Name: automated detection and analysis of exocytosis
Uploaded: 2021-09-11T14:30:00
Description: Watch this Scientific Journal Video about Automated Detection and Analysis of Exocytosis at JoVE.com

We thank Dustin Revell and Reginald Edwards for testing code and the GUI. Funding was provided by the National Institutes of Health supported this research: including R01NS112326 (SLG), R35GM135160 (SLG), and F31NS103586 (FLU).

NOTE:&#160;The original Exocytosis Analysis GUI was written and compiled in Matlab version 9.10 (2021a). New versions of MATLAB have required updates to the GUI, which are available for download from our website: https://guptonlab.web.unc.edu/code/
1. Choose datasets and directory
<ol>
	<li>To select datasets for analysis, click the Find Datasets button (Figure 2A, red box 1) to navigate to the folder where data are deposited (e...

<ol>
	<li>Miesenb&#246;ck, 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>Urbina, F. L., Gomez, S. M., Gupton, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+organization+of+exocytosis+emerges+during+neuronal+shape+change.">Spatiotemporal organization of exocytosis emerges during neuronal shape change.</a> Journal of Cell Biology. 217 (3), 1113-1128 (2018).</li><li>Urbina, F. L., 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=TRIM67+regulates+exocytic+mode+and+neuronal+morphogenesis+via+SNAP47.">TRIM67 regulates exocytic mode and neuronal morphogenesis via SNAP47.</a> Cell Reports. 34 (6), 108743 (2021).</li><li>Alabi, A. A., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+kiss-and-run:+Role+in+exocytosis,+endocytosis,+and+neurotransmission.">Perspectives on kiss-and-run: Role in exocytosis, endocytosis, and neurotransmission.</a> Annual Review of Physiology. 75, 393-422 (2013).</li><li>Albillos, A., 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=The+exocytotic+event+in+chromaffin+cells+revealed+by+patch+amperometry.">The exocytotic event in chromaffin cells revealed by patch amperometry.</a> Nature. 389 (6650), 509-512 (1997).</li><li>He, L., Wu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+debate+on+the+kiss-and-run+fusion+at+synapses.">The debate on the kiss-and-run fusion at synapses.</a> Trends in Neuroscience. 30 (9), 447-455 (2007).</li><li>Elhamdani, A., Azizi, F., Artalejo, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+patch+clamp+reveals+that+transient+fusion+(kiss-and-run)+is+a+major+mechanism+of+secretion+in+calf+adrenal+chromaffin+cells:+high+calcium+shifts+the+mechanism+from+kiss-and-run+to+complete+fusion.">Double patch clamp reveals that transient fusion (kiss-and-run) is a major mechanism of secretion in calf adrenal chromaffin cells: high calcium shifts the mechanism from kiss-and-run to complete fusion.</a> Journal of Neuroscience. 26 (11), 3030 (2006).</li><li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+forms+of+single-vesicle+astrocyte+exocytosis+imaged+with+total+internal+reflection+fluorescence+microscopy.">Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (10), 4212-4217 (2007).</li><li>Holroyd, P., Lang, T., Wenzel, D., De Camilli, P., Jahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+direct,+dynamin-dependent+recapture+of+fusing+secretory+granules+on+plasma+membrane+lawns+from+PC12+cells.">Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (26), 16806-16811 (2002).</li><li>Wang, C. 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=Different+domains+of+synaptotagmin+control+the+choice+between+kiss-and-run+and+full+fusion.">Different domains of synaptotagmin control the choice between kiss-and-run and full fusion.</a> Nature. 424 (6951), 943-947 (2003).</li><li>Winkle, C. C., Hanlin, C. C., Gupton, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+combined+methodologies+to+define+the+role+of+plasma+membrane+delivery+during+axon+branching+and+neuronal+morphogenesis.">Utilizing combined methodologies to define the role of plasma membrane delivery during axon branching and neuronal morphogenesis.</a> Journal of Visualized Experiments. (109), e53743 (2016).</li><li>Viesselmann, C., Ballweg, J., Lumbard, D., Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+and+primary+culture+of+embryonic+mouse+hippocampal+and+cortical+neurons.">Nucleofection and primary culture of embryonic mouse hippocampal and cortical neurons.</a> Journal of Visualized Experiments. (47), e2373 (2011).</li><li>Plooster, 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=TRIM9-dependent+ubiquitination+of+DCC+constrains+kinase+signaling,+exocytosis,+and+axon+branching.">TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching.</a> Molecular Biology of the Cell. 28 (18), 2374-2385 (2017).</li><li>Urbina, F. L., Gupton, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNARE-mediated+exocytosis+in+neuronal+development.">SNARE-mediated exocytosis in neuronal development.</a> Frontiers in Molecular Neuroscience. 13, 133 (2020).</li><li>Ripley, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second-order+analysis+of+stationary+point+processes.">The second-order analysis of stationary point processes.</a> Journal of Applied Probability. 13 (2), 255-266 (1976).</li><li>Liu, A., 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=pHmScarlet+is+a+pH-sensitive+red+fluorescent+protein+to+monitor+exocytosis+docking+and+fusion+steps.">pHmScarlet is a pH-sensitive red fluorescent protein to monitor exocytosis docking and fusion steps.</a> Nature Communication. 12 (1), 1413 (2021).</li></ol>

When using the exocytic detection and analysis software, please consider that the program only accepts lossless compression .tif files as input. The .tif image files may be 8-bit, 16-bit, or 32-bit grayscale (single channel) images. Other image formats must be converted into one of these types before input. For reference, examples used here are 16-bit grayscale images.
Inherent in the automated detection process, the timelapse image sets are processed for the automated background subtraction a...

The article <a href="http://www.jove.com/video/62400/">Automated Detection and Analysis of Exocytosis</a>&#160;has been retracted by the journal due to concerns regarding the validity of the presented data.

The article Automated Detection and Analysis of Exocytosis has been retracted by the journal due to concerns regarding the validity of the presented data.

Retraction: Automated Detection and Analysis of Exocytosis

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62400">Automated Detection and Analysis of Exocytosis</a>. The original Exocytosis Analysis GUI was written and compiled in Matlab version 9.10 (2021a). New versions of MATLAB have required updates to the GUI, which are available for download from our website: https://guptonlab.web.unc.edu/code/

Erratum: Automated Detection and Analysis of Exocytosis

VAMP-pHluorin constructs or transferrin receptor (TfR)-pHuji constructs are excellent markers of exocytic events, as these pH-sensitive fluorophores are quenched within the acid vesicle lumen and fluoresce immediately upon fusion pore opening between the vesicle and plasma membrane1. Following fusion pore opening, fluorescence decays exponentially, with some heterogeneity that reveals information about the fusion event. Here, a graphical user interface (GUI) application is described that automatically detects and analyzes exocytic events. This application allows the user to automatically detect exocytic events revealed by pH sensitive markers2 and generate features from each event that can be used for classification purposes3 (Figure 1A). In addition, analysis of exocytic event clustering using Ripley&#39;s K function is described.
The automated classification of exocytic events into different exocytic modes was recently reported3. Two modes of exocytosis, full-vesicle fusion (FVF) and kiss-and-run fusion (KNR) exocytosis have previously been described4,5,6,7. During FVF, the fusion pore dilates, and the vesicle is incorporated into the plasma membrane. During KNR, the fusion pore transiently opens and then reseals4,5,8,9,10. Four modes of exocytosis were identified in developing neurons, two related to FVF and two related to KNR. This work demonstrates that both FVF and KNR can be further subdivided into fusion events that proceed immediately to fluorescence decay (FVFi and KNRi) after fusion pore opening or exocytic events that exhibit a delay after fusion pore opening before fluorescence decay begins (FVFd and KNRd) (Figure 1B). The classifier identifies the mode of exocytosis for each fusion event. Here this analysis has been incorporated into a GUI that can be installed in MATLAB in Windows and Mac based operating systems. All analysis files can be found at&#160;https://drive.google.com/drive/folders/1VCiO-thMEd4jz-tYEL8I4N1Rf_zjnOgB?usp=sharing or 
https://github.com/GuptonLab.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>https://www.mathworks.com/products/matlab.html</td>
		</tr>
		<tr>
			<td>R</td>
			<td>R Core Team</td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td>Rstudio, PBC</td>
			<td></td>
			<td>https://rstudio.com/</td>
		</tr>
	</tbody>
</table>

automated detection and analysis of exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

Here the GUI (Figure 2A) was utilized to analyze exocytic events from three VAMP2-pHluorin expressing neurons at 3 DIV using TIRF (total internal reflection fluorescence) microscopy. E15.5 cortical neurons were isolated, followed by transfection with VAMP2-pHluorin and plating using the protocols as outlined in Winkle et al., 2016 and Viesselmann et al., 201111,12. The methodology of imaging parameters is as outlined in Urbina et al....

Watch this Scientific Journal Video about Automated Detection and Analysis of Exocytosis at JoVE.com

Automated Detection and Analysis of Exocytosis

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...

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