Hostname: page-component-cd9895bd7-gbm5v Total loading time: 0 Render date: 2024-12-26T22:11:42.212Z Has data issue: false hasContentIssue false
  • English
  • Français

Observer-Independent Quantification of Insulin Granule Exocytosis and Pre-Exocytotic Mobility by TIRF Microscopy

Published online by Cambridge University Press:  13 November 2013

Magnus Matz ,
Kirstin Schumacher ,
Kathrin Hatlapatka ,
Dirk Lorenz ,
Knut Baumann  and
Ingo Rustenbeck
Magnus Matz
Affiliation:
Institute of Medicinal and Pharmaceutical Chemistry, University of Braunschweig, Braunschweig D38106, Germany
Kirstin Schumacher
Affiliation:
Institute of Pharmacology and Toxicology, University of Braunschweig, Braunschweig D38106, Germany
Kathrin Hatlapatka
Affiliation:
Institute of Pharmacology and Toxicology, University of Braunschweig, Braunschweig D38106, Germany
Dirk Lorenz
Affiliation:
Institute of Analysis and Algebra, University of Braunschweig, Braunschweig D38106, Germany
Knut Baumann*
Affiliation:
Institute of Medicinal and Pharmaceutical Chemistry, University of Braunschweig, Braunschweig D38106, Germany
Ingo Rustenbeck*
Affiliation:
Institute of Pharmacology and Toxicology, University of Braunschweig, Braunschweig D38106, Germany
*
*Corresponding author. E-mail: k.baumann@tu-bs.de
**Corresponding author. E-mail: i.rustenbeck@tu-bs.de
Get access

Abstract

Total internal reflection fluorescence microscopy of fluorescently labeled secretory granules permits monitoring of exocytosis and the preceding granule behavior in one experiment. While observer-dependent evaluation may be sufficient to quantify exocytosis, most of the other information contained in the video files cannot be accessed this way. The present program performs observer-independent detection of exocytosis and tracking of the entire submembrane population of insulin granules. A precondition is the exact localization of the peak of the granule fluorescence. Tracking is based on the peak base radius, peak intensity, and the precrossing itineraries. Robustness of the tracking was shown by simulated tracks of original granule patterns. Mobility in the X–Y dimension is described by the caging diameter which in contrast to the widely used mean square displacement has an inherent time resolution. Observer-independent detection of exocytosis in MIN6 cells labeled with insulin-EGFP is based on the maximal decrease in fluorescence intensity and position of the centroid of the dissipating cloud of released material. Combining the quantification of KCl-induced insulin exocytosis with the analysis of prefusion mobility showed that during the last 3 s pre-exocytotic granules had a smaller caging diameter than control granules and that it increased significantly immediately before fusion.

Keywords

automated granule detectionfluorescence quantificationexocytosisinsulin secretionTIRF microscopy
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 1 , February 2014 , pp. 206 - 218
Copyright
Copyright © Microscopy Society of America 2014 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Atkins, P.W. & Paula, J. (2006). Physical Chemistry. Oxford, UK: Oxford University Press.Google Scholar
Bai, L., Wang, Y., Fan, J., Chen, Y., Ji, W., Qu, A., Xu, P., James, D.E. & Xu, T. (2007). Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action. Cell Metab 5, 47–57.CrossRefGoogle ScholarPubMed
Barg, S., Eliasson, L., Renström, E. & Rorsman, P. (2002). A subset of 50 secretory granules in close contact with L-type Ca2+ channels accounts for first-phase insulin secretion in mouse beta-cells. Diabetes 51(Suppl 1), S74–S82.Google Scholar
Becherer, U., Pasche, M., Nofal, S., Hof, D., Matti, U. & Rettig, J. (2007). Quantifying exocytosis by combination of membrane capacitance measurements and total internal reflection fluorescence microscopy in chromaffin cells. PLoS One 2, e505. CrossRefGoogle ScholarPubMed
Burchfield, J.G., Lopez, J.A., Mele, K., Vallotton, P. & Hughes, W.E. (2010). Exocytotic vesicle behaviour assessed by total internal reflection fluorescence microscopy. Traffic 11, 429–439.CrossRefGoogle ScholarPubMed
Dean, P.M. (1973). Ultrastructural morphometry of the pancreatic beta-cell. Diabetologia 9, 115–119.Google Scholar
Degtyar, V.E., Allersma, M.W., Axelrod, D. & Holz, R.W. (2007). Increased motion and travel, rather than stable docking, characterize the last moments before secretory granule fusion. Proc Nat Aca Sci USA 104, 15929–15934.CrossRefGoogle ScholarPubMed
Gerber, S.H. & Südhof, T.C. (2002). Molecular determinants of regulated exocytosis. Diabetes 51(Suppl 1), S3–S11.CrossRefGoogle ScholarPubMed
Hatlapatka, K., Matz, M., Schumacher, K., Baumann, K. & Rustenbeck, I. (2011). Bidirectional insulin granule turnover in the submembrane space during K(+) depolarization-induced secretion. Traffic 12, 1166–1178.CrossRefGoogle Scholar
Henquin, J.C. (2000). Triggering and amplifying pathways of regulation of insulin secretion by glucose. Diabetes 49, 1751–1760.CrossRefGoogle ScholarPubMed
Ivarsson, R., Obermüller, S., Rutter, G.A., Galvanovskis, J. & Renström, E. (2004). Temperature-sensitive random insulin granule diffusion is a prerequisite for recruiting granules for release. Traffic 5, 750–762.Google Scholar
Jaqaman, K., Loerke, D., Mettlen, M., Kuwata, H., Grinstein, S., Schmid, S.L. & Danuser, G. (2008). Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5, 6695–6702.CrossRefGoogle ScholarPubMed
Kasai, K., Fujita, T., Gomi, H. & Izumi, T. (2008). Docking is not a prerequisite but a temporal constraint for fusion of secretory granules. Traffic 9, 1191–1203.Google Scholar
Krzanowski, W.J. (2000). Principles of Multivariate Analysis: A User's Perspective. Oxford, UK: Oxford University Press.CrossRefGoogle Scholar
Lam, A.D., Ismail, S., Wu, R., Yizhar, O., Passmore, D.R., Ernst, S.A. & Stuenkel, E.L. (2010). Mapping dynamic protein interactions to insulin secretory granule behavior with TIRF-FRET. Biophys J 99, 1311–1320.Google Scholar
Lindau, M. & Neher, E. (1988). Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflugers Arch 411, 137–146.CrossRefGoogle ScholarPubMed
Luby-Phelps, K., Mujumdar, S., Mujumdar, R.B., Ernst, L.A., Galbraith, W. & Waggoner, A.S. (1993). A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm. Biophys J 65, 236–242.Google Scholar
Ma, L., Bindokas, V.P., Kuznetsov, A., Rhodes, C., Hays, L., Edwardson, J.M., Ueda, K., Steiner, D.F. & Philipson, L.H. (2004). Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion. Proc Natl Acad Sci USA 101, 9266–9271.CrossRefGoogle ScholarPubMed
Mather, P.M. (2004). Computer Processing of Remotely-Sensed Images: An Introduction. Hoboken, NJ: Wiley.Google Scholar
Mele, K., Coster, A., Burchfield, J.G., Lopez, J., James, D.E., Hughes, W.E. & Vallotton, P. (2009). Automatic identification of fusion events in TIRF microscopy image sequences. In Computer Vision Workshops (ICCV Workshop Proceedings), pp. 578–584, Tokyo: IEEEXplore.Google Scholar
Michael, D.J., Geng, X., Cawley, N.X., Loh, Y.P., Rhodes, C.J., Drain, P. & Chow, R.H. (2004). Fluorescent cargo proteins in pancreatic beta-cells: Design determines secretion kinetics at exocytosis. Biophys J 87, L03–L05.Google Scholar
Michael, D.J., Xiong, W., Geng, X., Drain, P. & Chow, R.H. (2007). Human insulin vesicle dynamics during pulsatile secretion. Diabetes 56, 1277–1288.CrossRefGoogle ScholarPubMed
Michalet, X. (2010). Mean square displacement analysis of single-particle trajectories with localization error: Brownian motion in an isotropic medium. Phys Rev E Stat Nonlin Soft Matter Phys 82, 041914. Google Scholar
Nagamatsu, S., Ohara-Imaizumi, M., Nakamichi, Y., Kikuta, T. & Nishiwaki, C. (2006). Imaging docking and fusion of insulin granules induced by antidiabetes agents: Sulfonylurea and glinide drugs preferentially mediate the fusion of newcomer, but not previously docked, insulin granules. Diabetes 55, 2819–2825.Google Scholar
Nesher, R. & Cerasi, E. (2002). Modeling phasic insulin release: Immediate and time-dependent effects of glucose. Diabetes 51(Suppl 1), S53–S59.Google Scholar
Nofal, S., Becherer, U., Hof, D., Matti, U. & Rettig, J. (2007). Primed vesicles can be distinguished from docked vesicles by analyzing their mobility. J Neurosci 27, 1386–1395.CrossRefGoogle ScholarPubMed
Ohara-Imaizumi, M., Aoyagi, K., Nakamichi, Y., Nishiwaki, C., Sakurai, T. & Nagamatsu, S. (2009). Pattern of rise in subplasma membrane Ca2+ concentration determines type of fusing insulin granules in pancreatic beta cells. Biochem Biophys Res Commun 385, 291–295.CrossRefGoogle ScholarPubMed
Ohara-Imaizumi, M., Nakamichi, Y., Tanaka, T., Ishida, H. & Nagamatsu, S. (2002). Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy: Distinct behavior of granule motion in biphasic insulin release. J Biol Chem 277, 3805–3808.Google Scholar
Ohara-Imaizumi, M., Nishiwaki, C., Kikuta, T., Nagai, S., Nakamichi, Y. & Nagamatsu, S. (2004). TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: Different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells. Biochem J 381, 13–18.CrossRefGoogle ScholarPubMed
Oheim, M., Loerke, D., Chow, R.H. & Stühmer, W. (1999). Evanescent-wave microscopy: A new tool to gain insight into the control of transmitter release. Philos Trans R Soc Lond B Biol Sci 354, 307–318.Google Scholar
Pedersen, M.G. & Sherman, A. (2009). Newcomer insulin secretory granules as a highly calcium-sensitive pool. Proc Natl Acad Sci USA 106, 7432–7436.CrossRefGoogle ScholarPubMed
Phillips, G.R. & Harris, J.M. (1990). Polynomial filters for data sets with outlying or missing observations: Application to charge-coupled-device-detected Raman spectra contaminated by cosmic rays. Anal Chem 62, 2351–2357.CrossRefGoogle Scholar
Pouli, A.E., Emmanouilidou, E., Zhao, C., Wasmeier, C., Hutton, J.C. & Rutter, G.A. (1998). Secretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaera. Biochem J 333, 193–199.CrossRefGoogle ScholarPubMed
Qian, H., Sheetz, M.P. & Elson, E.L. (1991). Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys J 60, 910–921.Google Scholar
Rorsman, P. & Renström, E. (2003). Insulin granule dynamics in pancreatic beta cells. Diabetologia 46, 1029–1045.Google Scholar
Saffman, P.G. & Delbrück, M. (1975). Brownian motion in biological membranes. Proc Natl Acad Sci USA 72, 3111–3113.Google Scholar
Savitzky, A. & Golay, M.J.E. (1964). Smoothing and differentiation of data by simplified least squares procedures. Anal Chem 36, 1627–1639.Google Scholar
Sebastian, R., Diaz, M.E., Ayala, G., Letinic, K., Moncho-Bogani, J. & Toomre, D. (2006). Spatio-temporal analysis of constitutive exocytosis in epithelial cells. IEEE/ACM Trans Comput Biol Bioinform 3, 17–32.CrossRefGoogle ScholarPubMed
Seksek, O., Biwersi, J. & Verkman, A.S. (1997). Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus. J Cell Biol 138, 131–142.Google Scholar
Shibasaki, T., Takahashi, H., Miki, T., Sunaga, Y., Matsumura, K., Yamanaka, M., Zhang, C., Tamamoto, A., Satoh, T., Miyazaki, J. & Seino, S. (2007). Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP. Proc Natl Acad Sci USA 104, 19333–19338.Google Scholar
Solimena, M. & Speier, S. (2010). Insulin release: Shedding light on a complex matter. Cell Metab 12, 5–6.CrossRefGoogle ScholarPubMed
Steyer, J.A. & Almers, W. (1999). Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy. Biophys J 76, 2262–2271.CrossRefGoogle ScholarPubMed
Takahashi, N., Hatakeyama, H., Okado, H., Noguchi, J., Ohno, M. & Kasai, H. (2010). SNARE conformational changes that prepare vesicles for exocytosis. Cell Metab 12, 19–29.Google Scholar
Tsuboi, T. & Rutter, G.A. (2003). Multiple forms of “kiss-and-run” exocytosis revealed by evanescent wave microscopy. Curr Biol 13, 563–567.Google Scholar
Verkman, A.S. (2002). Solute and macromolecule diffusion in cellular aqueous compartments. Trends Biochem Sci 27, 27–33.Google Scholar
Wang, Z. & Thurmond, D.C. (2009). Mechanisms of biphasic insulin-granule exocytosis—Roles of the cytoskeleton, small GTPases and SNARE proteins. J Cell Sci 122, 893–903.CrossRefGoogle ScholarPubMed
Willenborg, M., Belz, M., Schumacher, K., Paufler, A., Hatlapatka, K. & Rustenbeck, I. (2012). Ca(2+)-dependent desensitization of insulin secretion by strong potassium depolarization. Am J Physiol Endocrinol Metab 303, E223–E233.CrossRefGoogle ScholarPubMed
Supplementary material: File

Matz et al. Supplementary Material

Supplementary Material

Download Matz et al. Supplementary Material(File)
File 19.1 KB
Supplementary material: PDF

Matz et al. Supplementary Material

Figures

Download Matz et al. Supplementary Material(PDF)
PDF 411.7 KB
Supplementary material: PDF

Matz et al. Supplementary Material

Documentation

Download Matz et al. Supplementary Material(PDF)
PDF 422.3 KB

Matz et al. Supplementary Material

Video

Download Matz et al. Supplementary Material(Video)
Video 2.7 MB