Name: confocal imaging of neuropeptide y-phluorin: a technique to visualize insulin granule exocytosis in intact murine and human islets
Uploaded: 2017-09-13T13:00:00
Description: Watch this Scientific Journal Video about Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets at JoVE.com

The authors thank Marcia Boulina from the DRI imaging core facility for help with the microscopes. This work was supported by NIH grants 1K01DK111757-01 (JA), F31668418 (MM), R01 DK111538, R33 ES025673 and R56 DK084321 (AC).

The animal ethics committee of the University of Miami has approved all the experiments.
1. Viral Infection of Intact Isolated Human or Mouse Pancreatic Islets
<ol>
	<li>Islet culture: prepare the islet culture media: Connaught Medical Research Laboratories (CMRL) 1066, 10% (v/v) FBS and 2 mM L-Glutamine.
	<ol>
		<li>Human pancreatic islets are obtained from the Integrated Islet Distribution Program (NIDDK, NIH). Upon arrival, transfer the islets (~ 500 islet equivalents) to 35-mm non-tissue...

<ol>
	<li>Roder, P. V., Wong, X., Hong, W., Han, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+insulin+granule+biogenesis+and+exocytosis.">Molecular regulation of insulin granule biogenesis and exocytosis.</a> Biochem J. 473 (18), 2737-2756 (2016).</li><li>Rutter, G. A., Pullen, T. J., Hodson, D. J., Martinez-Sanchez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta-cell+identity,+glucose+sensing+and+the+control+of+insulin+secretion.">Pancreatic beta-cell identity, glucose sensing and the control of insulin secretion.</a> Biochem J. 466 (2), 203-218 (2015).</li><li>Rorsman, P., Renstrom, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+granule+dynamics+in+pancreatic+beta+cells.">Insulin granule dynamics in pancreatic beta cells.</a> Diabetologia. 46 (8), 1029-1045 (2003).</li><li>Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet.">Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet.</a> Science. 297 (5585), 1349-1352 (2002).</li><li>Ramirez, D. M., Khvotchev, M., Trauterman, B., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vti1a+identifies+a+vesicle+pool+that+preferentially+recycles+at+rest+and+maintains+spontaneous+neurotransmission.">Vti1a identifies a vesicle pool that preferentially recycles at rest and maintains spontaneous neurotransmission.</a> Neuron. 73 (1), 121-134 (2012).</li><li>Michael, D. J., Xiong, W., Geng, X., Drain, P., Chow, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+insulin+vesicle+dynamics+during+pulsatile+secretion.">Human insulin vesicle dynamics during pulsatile secretion.</a> Diabetes. 56 (5), 1277-1288 (2007).</li><li>Ohara-Imaizumi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+exocytosis+and+endocytosis+of+insulin+secretory+granules+in+the+pancreatic+beta-cell+line+MIN6+using+pH-sensitive+green+fluorescent+protein+(pHluorin)+and+confocal+laser+microscopy.">Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.</a> Biochem J. 363 (Pt 1), 73-80 (2002).</li><li>Whim, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta+cells+synthesize+neuropeptide+Y+and+can+rapidly+release+peptide+co-transmitters.">Pancreatic beta cells synthesize neuropeptide Y and can rapidly release peptide co-transmitters.</a> PLoS One. 6 (4), e19478 (2011).</li><li>Tsuboi, T., Rutter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forms+of+&amp;#34;kiss-and-run&amp;#34;+exocytosis+revealed+by+evanescent+wave+microscopy.">Multiple forms of &amp;#34;kiss-and-run&amp;#34; exocytosis revealed by evanescent wave microscopy.</a> Curr Biol. 13 (7), 563-567 (2003).</li><li>Zhu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin+I+and+IX+function+redundantly+in+controlling+fusion+pore+of+large+dense+core+vesicles.">Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles.</a> Biochem Biophys Res Commun. 361 (4), 922-927 (2007).</li><li>Aoki, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duration+of+fusion+pore+opening+and+the+amount+of+hormone+released+are+regulated+by+myosin+II+during+kiss-and-run+exocytosis.">Duration of fusion pore opening and the amount of hormone released are regulated by myosin II during kiss-and-run exocytosis.</a> Biochem J. 429 (3), 497-504 (2010).</li><li>Felmy, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+cargo+release+from+dense+core+granules+by+size+and+actin+network.">Modulation of cargo release from dense core granules by size and actin network.</a> Traffic. 8 (8), 983-997 (2007).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Gandasi, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+Red+Fluorescence+Proteins+as+Markers+for+Secretory+Granule+Exocytosis.">Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis.</a> PLoS One. 10 (6), e0127801 (2015).</li><li>Almaca, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+coordination+of+insulin+granule+exocytosis+in+intact+human+pancreatic+islets.">Spatial and temporal coordination of insulin granule exocytosis in intact human pancreatic islets.</a> Diabetologia. 58 (12), 2810-2818 (2015).</li><li>Do, O. H., Low, J. T., Thorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lepr(db)+mouse+model+of+type+2+diabetes:+pancreatic+islet+isolation+and+live-cell+2-photon+imaging+of+intact+islets.">Lepr(db) mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets.</a> J Vis Exp. (99), e52632 (2015).</li><li>Hanna, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kiss-and-run+exocytosis+and+fusion+pores+of+secretory+vesicles+in+human+beta-cells.">Kiss-and-run exocytosis and fusion pores of secretory vesicles in human beta-cells.</a> Pflugers Arch. 457 (6), 1343-1350 (2009).</li><li>Carter, J. D., Dula, S. B., Corbin, K. L., Wu, R., Nunemaker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+rodent+islet+isolation+and+assessment.">A practical guide to rodent islet isolation and assessment.</a> Biol Proced Online. 11, 3-31 (2009).</li><li>Weber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+transfection+of+isolated+pancreatic+islets:+a+study+of+programmed+cell+death+(apoptosis)+and+islet+function.">Adenoviral transfection of isolated pancreatic islets: a study of programmed cell death (apoptosis) and islet function.</a> J Surg Res. 69 (1), 23-32 (1997).</li><li>Michael, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+cargo+proteins+in+pancreatic+beta-cells:+design+determines+secretion+kinetics+at+exocytosis.">Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis.</a> Biophys J. 87 (6), L03-L05 (2004).</li><li>Serre-Beinier, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cx36+makes+channels+coupling+human+pancreatic+beta-cells,+and+correlates+with+insulin+expression.">Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression.</a> Hum Mol Genet. 18 (3), 428-439 (2009).</li><li>Rutter, G. A., Hodson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+cell+connectivity+in+pancreatic+islets:+a+type+2+diabetes+target?.">Beta cell connectivity in pancreatic islets: a type 2 diabetes target?.</a> Cell Mol Life Sci. 72 (3), 453-467 (2015).</li><li>Shen, Y., Rosendale, M., Campbell, R. E., Perrais, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pHuji,+a+pH-sensitive+red+fluorescent+protein+for+imaging+of+exo-+and+endocytosis.">pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis.</a> J Cell Biol. 207 (3), 419-432 (2014).</li><li>Speier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+in+vivo+imaging+of+pancreatic+islet+cell+biology.">Noninvasive in vivo imaging of pancreatic islet cell biology.</a> Nat Med. 14 (5), 574-578 (2008).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion+from+beta+cells+in+intact+mouse+islets+is+targeted+towards+the+vasculature.">Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature.</a> Diabetologia. 57 (8), 1655-1663 (2014).</li></ol>

This manuscript describes a technique that can be used to visualize exocytosis of insulin granules in beta cells within intact pancreatic islets by confocal microscopy. It uses NPY-pHluorin as the fluorescent reporter cloned into adenovirus to ensure a high transfection efficiency.
Although the method was highly efficient in our hands, it might require some modifications that primarily depend on two parameters: 1) the quality of islet preparation, and 2) the titer of viral stock with optimizat...

Insulin is produced by the beta cells of the pancreatic islet and it is a key regulator of glucose metabolism1. Death or dysfunction of beta cells disturbs glucose homeostasis and leads to diabetes2. Insulin is packed in dense-core granules that are released in a Ca2+-dependent manner3. Elucidating how insulin granule exocytosis is regulated is essential to fully understand what determines insulin secretion and opens new avenues for the identification of novel therapeutic targets for the treatment of diabetes.
Insulin exocytosis has been extensively studied using electrophysiological approaches, such as membrane capacitance measurements, and microscopic approaches in combination with fluorescent molecules. Membrane capacitance measurements have good temporal resolution and allow single cell recordings. However, changes in the capacitance reflect the net surface change of the cell and do not capture individual fusion events or distinguish insulin granule fusion from other non-insulin secretory vesicles3. Microscopic approaches, such as two-photon or total internal reflection fluorescence (TIRF) microscopy in combination with fluorescent probes and vesicle cargo proteins, provide additional detail. These techniques capture single exocytotic events and also the pre- and post-exocytotic stages and can be used for studying exocytotic patterns in populations of cells3.
Fluorescent reporters can be of three types: 1) extracellular, 2) cytoplasmic, or 3) vesicular. 1) Extracellular reporters are polar tracers (e.g., dextrans, sulforhodamine B (SRB), lucifer yellow, pyranine) that can be introduced through the extracellular milieu4. The use of polar tracers allows for the investigation of the fusion pore in a population of cells and captures various intercellular structures such as blood vessels. However, they do not report on vesicle cargo behavior. 2) Cytoplasmic reporters are fluorescent probes coupled to membrane-associated proteins that face the cytoplasm and are involved in docking and exocytosis. Examples include members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family that have been successfully used in neuroscience for studying neurotransmitter release5. Such proteins have multiple binding partners and are not insulin-granule specific. 3) Vesicular reporters are fluorescent probes fused to vesicular cargo proteins that allow for the investigation of cargo-specific vesicle behavior. Insulin-granule specific cargo proteins include insulin, c-peptide, islet amyloid polypeptide, and NPY among others6,7. NPY is only present in insulin containing granules, and is co-released with insulin, making it an excellent partner for a fluorescent reporter8.
The fusion of different fluorescent proteins to NPY has been previously employed to study various aspects of exocytosis in neuroendocrine cells, such as the requirement of specific synaptotagmin isoforms9,10 and how the time-course of release depends on the actin cytoskeleton and on myosin II11,12. In this study, we chose pHluorin as the fluorescent reporter, which is a modified GFP that is non-fluorescent at the acidic pH inside dense core granules but becomes brightly fluorescent upon exposure to the neutral extracellular pH13. Mature insulin granules have an acidic pH below 5.5. Once the granule fuses with the plasma membrane and opens, its cargo is exposed to the neutral extracellular pH of 7.4, allowing the use of the pH-sensitive proteins pHluorin as a reporter7,14.
In view of the pH sensitive nature of pHluorin and the selective expression of NPY in insulin granules, the NPY-pHluorin fusion construct can be used to study various properties of insulin granule exocytosis. The viral delivery of the fusion construct ensures high transfection efficiency and works on primary beta cells or cell lines as well as on isolated islets. This method can also be used as a guideline for studying exocytosis in any other cell type with NPY-containing vesicles. It can also be combined with any transgenic mouse model to study effects of certain conditions (knockdowns, overexpression, etc.) on exocytosis. This technique has been previously used to characterize spatial and temporal patterns of insulin granule secretion in beta cell populations in human islets15.

<table width="845">
	<tbody>
		<tr height="63">
			<td height="63" style="width:280px;height:63px;">Upright laser-scanning confocal microscope</td>
			<td style="width:232px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:167px;">TCS-SP5</td>
			<td style="width:167px;">includes LAS AF, the image acquisition software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">RC-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber platform</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">PH-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">22x40 glass coverslips</td>
			<td style="width:232px;">Daiggerbrand</td>
			<td style="width:167px;">G15972H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum silicone grease</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">Z273554-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Multichannel perfusion system</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">VC-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Single inline solution heater</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">SH-27B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Temperature controller</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">TC-324C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Peristaltic Suction pump</td>
			<td style="width:232px;">Pharmacia</td>
			<td style="width:167px;">P-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:280px;height:42px;">35 mm Petri dish, non-tissue culture treated</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">10861-586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">CMRL Medium, no glutamine</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">11530037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">FBS, heat inactivated</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">16140071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">L-Glutamine 200mM</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">5M NaCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">S5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3M KCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">60135</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M CaCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">21115</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M MgCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">M1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Bovine Serum Albumin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">1M HEPES solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum filter</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">431098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">D-Glucose</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Poly-D-lysine hydrobromide</td>
			<td style="width:232px;">Sigma-aldrich</td>
			<td style="width:167px;">P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Di-8-ANNEP</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">D3167</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3-isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">I5879</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Forskolin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">F3917</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

confocal imaging of neuropeptide y-phluorin: a technique to visualize insulin granule exocytosis in intact murine and human islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

The entire workflow of the technique is shown in Figure 1. Briefly, mouse or human islets can be infected with adenovirus encoding NPY-pHluorin and imaged, after few days in culture, under a confocal microscope. As granules fuse with plasma membrane and open, an increase in fluorescence is observed and can be quantified (Figure 1). To determine if NPY-pHluorin is indeed a suitable tool to monitor insulin granule dynamics, infecte...

Watch this Scientific Journal Video about Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets at JoVE.com

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

The overall goal of this assay is to visualize insulin granule exocytosis in real time in intact pancreatic islets by confocal microscopy. This method will answer key questions in the diabetes field. For example, what regulates insulin exocytosis?This is critical because granule fusion is the single most important event in the life of a better cell. The main advantage of this technique is that we can use intact islets, we can see fusion events in cells within their native environment. Madina Mahkmutova, a PhD student from our laboratory will be demonstrating the technique.The begin, upon arrival of pancreatic islets, transfer them to 50 milliliter falcon tubes and centrifuge the islets suspension at 1, 000 rotations per minute for five minutes. The islets will collect at the bottom of the tube, transfer the islets in two milliliters of CMRL culture medium to 35 millimeter non-tissue culture treated petri dishes and incubate them at 37 degree celsius and 5%carbon dioxide for 24 hours before viral infection. Following their isolation, re-suspend approximately mouse pancreatic islet equivalents in two milliliters of CMRL culture medium and transfer them to 35 millimeter non-tissue culture petri dishes.Incubate the tissue at 37 degree celsius and 5%carbon dioxide for 24 hours before viral infection. To carry out viral infection, add five to 10 microliters of stock virus to each 35 millimeter petri dish containing human or mouse islets in two milliliters of CMRL culture medium. After culturing the islets for 24 hours, aspirate the medium and replace it with two milliliters of CMRL culture medium without virus.Culture the islets for four to six days, replacing the medium every three days. Prepare extracellular solution by combining the reagents listed here and then filter sterilize the buffer. Make basal glucose medium to a three millimolar final glucose concentration, by adding 75 microliters of two molar glucose stock to 50 milliliters of extracellular solution.Then to prepare hyperglycemic medium to a 16 millimolar final glucose concentration, add 400 microliters of two molar glucose stock to 50 milliliters of extracellular solution. Dilute any additional stimuli and extracellular solution containing three millimolar glucose. Before starting an experiment, pre treat cover slips with 30 microliters of one milligram per milliliter poly-d-lysine solution and incubate for one hour.Then use water to thoroughly rinse the cover slips. At least one hour before the experiment, transfer a group of islets to a 35 millimeter petri dish containing extracellular solution with three millimolar glucose. Keep the remaining islets at 37 degrees celsius and 5%carbon dioxide.30 minutes before starting an experiment, attach the cover slip to the imaging chamber by using silicon grease to seal it. Then fix the imaging chamber to the imaging platform. Next, using a pipette, transfer 20 to 30 islets to the poly-d-lysine treated area of the cover slip and let the islets adhere to the surface for 20 minutes.This step is critical for the success of the experiment and we collect approximately 20 to 30 islets in less than 10 microliters of buffer and wait for 20 minutes. It is important to not let the cover slip dry completely, to avoid islet damage. While the islets are adhering to the cover slip, prepare the perfusion system by using water to thoroughly rinse it.Then add each solution to a different channel, according to this table. Remove all the bubbles was from the system by opening each channel separately and letting the solution flow for a few minutes. Ensure that the flow is consistent and the tubing is not leaking.Next, connect to the single inline solution heater to the perfusion outlet tube and adjust the temperature of the out flowing buffer to 37 degree celsius. Then, prepare the suction pump. Connect the tubing, turn on the pump and make sure the liquid is being aspirated.Place the imaging platform with the islets onto the microscope stage and connect it to the perfusion system and suction pump. Then, gently fill up the imaging chamber with the extracellular solution containing three millimolar glucose. To carry out confocal imaging, locate the islets in the microscope field with low magnification.Once focused on the islets, switch to higher magnification objectives. Open the acquisition software and activate the resident scanning mode. Select xyzt imaging, then to configure the acquisition settings, turn on the argon laser and set it to 30%and then the 488 nanometer laser line, adjusting the laser power to around 50%for pHluorin excitation.Collect emission at 505 to 555 nanometers, using the EGFP emission spectrum as guide. Choose a resolution of 512 by 512 pixels. Start imaging by pressing the live button and adjust the gain levels.Set the beginning end of the z stack by focusing on the top of islet and choosing begin and then moving to the last plane that can be focused and choosing n. Choose a z stack size of five micrometers. Set the time interval for acquisition of each z stack close to 1.5 to 2 seconds and choose the option, acquire until stopped for continuous imaging.Then, press the start button to initialize imaging. Refer to the text protocol for stimulation protocols to induce granule exocytosis. Using the resident scanner, confocal images of the islet are acquired as timelapse recordings in less than two seconds.In addition, this technique can be combined with dyes for functional imaging, like membra potential or cytosolic free calcium dyes. To determine if NP Y-pHluorin is indeed a suitable tool to monitor insulin granule dynamics, infected islets were immunostained with antibodies against GFP for NP Y-pHluorin and the islet hormones insulin, somatostatin or glucagon. Most cells expressing NP Y-pHluorin were beta cells as they also expressed insulin.Only a few glucagon positive alpha cells or somatostatin positive delta cells were GFP labeled. Importantly in infected cells, the NP Y fusion co-localized with insulin. This figure demonstrates that pHluorin is an effective pH sensor.As increasing the intercellular pH with 50 millimolar ammonium chloride resulted in a greater than 500%increase in the intercellular fluorescence. As the pH inside the granule increases upon fusion with the plasma membrane in the opening of the fusion pore, granules containing NP Y-pHluorin become visible under conditions that stimulate insulin exocytosis such as, membrane de-polarization with potassium chloride. Using confocal timelapse imaging of NP Y-pHluorin infected islets, single secretarial events in beta cells within intact islets can be visualized.These occur in response to direct membrane de-polarization with potassium chloride or several other physiological stimuli. At the basal extracellular glucose concentration, little secretarial activity is observed. However, granule fusion with the plasma membrane is triggered in response to stimulation with high glucose.Following this procedure, other methods like immunohistochemistry can be performed to answer additional questions like, why granule fusion occurs in specific regions of the cell. After its development, this technique has been used to explore the temporal pattern of insulin granule secretion in beta cell populations, in human islets.

confocal-imaging-neuropeptide-y-phluorin-technique-to-visualize

Research

JoVE Journal

Biology

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 ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

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.

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 ...