We greatly acknowledge Thierry Galli (Center of Psychiatry and Neurosciences, INSERM) for providing the VAMP7-pHluorin plasmid. We thank Tarn Duong for advice on statistical analysis and members of the GOUD laboratory for fruitful discussions. The authors greatly acknowledge the Cell and Tissue Imaging Facility (PICT-IBiSA @Burg, PICT-EM @Burg and PICT-IBiSA @Pasteur) and Nikon Imaging Center, Institut Curie (Paris), member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04). H.L. was supported by the Association pour la Recherche sur le Cancer (ARC) and P.M. received funding from the European Union&#8217;s Horizon 2020 research and innovation programme under Marie Sk&#322;odowska-Curie grant agreement No 666003. This work was supported by grants from INFECT-ERA (ANR-14-IFEC-0002-04), the Labex CelTisPhyBio (ANR-10-LBX-0038) and Idex Paris Sciences et Lettres (ANR-10-IDEX-0001-02 PSL), as well as the Centre National de la Recherche Scientifique and Institut Curie.&#160;

1. Preparation of micropatterned cells
<ol>
	<li>Transfection of cells
	<ol>
		<li>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.</li>
		<li>On the day of transfection, prepare the transfection mixture with VAMP7-pHluorin plasmid (100 &#181;L of buffer, 0.8 &#181;g of DNA, 3 &#181;L of transfection mixture). Incubate for 10 min. 
		NOTE: VAMP7 is a lysosomal v-SNARE, fused with a luminal pHluorin tag. The pHluorin probe is quen...

<ol>
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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&#8217;s K function and a statistical test based on the nearest neighbor distance, we confirmed that secretion from lysosomes is not a random process8,9. Both statistical analyses convincin...

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 &#8220;hot spots&#8221; 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&#8217;s K function have been proposed to explore these questions7,8,9. 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 process8,9 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.

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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">Chamlide Magnetic Chamber</td>
			<td style="width:259px;">Chamlide</td>
			<td style="width:207px;"></td>
			<td style="width:803px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">DMEM/F12</td>
			<td style="width:259px;">Gibco</td>
			<td style="width:207px;">21041-025</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">Fibrinogen</td>
			<td>Molecular Probes, Invitrogen</td>
			<td style="width:207px;">F35200</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">Fibronectin bovine plasma</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:207px;">F1141</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">HEPES (1M)</td>
			<td style="width:259px;">Gibco</td>
			<td style="width:207px;">15630-056</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">hTert RPE1 cell line</td>
			<td style="width:259px;">https://www.atcc.org</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">ImageJ</td>
			<td style="width:259px;">http://rsbweb.nih.gov/ij/</td>
			<td style="width:207px;">n/a</td>
			<td>Authored by W. Rasband, NIH/NIMH</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:244px;">JetPRIME Transfection reagent</td>
			<td style="width:259px;">Polyplus</td>
			<td style="width:207px;">114-07</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">Penicilin/Streptomycin</td>
			<td style="width:259px;">Gibco</td>
			<td style="width:207px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">Photomask</td>
			<td style="width:259px;">Delta Mask</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">PLL-g-PEG solution</td>
			<td style="width:259px;">Surface Solutions</td>
			<td style="width:207px;">PLL(20)-g[3.5]- PEG(2)</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">R Software</td>
			<td style="width:259px;">https://www.r-project.org/</td>
			<td style="width:207px;">n/a</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">Trypsin (TrypLE Express 1X)</td>
			<td>Gibco</td>
			<td style="width:207px;">12605-010</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">UV ozone oven</td>
			<td style="width:259px;">Jelight Company Inc</td>
			<td style="width:207px;">342-220</td>
			<td></td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;width:244px;">VAMP7-pHFluorin plasmid</td>
			<td style="width:259px;">n/a</td>
			<td style="width:207px;">n/a</td>
			<td style="width:803px;">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.</td>
		</tr>
	</tbody>
</table>

quantifying spatiotemporal parameters of cellular exocytosis in micropatterned cells

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&#8217;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.

The spatiotemporal characteristics of exocytosis events were analyzed from lysosomes visualized by VAMP7-pHluorin10,11 in hTert-RPE1 cells. hTert-RPE1 cells are nontransformed cells that adopt well to micropatterning and have been extensively used in previous micropattern-based studies4,14. VAMP7 is a lysosomal v-SNARE15 that was tagged with the super ecliptic pHluorin at its N-ter...

Watch this Scientific Journal Video about Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells at JoVE.com

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

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.

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

This protocol demonstrates how to investigate cell secretion using live imaging by TOF microscopy and provides tools for detecting clustering events or regions of high activity. Using this protocol, we can generate cell cultures on micropattern surfaces that normalize cell division and thus facilitate the special ranges of secretory events. Cells secrete a variety of macromolecules that play important roles in immune regulation.In cancer, deregulated secretion affects the release of proteolytic enzymes that facilitate invasion. The quantification of secretion will enable us to better understand degraded secretion in cancer and other dynamic processes such has antigen presentation. Although our imaging and analysis protocol is straightforward, it requires the generation of single cells that are well spread on a piece of micropattern.In this video, we will demonstrate how to culture cells on micropattern coverslips and how to visualize and analyze secretory events using spacial statistical tools. To prepare coverslips for micropatterning, activate ethanol sterilized 25 millimeter diameter glass coverslips under deep ultraviolet light for five minutes. While the coverslips are being activated, add one 30 microliter drop of polylysine graft polyethylene glycol per coverslip onto a piece of paraffin film within a humid chamber.At the end of the activation, place the coverslips activated surface side down onto the drops and cover the humid chamber for a one-hour incubation at room temperature. At the end of the incubation, wash the coverslips two times in distilled water. While the coverslips are drying, wash a quartz photo mask one time with distilled water and one time with alcohol.Dry the photo mask with filtered air flow and expose the photo mask chrome coated side up to deep ultraviolet light for five minutes. At the end of the illumination, add a 10 microliter drop of water onto the chrome coated side of each photo mask and place the polyethylene glycol side of each coverslip onto each drop of water. After removing any excess water, place a film cover over the slides to avoid dehydration.Expose the non-chrome coated sides of the photo masks to deep ultraviolet light for an additional five minutes before adding excess water to the photo masks to remove the coverslips. This step will produce the micropattern imprints on the surface. Then incubate the coverslips in an extracellular matrix protein solution on paraffin film in a humid chamber for one hour under a laminar flow hood.For cell seeding onto the prepared micropattern surface, at the end of the incubation, place the coverslips into a magnetic coverslip holder micropattern side up and immediately add pattern medium to the coverslips. After adding the seal, immobilize the coverslips with the magnetic device before filling the coverslip holder with additional pattern medium. Then close the holder with a glass lid.When the cells are ready, add 5 times 10 to the 5th of the transfected immortalized retinal pigment epithelial cells to the coverslips and incubate the cells in the holder for 10 minutes in the cell culture incubator. At the end of the incubation, use one pipette to aspirate the old medium while using a second pipette to wash the cells five times with fresh medium per wash to remove the non-attached cells and any residual fetal bovine serum. After the last wash, return the holder to the incubator for three hours to allow full cell spreading.To image exocytosis events, place the coverslip holder under a total internal reflection fluorescence microscope, and search for a cell expressing VAMP7-pHluorin that is fully spread. Change the angle of the laser until a total internal reflection fluorescence angle that allows the visualization of VAMP7-pHluorin exocytosis events is reached and perform a five-minute acquisition at a frequency compatible with the exocytosis rate and timescale. To obtain the exocytosis coordinates, open the acquired exocytosis event movie in Fiji and manually identify the exocytosis events by the appearance of a bright signal that spreads outward.Use the point tool to mark the center of the exocytosis event and use analyze and measure to measure the x and y-coordinates as well as the temporal coordinate. Save the measurements of one cell in one text file. Open text file in a spreadsheet and remove all but the slice x and y-coordinate columns.Next, use the oval tool and measure to measure the center and diameter and to obtain the x and y-coordinates and Feret's diameter of each cell. Save the measurements of all cells in one text file. Open the text file in a spreadsheet and remove all but the x and y-coordinate and Feret's diameter columns.Then add the radius measurement for each cell, then obtain the radius from the Feret's diameter for each cell. Use the straight line tool to measure the thickness of the micropattern ring of each cell. Save this measurement for all cells in one text file, then divide the adhesion length by the cell radius to calculate the normalized adhesion length.For single cell spatial analysis, first install the package of RStudio and load the package in RStudio. Run the package with the ESA function. A user interface will open, then select the directory for the dataset TXT files and a directory for the output plots.The script will automatically start and perform the analysis, providing PDF files of the corresponding plots and TXT files containing the numerical results. Inside the cell, the floor in probe is quenched by the low pH of the lysosome, but during exocytosis, pHluorin begins to emit a signal as the pH increases due to proton release. The pHluorin signal exhibits a peak during exocytosis that represents the fast release of lysosomal protons, followed by an exponential signal decay that represents the 2D diffusion of the probe at the plasma membrane.Typically, transfect immortalized human retinal pigmented epithelial cells demonstrate an important lysosomal secretion activity with an average exocytosis rate of 0.28 Hertz. It is possible to visualize the 2D distribution of exocytosis by kernel density estimation to reveal differences in local densities. There are three possible deviations from complete spatial randomness:clustering, dispersion, or a mixture of clustering and dispersion.Ripley's K function can be used to assess these deviations. Of note, exocytosis events in the non-adhesive area are also clustered indicating that adhesion molecules are not the only structures that induce secretory hotspots at plasma membranes. Plotting the histogram of exocytosis events according to the modulus for a representative cell reveals a peak around the border between the adhesive and non-adhesive areas.Paired analysis demonstrates that the surface density is lower in the adhesion area than in the non-adhesion area, potentially due to the strong decrease of exocytosis at the cell periphery. Combining cell adhesion on micropatterns with statistical analysis facilitates the ability to pinpoint how complex cellular processes such as secretion are regulated in space. More future work is needed to determine which molecular mechanism that underlie the clustering of secretory activity.

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6.0K Views.  Institut Curie, 75005 Paris, France. This protocol demonstrates how to investigate cell secretion using live imaging by TOF microscopy and provides tools for detecting clustering events or regions of high activity. Using this protocol, we can generate cell cultures on micropattern surfaces that normalize cell division and thus facilitate the special ranges of secretory events. Cells secrete a variety of macromolecules that play important roles in immune regulation.In cancer, deregulated secretion affects the release of pr...