Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Summary

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

Abstract

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley’s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Introduction

Exocytosis is a universal cellular process in which a vesicle fuses with the plasma membrane and releases its content. The vesicle can either fuse totally with the plasma membrane (full fusion) or create a fusion pore that stays open during a limited time (kiss-and-run)¹. For instance, newly synthesized proteins are released into the extracellular medium from vesicles that come from the Golgi complex. This biosynthetic, anterograde pathway is primordial, especially in multicellular organisms, to secrete signaling peptides (e.g., hormones, neurotransmitters) and extracellular matrix components (e.g., collagen), as well as to traffic transmembrane proteins to the plasma membrane. Additionally, secretions can occur from different endosomes: 1) recycling endosomes in order to reuse transmembrane proteins; 2) multivesicular bodies (MVBs) to release exosomes; and 3) lysosomes for the release of proteolytic enzymes. Endosomal secretion has been shown to be important for neurite outgrowth, pseudopodia formation, plasma membrane repair, and ATP-dependent signaling².

To study exocytosis at the single cell level, several techniques have been employed. Patch-clamp allows for the detection of single exocytosis events with a high temporal resolution in a wide variety of living cells³. However, this method does not provide information on the localization of exocytosis events, nor from which compartment it occurs. Electron microscopy allows direct visualization of exocytic events with high spatial resolution, and in combination with immunolabeling provides information about the specificity of the compartments and molecules involved. A disadvantage of this approach is the lack of information on the dynamics of the process, as well as its inability to perform high-throughput studies. Light microscopy approaches such as total internal reflection fluorescence microscopy (TIRFM), which exploits the evanescent field to illuminate fluorophores at the vicinity of the coverslip (100 nm), provides good temporal and spatial resolution to study exocytosis events. However, this method is only compatible with adherent cells and can only be applied to the ventral/inferior part of cells.

Of note, the plasma membrane reveals significant heterogeneity based on adhesive complexes that are present only in restricted areas. This heterogeneity restricts, for instance, the uptake of different ligands⁴. Similarly, it has recently been reported that secretion from the Golgi complex is concentrated at “hot spots” in the plasma membrane⁵. Moreover, it is known that certain cargos are secreted through focal-adhesion-associated exocytosis⁶. Thus, special attention should be paid to the question of whether exocytosis events are randomly distributed in space, or whether they are concentrated at specific areas of the plasma membrane. Several statistical tools based on Ripley’s K function have been proposed to explore these questions^7,8,9. Our approach combines these tools with micropatterning to control cell shape and plasma membrane heterogeneity. In addition to providing a means to distinguish between adhesive and nonadhesive areas, this technique also allows comparison across different cells and conditions and increases the power of statistical analyses.

Here we employ a variety of statistical tools to study the spatial distribution of exocytosis events from the lysosomal compartment monitored by TIRFM live cell imaging of VAMP7-pHluorin in ring-shaped micropattern-normalized hTert-RPE1 cells. It was confirmed that secretion from lysosomes is not a random process^8,9 and that exocytosis events exhibit clustering. Of note, we found that exocytosis events are also clustered in nonadhesive areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Nevertheless, cell adhesion did enhance clustering. Consistently, our analysis identified enriched areas of exocytosis that were located at the border between the adhesive and nonadhesive areas.

Protocol

1. Preparation of micropatterned cells

1. Transfection of cells
   1. One day before transfection, seed 2.5 x 10⁶ hTERT-RPE1 cells into one well of a 12 well plate (2 x 2 cm) in 1 mL of medium.
   2. On the day of transfection, prepare the transfection mixture with VAMP7-pHluorin plasmid (100 µL of buffer, 0.8 µg of DNA, 3 µL of transfection mixture). Incubate for 10 min.
      NOTE: VAMP7 is a lysosomal v-SNARE, fused with a luminal pHluorin tag. The pHluorin probe is quenched by low pH, but during exocytosis protons are released and pHluorin starts emitting a signal^10,11.
   3. Add the transfection mix to the cells in their medium.
   4. Change the medium 4 h after adding the transfection mix on the cells.
   5. Use the cells for experiments during the next 24–48 h.
2. Micropattern preparation (photolithography method)
   1. Wash the coverslips (25 mm of diameter) in ethanol and let them dry for 5 min.
   2. Activate coverslips by illumination under deep UV for 5 min.
   3. Create a humid chamber by thoroughly humidifying a paper towel on which a paraffin film is placed. Add drops (30 μL for 22 mm coverslip) of Poly-L-Lysine-graft-Polyethylene Glycol (PLL-g-PEG) solution (0.1 mg/mL, 10 mM HEPES, pH = 7.4) and place coverslips with the activated surface on them. Close the humid chamber with a top and incubate coverslips for 1 h.
   4. Wash coverslips 2x in PBS and 1x in distilled water and let them dry.
   5. Wash the quartz photomask with distilled water and then with ethanol or propanol. Dry the photomask with filtered airflow.
      NOTE: The quartz photomask is coated on one side with antireflective chrome that contains holes in the form of micropatterns. A photomask containing ring-shaped micropatterns of 37 µm is used in this protocol. When deep UV is shined on the photomask, the light can only pass through these holes¹².
   6. Expose the photomask (chrome-coated side) to deep UV for 5 min to clean the surface.
   7. Add small water drops (10 µL for a 20 mm coverslip) on the chrome-coated side of the photomask. Place the coverslip with their PLL-g-PEG-treated side on the drop and dry the extra water. Make sure that no air bubbles form between the mask and the coverslips.
      NOTE: The capillary force of the water will immobilize the coverslips.
   8. Expose the photomask to deep UV for 5 min with the non-chrome-coated side up (the coverslips are attached on the lower surface).
      NOTE: The light can only pass through the holes and modify the PLL-g-PEG-treated surface of coverslips below the photomask.
   9. Remove the coverslips from the photomask by adding excess water.
      NOTE: Coverslips should quickly float off.
   10. Incubate the coverslips in a solution of extracellular matrix proteins (50 μg/mL of fibronectin, 5 μg/mL of fluorescent fibrinogen diluted in water) on paraffin film in a humid chamber (as in step 1.2.3) for 1 h under a laminar flow hood to avoid contamination.
       NOTE: The experiment can be paused at this point by storing the coverslips in PBS at 4 °C.
3. Cell seeding on micropatterned surfaces
   1. Use a magnetic coverslip holder that fits the size of the micropatterned coverslips to mount the coverslips. On the day of acquisition, heat the coverslip holder to 37 °C to avoid thermal shock for the cells during subsequent steps.
   2. Prepare the pattern medium by supplementing DMEM/F12 medium with 20 mM HEPES and 2% of penicillin/streptomycin.
   3. Place coverslips into the holder with the micropatterned side up and add pattern medium as soon as the coverslip is on the holder base. Add the seal and immobilize with the magnetic device. Fill the coverslip holder with the pattern medium and close it with the glass lid.
      NOTE: Be quick, to not allow the coverslip to dry. Do not wash the coverslip holder with ethanol between experiments, because the seal might retain some ethanol, which can react with PLL-g-PEG and result in cell stress. Wash the coverslip holder only with soapy water. Moreover, the joint can be incubated in the pattern medium at 37 °C for 1 h to dilute residual product.
   4. Collect transfected hTERT-RPE1 cells by trypsinization (0.5 mL for one 12 well plate) and add 1 mL of 10% FBS DMEM/F12 medium.
   5. Add 0.5 x 10⁶ transfected hTERT-RPE1 cells to the coverslip holder and reclose it. Incubate for 10 min in the incubator.
   6. Wash the coverslip holder 5x with pattern medium to remove nonattached cells and residual FBS by adding the pattern medium with one pipette and aspirating the medium with another pipette to create a washing flow. Always keep a small volume of pattern medium in the coverslip holder to avoid drying of the cells on the micropatterned coverslip, which will lead to cell death.
   7. Incubate in the incubator for 3 h to allow full cell spreading.

2. Acquisition of exocytosis data

1. Imaging of exocytosis events
   1. Place coverslip holder under a TIRM. The signal has to be detected by a sensitive camera set up with the best imaging format available.
      NOTE: In this experiment, a 100x lens objective and an EMCCD camera with 512 x 512 pixel detection region was used giving rise to a pixel size of 160 nm.
   2. Search for a cell expressing VAMP7-pHluorin that is fully spread (Figure 1A).
      NOTE: Cells expressing VAMP7 are clearly identifiable, because they exhibit a green signal.
   3. Change the angle of the laser until a TIRF angle that allows the visualization of VAMP7-pHluorin exocytosis events is reached. Perform a 5 min acquisition at a frequency compatible with the exocytosis rate and time scale (typically 3 Hz, Figure 1D) using the microscope software.
      NOTE: hTERT-RPE1 cells have a lysosomal secretory rate of around 0.3 Hz on micropatterns. Lysosomal exocytosis has a typical duration of 1 s. It is characterized by a peak intensity followed by an exponential decay. The diffusion of the probe should be evident at this time (Figure 1B, C).
   4. For each cell, also perform an acquisition of the micropattern using the microscope software (Figure 1A).
2. Acquisition of exocytosis coordinates
   1. Open the acquired movie with ImageJ/FIJI. Use File | Import | Image Sequence. Find exocytosis events by eye. An exocytosis event is characterized by the appearance of a bright signal that spreads outwards (Figure 1).
   2. Use the point tool to mark the center of the exocytic event. Use Analyze | Measure to measure X and Y coordinates, as well as the temporal coordinate (slice number). Perform these measurements for all exocytosis events of the movie.
   3. Save the results (Results | File | Save As). Prepare a text file for each analyzed cell named “Results(cell_name).txt” that contains the slice, X coordinates, and Y coordinates for all exocytosis events in that order.
      
      The text file is supposed to look like this:
      ID         X          Y          Feret's diameter         Radius
      RPE1_WT_Cell1                      167      136      230      115
      RPE1_WT_Cell2                      164      160      230      115
      
      NOTE: Be careful to replace all commas with points.
   4. Measure the center and diameter of each cell using the “Oval Tool”. Fit a perfect circle (do not use an oval) and use “Measure” to obtain the X and Y coordinates and Feret's diameter. Save each cell’s identity (ID), X and Y coordinates, Feret’s diameter, and radius (diameter/2) in a text file named “Spherical parameter.txt”.
      
      The text file is supposed to look like this:
      ID         X          Y          Feret's diameter         Radius
      RPE1_WT_Cell1                      167      136      230      115
      RPE1_WT_Cell2                      164      160      230      115
      
      NOTE: Be careful to replace all commas with points.
   5. Measure the thickness of the micropattern ring (adhesion length) with the straight tool and save the cell ID, cell radius (from the file: “Spherical parameter.txt”), and adhesion length in a text file named “Pattern parameter.txt”. Calculate the normalized adhesion length by dividing the adhesion length by cell radius.
      NOTE: Be careful to replace all commas by points.
      
      The file should look like this:
      ID         Cell radius       adhesion length          Normalized adhesion length
      RPE1_WT_Cell1                      115      34        0.295652174
      RPE1_WT_Cell2                      115      35        0.304347826

3. Single cell spatial analysis

1. R package and installation
   NOTE: The R package for this analysis takes advantage of the Spatstat package¹³ to compute the two-dimensional (2D) density and Ripley’s K function. The code is open-source and uses text files that have been previously described.
   1. Download and install R from https://www.r-project.org/ (version 3.5.2 was used in this analysis).
   2. Download the package (and the demo dataset) from: https://github.com/GoudTeam/JoVE-paper
   3. Install the package on R Studio using “Tools” using “Install Packages”. Select “Package Archive File (.zip; .tar.gz)” for the category “Install from:” and choose the package file. Press “Install”.
   4. Load the package with the function “library("ExocytosisSpatialAnalysis")” by writing this command in R studio and pressing “Enter”.
   5. Run the package with the function “ESA()” by writing this command in R studio and pressing “Enter”.
      NOTE: A user interface will open.
   6. Select the directory for the dataset (.txt files) and a directory for output plots.
      NOTE: Parameters of the analysis (see text below) can be changed through a user interface.
   7. This script will automatically start and perform the analysis. It provides .pdf files of corresponding plots and .txt files containing numerical results. 

Results

The spatiotemporal characteristics of exocytosis events were analyzed from lysosomes visualized by VAMP7-pHluorin^10,11 in hTert-RPE1 cells. hTert-RPE1 cells are nontransformed cells that adopt well to micropatterning and have been extensively used in previous micropattern-based studies^4,14. VAMP7 is a lysosomal v-SNARE¹⁵ that was tagged with the super ecliptic pHluorin at its N-ter...

Discussion

We monitored exocytosis events from the lysosomal compartment by TIRFM live cell imaging of VAMP7-pHluorin in ring-shaped micropattern-normalized cells and performed a rigorous statistical analysis of the spatial parameters of exocytosis events. Employing the transformed Ripley’s K function and a statistical test based on the nearest neighbor distance, we confirmed that secretion from lysosomes is not a random process^8,9. Both statistical analyses convincin...

Materials

Name	Company	Catalog Number	Comments
Chamlide Magnetic Chamber	Chamlide
DMEM/F12	Gibco	21041-025
Fibrinogen	Molecular Probes, Invitrogen	F35200
Fibronectin bovine plasma	Sigma	F1141
HEPES (1M)	Gibco	15630-056
hTert RPE1 cell line	https://www.atcc.org
ImageJ	http://rsbweb.nih.gov/ij/	n/a	Authored by W. Rasband, NIH/NIMH
JetPRIME Transfection reagent	Polyplus	114-07
Penicilin/Streptomycin	Gibco	15140-122
Photomask	Delta Mask
PLL-g-PEG solution	Surface Solutions	PLL(20)-g[3.5]- PEG(2)
R Software	https://www.r-project.org/	n/a
Trypsin (TrypLE Express 1X)	Gibco	12605-010
UV ozone oven	Jelight Company Inc	342-220
VAMP7-pHFluorin plasmid	n/a	n/a	Paper reference :http://www.ncbi.nlm.nih.gov/pubmed/?term=Role+of+HRB+in+clathrin-dependent+endocytosis. J Biol Chem. 2008 Dec 5;283(49):34365-73. doi: 10.1074/jbc.M804587200. Role of HRB in clathrin-dependent endocytosis. Chaineau M, Danglot L, Proux-Gillardeaux V, Galli T.