Abstract
Due to the ultra-thin optical sectioning capability of exclusively illuminating space at the interface where total internal reflection occurs, the TIRF microscope has been indispensable for monitoring biological processes adjacent to the plasma membrane with excellent signal-to-noise ratio. Insulin-containing granules fuse with the plasma membrane to release contents within hundreds of milliseconds, which involves well-orchestrated assembly of SNARE complex and associated proteins. A video-rate multiple-color TIRF microscope offers the unique opportunity to visualize single secretory granule docking and fusion dynamics and can also map its regulators with high spatiotemporal resolution. Here, we describe the basic principles and practical implementation of a fast dual-color TIRF microscope, detailing a how-to guide on imaging and analysis of insulin granule dynamics in human β-cells.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gaisano HY (2017) Recent new insights into the role of SNARE and associated proteins in insulin granule exocytosis. Diabetes Obes Metab 19(Suppl 1):115–123. https://doi.org/10.1111/dom.13001
Thurmond DC, Gaisano HY (2020) Recent insights into Beta-cell exocytosis in type 2 diabetes. J Mol Biol 432(5):1310–1325. https://doi.org/10.1016/j.jmb.2019.12.012
Liang T, Qin T, Kang F, Kang Y, Xie L, Zhu D, Dolai S, Greitzer-Antes D, Baker RK, Feng D, Tuduri E, Ostenson CG, Kieffer TJ, Banks K, Pessin JE, Gaisano HY (2020) SNAP23 depletion enables more SNAP25/calcium channel excitosome formation to increase insulin exocytosis in type 2 diabetes. JCI Insight 5(3):e129694. https://doi.org/10.1172/jci.insight.129694
Axelrod D (2008) Chapter 7 Total internal reflection fluorescence microscopy. In: Biophysical tools for biologists, Volume Two: In Vivo Techniques. Methods in Cell Biology. Elsevier Science, Amsterdam, pp 169–221. https://doi.org/10.1016/s0091-679x(08)00607-9
Axelrod D (2001) Total internal reflection fluorescence microscopy in cell biology. Traffic (Copenhagen, Denmark) 2(11):764–774. https://doi.org/10.1034/j.1600-0854.2001.21104.x
Lipson A, Lipson SG, Lipson H (2010) Optical physics. Cambridge University Press, Cambridge
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019
Gandasi NR, Vesto K, Helou M, Yin P, Saras J, Barg S (2015) Survey of red fluorescence proteins as markers for secretory granule exocytosis. PLoS One 10(6):e0127801. https://doi.org/10.1371/journal.pone.0127801
Li WH (2017) Probes for monitoring regulated exocytosis. Cell Calcium 64:65–71. https://doi.org/10.1016/j.ceca.2017.01.002
Zhu D, Koo E, Kwan E, Kang Y, Park S, Xie H, Sugita S, Gaisano HY (2013) Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells. Diabetologia 56(2):359–369. https://doi.org/10.1007/s00125-012-2757-0
Aoki R, Kitaguchi T, Oya M, Yanagihara Y, Sato M, Miyawaki A, Tsuboi T (2010) Duration of fusion pore opening and the amount of hormone released are regulated by myosin II during kiss-and-run exocytosis. Biochem J 429(3):497–504. https://doi.org/10.1042/bj20091839
Gandasi NR, Barg S (2014) Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site. Nat Commun 5:3914. https://doi.org/10.1038/ncomms4914
Xie L, Zhu D, Dolai S, Liang T, Qin T, Kang Y, Xie H, Huang YC, Gaisano HY (2015) Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells. Diabetologia 58(6):1250–1259. https://doi.org/10.1007/s00125-015-3545-4
Acknowledgments
This work was supported by operating grants from the Canadian Institute for Health Research PJT-159741 and PJT-148652, equipment and human islets from the Canadian Foundation for Innovation Projects no 19442 (Center for Diet, Digestive Tract and Disease) and 33603 (Centre for Islet Research and Therapeutics), respectively, and a Tier 1 Canada Research Chair.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kang, F., Gaisano, H.Y. (2022). Imaging Insulin Granule Dynamics in Human Pancreatic β-Cells Using Total Internal Reflection Fluorescence (TIRF) Microscopy. In: Shen, J. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 2473. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2209-4_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2209-4_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2208-7
Online ISBN: 978-1-0716-2209-4
eBook Packages: Springer Protocols