neurotransmitter receptors to endosomes and lysosomes are usually in the range of 30–60 min. For the colocalization with endosomal compartments, from start to finish including the EEA-1 immunostaining component of the experiment takes 24 hours, including the overnight primary antibody incubation, followed by completion of the immunostaining the following day and then fixed sample confocal image acquisition at a later date. The lysosomal colocalization assay for a single dish in a treatment group is completed in approximately one and a half hours.

TABLE 1:

Troubleshooting Guide for Protocols 1–3

Protocol	Problem	Possible cause	Evaluation	Solution
Basic Protocol 1	neuronal clumping	Error with preparation of poly-D-lysine or poor glass quality Error with preparation of neuronal media	Ensure sufficient time has occurred for poly-D-lysine coating on coverslips (overnight at RT), without actual drying or evaporation. Ensure media is prepared correctly	Lower sash and turn off blower in biosafety cabinet, avoiding this problem without compromising sample sterility. Ensure media is prepared correctly.
		Poor neuronal health	Evaluate steps of dissociation and transfection, include plating of non-transfected neurons and GFP transfected neurons to evaluate possible contributions including constructs	Work quickly but carefully throughout dissociation and transfection protocol, minimizing time neurons are in nucleofection buffer before returning to media.
Basic Protocol 2 or 3	Weak signal from surface labeling	FAP and pHluorin tag insertion site is non-optimal, decreasing expression	Confirm surface and total expression by standard immunofluorescence using anti-GFP antibodies	Engineer FAP tag into another location.
			Confirm surface and total expression by biochemical methods and western blotting and anti-GFP antibodies
			Note: these experiments can be performed in HEK-293 cells for easier evaluation of constructs
Basic Protocol 2–3	Weak signal of receptors colocalized with early-endosomes or lysosomes	Low or slow levels of receptor turnover	Perform assay with multiple time-point analysis to evaluate receptor turnover	Length of lysosomal targeting assay can be increased.
			Alternative method: Use cell surface biotinylation and western blotting to evaluate speed of receptor endocytosis and degradation	Lysosomal inhibitor leupeptin can be adA1:E9ded to increase detection in this intracellular compartment.