We thank Dr. U. Ashery for the generous gift of cDNA. We thank Drs. G. Mass, L. Mittleman, M. Shaharbani, and Y.Zilberstein from the &#160;Sackler&#160;Cellular &#38; Molecular Imaging Center for their invaluable assistance with microscopy.&#160;This work was supported by the United States-Israel Binational Science Foundation (grant 2013263 to R. Sagi-Eisenberg, I. Hammel, and S.J.Galli) and grant 933/15 from the Israel Science Foundation, founded by the Israel Academy for Sciences (to R.Sagi-Eisenberg) and NIH grants U19 AI 104209 and R01 AR067145 (to S.J. Galli).

1. Preparations
<ol>
	<li>Preparation of RBL culture media
	<ol>
		<li>Mix 500 mL of low glucose Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 56 mL of fetal bovine serum (FBS), 5.5 mL of penicillin-streptomycin-nystatin solution, 5.5 mL of L-Glutamine 200 mM solution. This results in low glucose DMEM supplemented with 10% FBS, 100 &#181;g/mL streptomycin, 100 units/mL penicillin, 12 units/mL nystatin, and 2 mM L-glutamine.</li>
		<li>Filter the media by using a top-vacuum filter of 0.22 &#181;m pore size and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Maintenance of RBL cells.
	<ol>
		<li>Grow RBL cells to a maximum confluency of 90% in a 10 cm dish. If the cell culture is healthy, the cells should have a spindle shape with occasional protrusions.</li>
		<li>For cell splitting, detach the cells from the dish by aspirating the media and replacing with 2 mL of trypsine/EDTA solution B. Incubate for 5-10 minutes in a humidified atmosphere of 5% CO2 at 37 &#176;C.</li>
		<li>Once the cells have detached, neutralize the trypsin by adding 2 mL of culture media, using a pipette, and split the cells in a 1:2 - 1:10 ratio.</li>
	</ol>
	</li>
	<li>Preparation of 20x Tyrode&#39;s buffer
	<ol>
		<li>Prepare a stock 20x solution of 54 mM KCl, 20 mM MgCl2, 2.74 M NaCl, and 8 mM NaH2PO4 in double distilled water (DDW). Mix well and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Culture of RBL Cells for Live Cell Microscopy
<ol>
	<li>Preparation of FITC-dextran solution.
	<ol>
		<li>Mix 1 mg of FITC-dextran powder (150 K) per 1 mL of culture media (see step 1.1.1). For a full chambered coverglass, prepare 3 mL.</li>
		<li>Using a cellulose acetate syringe filter unit with 0.22 &#181;m pore size, filter the dissolved FITC-dextran.</li>
		<li>Add mouse IgE to a concentration of 1 &#181;g/mL.</li>
	</ol>
	</li>
	<li>Seeding RBL cells for imaging
	<ol>
		<li>The day before imaging, aspirate the media from the culture dish and replace with 2 mL of trypsine/EDTA solution B. Incubate for 5 - 10 minutes in a humidified atmosphere of 5% CO2 at 37 &#176;C. Once the cells have detached, neutralize the trypsin by adding 2 mL of culture media.</li>
		<li>Count RBL cells using a hemocytometer and adjust the volume accordingly with culture media to get a cell concentration of 7.5 x 105/mL.</li>
		<li>Add 10 &#181;L of cell suspension to a chambered coverglass pre-filled with fresh FITC-dextran supplemented culture media (this results in seeding of 7.5 x 103 cells in a chamber).</li>
		<li>Grow RBL cells overnight in a humified atmosphere of 5% CO2 at 37 &#176;C. The cells should remain in a sub-confluent level in order to make sure that the cells are separated and that it is easy to identify each cell individually under the microscope.</li>
	</ol>
	</li>
	<li>Transfection of RBL cells - optional.
	<ol>
		<li>For imaging exocytic events in combination with other fluorescently tagged proteins, see transfection protocol for RBL cells in Azouz et al.67</li>
	</ol>
	</li>
</ol>
3. Live Cell Microscopy of Exocytosis
<ol>
	<li>Preparation of solutions:
	<ol>
		<li>Prepare a final Tyrode&#39;s buffer solution by diluting the stock solution in DDW in a 1:20 dilution and supplement with 20 mM Hepes pH 7, 1.8 mM CaCl2, 1 mg/mL BSA, and 5.6 mM glucose.</li>
		<li>Freshly prepare a 20x secretagogue reagent in Tyrode&#39;s buffer [1 &#181;g/mL dinitrophenyl conjugated to human serum albumin (DNP-HAS (Ag)) in our case, for a 50 ng/mL 1x concentration].</li>
		<li>Prepare a 400 mM ammonium chloride solution by dissolving powder in Tyrode&#39;s buffer.</li>
	</ol>
	</li>
	<li>Preparation of the cells.
	<ol>
		<li>Wash the chambered coverglass 3 times by aspirating the media from the chamber and refilling it with 300 &#181;L of Tyrode&#39;s buffer, prewarmed to 37 &#176;C. Finally, replenish the chamber with 300 &#181;L of Tyrode&#39;s buffer, prewarmed to 37 &#176;C.</li>
		<li>Place the chambered coverglass in the microscope&#39;s incubator chamber. Make sure that the chamber is stable.</li>
	</ol>
	</li>
	<li>Setting up the microscope
	<ol>
		<li>To choose a region of interest to track, turn on the fluorescent light source (traditionally a mercury lamp) and choose the appropriate fluorescence filter (choose the filter for green fluorophores for viewing FITC-dextran). Once the region of interest is in focus and is in the middle of the field of view, turn off the light source in order to avoid photo-bleaching and toxicity. 
		NOTE: Some of the FITC-dextran incorporated in the cells may retain fluorescence. This is because FITC-dextran may also be sorted to non-lysosomal compartments such as endosomes.</li>
		<li>Turn on the appropriate lighting for FITC excitation. If using a laser-based microscope, turn on the 488 nm laser. Emission should be gathered around 500 - 550 nm (FITC emission peaks at 510 - 520 nm).</li>
		<li>When using a confocal microscope, open the pinhole to the maximum. This will allow using lower laser power to avoid bleaching and toxicity and will ensure the capture of exocytosis events from all planes of the cell.</li>
		<li>Calibrate the time interval between image acquisitions.
		<ol>
			<li>Different cells may vary in their kinetics of regulated exocytosis68. For specific imaging of compound exocytosis, we recommend a time interval of at least 5 seconds. However, as a rule of thumb, to allow for fast imaging, make sure that the acquisition time of a single frame is fast.</li>
			<li>When using a laser-scanning confocal microscope, set the scan direction to bi-directional, do not allow for averaging, and set resolution at 512 x 512 (recommended; the latter two provisions will also help to minimize bleaching and toxicity).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Imaging of exocytosis.
	<ol>
		<li>Image cells for desired durations, depending on the type of cell or secretagogue. In RBL cells triggered with IgE/Ag, most exocytic events occur within 15 - 20 min of activation.</li>
		<li>For activation of the cells, add 16 &#181;L from the 20x secretagogue solution to the chamber.</li>
	</ol>
	</li>
	<li>Confirming FITC-dextran presence in the cells.
	<ol>
		<li>To confirm the presence and localization of FITC-dextran to the SGs, add 16 &#181;L of ammonium chloride solution (be careful not to move the chamber in order not to lose focus) to the chamber gently (this will make a final concentration of 20 mM). This will induce alkalization of the SGs and dequenching of FITC fluorescence, which will occur within seconds.</li>
	</ol>
	</li>
</ol>

The authors declare no competing financial interest

Here we describe how tracing the fluorescence of FITC-dextran loaded into SGs can be used to specifically capture compound exocytosis events. This was achieved by setting the microscope to acquire an image every 15 seconds, thus ensuring that only long-lasting events will be recorded over time, and therefore excluding short events that would correspond to full exocytosis or kiss-and-run exocytosis. To establish the method, we showed that knockdown of the Rab5 isoforms that are expressed in RBL cells, and are pivotal for ...

Regulated exocytosis is the primary mechanism by which readily made cargo is released from a secretory cell in response to a specific trigger. The cargo is pre-formed and sequestered into secretory vesicles, in which it is stored until a trigger relays the signal for the release of the SGs&#39; content. Different types of signals may result in different modes of regulated exocytosis, or different modes of regulated exocytosis may occur simultaneously. Three main modes of regulated exocytosis are known: full exocytosis, which involves full fusion of a single secretory granule with the plasma membrane; kiss-and-run exocytosis, which involves transient fusion of the secretory granule with the plasma membrane followed by its recycling; and compound exocytosis, which is characterized by homotypic fusion of several SGs prior (i.e., multigranular exocytosis) or sequential (i.e., sequential exocytosis) to fusion with the plasma membrane1. Compound exocytosis is considered the most extensive mode of cargo release2, as it allows for the fast secretion of cargo, including that from SGs that are located distal from the plasma membrane. Compound exocytosis has been documented in both exocrine and endocrine cells3,4,5,6,7,8,9, as well as in immune cells. In immune cells, such as eosinophils10,11,12 and neutrophils13, compound exocytosis allows the fast and robust release of mediators that are required to kill invading pathogens such as bacteria or parasites. Mast cells (MCs) deploy compound exocytosis for the efficient release of pre-stored inflammatory mediators during innate immune responses, anaphylaxis,&#160;and other allergic reactions14,15,16,17. Since the different modes of exocytosis may occur simultaneously18,19, it has become a challenge to distinguish between them in real time or to identify their respective fusion machineries, hence elucidating their underlying mechanisms.
Here we present a method, based on live cell imaging of cell loaded FITC-dextran, that allows real time tracking of exocytic events and distinguishing between their different modes. In particular, our method allows exclusive monitoring of compound exocytosis.
FITC-dextran is a conjugate of the pH-sensitive fluorophore FITC with the glucan polysaccharide dextran. Fluorescently labeled dextrans have been shown to enter the cell by micropinocytosis20,21 and macropinocytosis22,23. As endocytic compartments mature into lysosomes, it has been shown that FITC-dextran accumulates in the lysosome with no apparent degradation. However, since FITC is a highly pH-sensitive fluorophore24, and the lysosome lumen is acidic, FITC-dextran fluorescence quenches upon reaching the lysosome24. Thus, establishing dextrans as lysosome targeted cargo, taken together with the pH sensitivity of FITC, have laid the foundation for the use of FITC-dextran in studies of lysosome exocytosis25,26,27,28,29.
In several cell types, including MCs, neutrophils, eosinophils, cytotoxic T cells, melanosomes, and others, the SGs display lysosomal features and are classified as lysosome-related organelles (LROs) or secretory lysosomes30,31. Since LROs have an acidic luminal pH, FITC-dextran can be used to visualize their exocytosis, as a result of higher pH associated with the exteriorization of the LROs. Indeed, FITC-dextran has been used to monitor exocytosis in MCs18,32,33. In this method, FITC-dextran is added to the cell culture, taken up by the cells by pinocytosis and sorted into the SGs. As it is in lysosomes, FITC fluorescence is quenched in the SGs when they are within the cell. However, upon SG fusion with the plasma membrane and consequent exposure to the external milieu, the FITC-dextran regains its fluorescence as the SG pH rises, allowing the simple tracking of exocytic events by live cell microscopy. Here, we adjusted this method to enable unique tracking of compound exocytosis.
Two other methods have been used previously to track compound exocytosis. Electron microscopy was the first method to characterize exocytic structures that suggested the occurrence of different modes of exocytosis. In particular, observations of &#34;secretory tunnels&#34; in pancreatic acinar cells34 and MCs35,36,37 gave rise to the hypothesis of compound exocytosis. However, while the high resolution of electron microscopy has the power to reveal fused vesicles, it cannot track the dynamics of their fusion and hence can&#39;t define whether they correspond to SG fusion during compound exocytosis or fusion of recaptured granules following their endocytosis. This obstacle is overcome in other methods that can measure exocytosis in living cells, such as patch clamp measurements of the plasma membrane capacitance11,13,38,39 or amperometry40 of the media. However, patch clamping requires a special set-up and may not be suitable for all cell types. Amperometry measurements are able to track exocytosis only if the cargo is released in very close proximity to the electrode. Therefore, using live cell imaging offers an advantage over these methods, as it not only allows for real time tracking of exocytosis, but it also allows quick and simple acquisition of data from the whole cell.
The tracking of FITC-dextran by live cell microscopy also offers some advantages to other live cell imaging-based methods. For example, a widely used method is total internal reflection fluorescence microscopy (TIRFM) of cells loaded with a fluorescent SG probe or expressing a fluorescent protein-tagged SG cargo or membrane protein26,41,42,43. The strength of this method lies in its ability to monitor exclusively events that occur close to the plasma membrane (herein referred to as footprint), hence exocytic events. However, this is also the drawback of this method because only the cell fraction that is adjacent to the coverglass and close to the microscope lens can be imaged44. Whether such footprints indeed represent the entire cell membrane surface is still debatable45,46,47. In this regard, using a pH-sensitive dye such as FITC-dextran and a standard fluorescence microscope or a confocal microscope with an open pinhole allows imaging of the whole cell, thus capturing the total exocytic events that occur in that cell.
Additional pH-sensitive reporters that are used to study regulated exocytosis by whole cell imaging or TIRFM include SG cargo or SG membrane protein fused to phlourin, a pH-sensitive GFP variant. Examples include NPY-phlourin-, &#946;-hexoseaminidase-phlourin, and synapto-phluorin48,49,50,51,52. While expression of these probes may represent more closely the endogenous composition of the SGs, it entails transfection of the cells, and may therefore be less suitable for cells that are difficult to transfect. Therefore, when studying cells that are difficult to transfect or under experimental conditions that require multiple genomic manipulations, the use of a compound that can be simply supplemented into the cell culture medium, such as FITC-dextran, is advantageous. FITC-dextran also offers an advantage over acridine orange (AO), another pH-sensitive dye that has been used for the tracking of exocytosis by live cell microscopy53,54,55,56,57,58. AO has been shown to induce photolysis of vesicles that result in false flashes, which do not correspond to actual secretion processes27. In contrast, FITC-dextran reflects better secretion events, probably due to its low photo-induced production of reactive oxygen27.
Notably, an alternative approach for studying exocytosis is by tracking the influx of a dye, from the external medium into the SG through the fusion pore that opens during this process. In this case, the dye is added to the external medium alongside the secretory trigger. Then, when the fusion pore opens, the dye diffuses into the SG59,60. A clear advantage of this method is that it also offers the ability to estimate the fusion pore size, by the use of dyes of variable size. For example, dextrans of different molecular weight (MW), conjugated to different fluorophores, can be used as extracellular dyes whereby the maximum size of dextran that can penetrate the SG would correspond to the size of the fusion pore59,61,62,63,64. In addition, this approach does not require the use of a pH-sensitive probe. However, a significant disadvantage is that the signal to noise ratio is very low, since a large amount of dye is present in the media during acquisition of images, resulting in high background.
Overall, the use of FITC-dextran as a marker for exocytosis overcomes several drawbacks in previously reported methods, such as signal to noise ratio, toxicity, dynamic tracking and complexity.
Here we describe the use of FITC-dextran to monitor compound exocytosis in the RBL-2H3 mast cell line (herein referred to as RBL, primarily established by Eccleston et al.65 and further cloned by Barsumian et al.66), in response to immunoglobulin E (IgE)/antigen (Ag) activation.

<table><tbody><tr><td>DMEM low glucose</td><td>Biological Inductries</td><td>01-050-1A</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>12657</td><td></td></tr><tr><td>Pen-strep-nystatin solution</td><td>Biological Inductries</td><td>03-032-1B</td><td></td></tr><tr><td>L-Glutamine 200 mM solution&amp;nbsp;</td><td>Biological Inductries</td><td>03-020-1A</td><td></td></tr><tr><td>FITC dextran 150K</td><td>Sigma-Aldrich</td><td>46946-500MG-F</td><td></td></tr><tr><td>Trypsin/EDTA Solution B</td><td>Biological Inductries</td><td>03-052-1A</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Top-vacuum filter of 0.22 &amp;micro;m pore size&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CLS430769-1EA</td><td></td></tr><tr><td>Cellulose acetate syringe filter unit, 0.22 &amp;micro;m pore size</td><td>Sartorius</td><td>16534K</td><td></td></tr><tr><td>Chambered coverglass&amp;nbsp;</td><td>Thermo scientific</td><td>155411</td><td></td></tr><tr><td>Corning tissue-culture treated culture dishes</td><td>Sigma-Aldrich</td><td>CLS430167</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A4503</td><td></td></tr><tr><td>Anti-DNP monoclonal IgE</td><td>Sigma-Aldrich</td><td>D8406</td><td></td></tr><tr><td>DNP-HSA (Ag)</td><td>Sigma-Aldrich</td><td>A6661&amp;nbsp;</td><td>Avoid direct light exposure</td></tr><tr><td>Hepes buffer 1M, pH 7.4</td><td>Biological Inductries</td><td>03-025-1B</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>MERK</td><td>102382</td><td></td></tr><tr><td>Glucose</td><td>BDH Laboratories</td><td>284515V</td><td></td></tr><tr><td>Ammonium chloride</td><td>MERK</td><td>1145</td><td></td></tr><tr><td>PIPES dipotassium salt</td><td>Sigma-Aldrich</td><td>108321-27-3</td><td></td></tr><tr><td>Calcium acetate hydrate</td><td>Sigma-Aldrich</td><td>114460-21-8</td><td></td></tr><tr><td>Magnesium acetate tetrahydrate</td><td>Sigma-Aldrich</td><td>M5661</td><td></td></tr><tr><td>Potassium glutamate (L-Glutamic acid potassium salt monohydrate)</td><td>Sigma-Aldrich</td><td>G1501</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>MERK</td><td>6346</td><td></td></tr><tr><td>NaCl</td><td>MERK</td><td>106404</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>MERK</td><td>105833</td><td></td></tr><tr><td>KCl</td><td>MERK</td><td>104936</td><td></td></tr><tr><td>Electroporator&amp;nbsp;</td><td>BTX</td><td>ECM830</td><td></td></tr><tr><td>Confocal microscope, Zeiss</td><td>Zeiss</td><td>LSM 5 Pascal, Axiovert 200M</td><td>Used in Figure 3. Equipped with an electronic temperature-controlled&lt;br/&gt; airstream incubator</td></tr><tr><td>Confocal microscope, Zeiss</td><td>Zeiss</td><td>LSM 800, Axio Observer.Z1 /7</td><td>Used in Figure 2. Equipped with a GaAsP detector an electronic temperature-controlled&lt;br/&gt; airstream incubator</td></tr><tr><td>Confocal microscope, Leica</td><td>Leica</td><td>SP5</td><td>Used in Figure 1. Equipped with a leica HyD detector and an top-stage incubator (okolab)</td></tr><tr><td>RBL-2H3 cells</td><td></td><td></td><td>RBL-2H3 cells were cloned in the lab of Reuben P. Siraganian. See reference 67</td></tr></tbody></table>

imaging fitc-dextran as a reporter for regulated exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Figure 1a represents schematically how FITC-dextran may act as a reporter for regulated exocytosis and recapitulate the different modes of exocytic events. First, cells are incubated with FITC-dextran, which is internalized by pinocytosis and sorted to the SGs. Since the SGs of MCs are LROs, their low pH dampens the fluorescence of FITC, shown here as black granules (A-C, I). When cells are triggered by a secretagogue and the SGs fuse with the plasma membrane...

Watch this Scientific Journal Video about Imaging FITC-dextran as a Reporter for Regulated Exocytosis at JoVE.com

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

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Endocytosis and Exocytosis

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12.5K Views.  Tel Aviv University. Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

Video: Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Neuroscience

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

An Introduction to Endocytosis and Exocytosis

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes involve lipid membrane-bound sacs called vesicles. Knowledge of the molecular architecture and mechanisms of both is key to understanding normal cell physiology, as well as the disease states that arise when they become defective. This video will first briefly review a few pivotal discoveries in the history of endo- and exocytosis research. Next, some key questions will be examined, followed by a discussion of the prominent methods used to investigate these problems, including cell labeling, fusion assays, and fluorescence imaging. Finally, it will explore current research being conducted by scientists in the field today.

Endocytosis and Exocytosis; Clathrin Coated Pits and SNAREs

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes ...