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Cited by 23Cellular transport and secretionAdvanced Fluorescence Microscopy TechniquesPancreatic function and diabetes

Mapping Dynamic Protein Interactions to Insulin Secretory Granule Behavior with TIRF-FRET

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Lam, Alice D., Ismail, Sahar, Wu, Ray, Yizhar, Ofer, Passmore, Daniel R., Ernst, Stephen A., Stuenkel, Edward L.·2010·Biophysical Journal
DOI 10.1016/j.bpj.2010.06.014PMC PMC2920663
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Abstract

Biological processes are governed by extensive networks of dynamic molecular interactions. Yet, establishing a spatial and temporal map of these interactions and their direct relationship to specific cell functions has remained a challenge. Here, we implement sensitized emission Förster resonance energy transfer (FRET) stoichiometry under total internal reflection fluorescence (TIRF) microscopy. We demonstrate through quantitative analysis and modeling that evanescent fields must be precisely matched between FRET excitation wavelengths to isolate dynamic interactions between bimolecular FRET pairs that are not entirely membrane-delimited. We then use TIRF-FRET to monitor the behavior of individual insulin-containing secretory granules at the plasma membrane of living cells, while simultaneously tracking the dynamic interaction between the GTPase Rab27A and its effector Slp4A, on those same granules. Notably, insulin granules that underwent exocytosis demonstrated a specific increase in Rab27A-GTP/Slp4A FRET in the 5 s before membrane fusion, which coincided temporally with an increase in granule displacement and mobility. These results demonstrate an initial spatiotemporal mapping of a dynamic protein-protein interaction on individual secretory granules that is linked to a specific granule behavior in living cells.

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  • Rab27A is associated with insulin secretory granules and is involved in granule docking, influences insulin exocytosis, and is essential for replenishment and release of an immediately releasable pool of secretory granules.

    “Rab27A is associated with the insulin secretory granules (362, 724, 766) and the data suggest it is involved in granule docking with the plasma membrane (338), influences insulin exocytosis (444) and is essential for the replenishment and release of an immediately releasable pool of secretory granules (110).”

    From Pancreatic β-Cell Electrical Activity and Insulin Secretion: Of Mice and Men (2018) · cites this paper as [362]

    Citation (110) is attached to the claim about replenishment and release of an immediately releasable pool. The Results state that Rab27A regulates granule priming and that Rab27A-GTP facilitates insulin secretion, but they do not establish that Rab27A is essential for replenishing or releasing that specific pool. No page number is available in our copy. (LLM, conf=0.84)

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  1. [1]1. Sørensen, J.B. Formation, stabilization and fusion of the readily releasable pool of secretory vesicles Pflugers Arch. 2004; 448:347-362 Crossref PubMed Google Scholar
  2. [2]2. Jahn, R. ∙ Lang, T. ∙ Südhof, T.C. Membrane fusion Cell. 2003; 112:519-533 Full Text Full Text (PDF) Scopus (1280) PubMed Google Scholar
  3. [3]3. Jahn, R. ∙ Scheller, R.H. SNAREs—engines for membrane fusion Nat. Rev. Mol. Cell Biol. 2006; 7:631-643 Crossref Scopus (2072) PubMed Google Scholar
  4. [4]4. Rizo, J. ∙ Rosenmund, C. Synaptic vesicle fusion Nat. Struct. Mol. Biol. 2008; 15:665-674 Crossref Scopus (423) PubMed Google Scholar
  5. [5]5. Oheim, M. ∙ Loerke, D. ... The last few milliseconds in the life of a secretory granule. Docking, dynamics and fusion visualized by total internal reflection fluorescence microscopy (TIRFM) Eur. Biophys. J. 1998; 27:83-98 Crossref Scopus (165) PubMed Google Scholar
  6. [6]6. Steyer, J.A. ∙ Almers, W. Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy Biophys. J. 1999; 76:2262-2271 Full Text Full Text (PDF) Scopus (200) PubMed Google Scholar
  7. [8]8. Periasamy, A. ∙ Day, R.N. Molecular Imaging: FRET Microscopy and Spectroscopy Oxford University Press, New York, 2005 Google Scholar
  8. [9]9. Oheim, M. Imaging transmitter release. II. A practical guide to evanescent-wave imaging Lasers Med. Sci. 2001; 16:159-170 Crossref Scopus (34) PubMed Google Scholar
  9. [10]10. Jaiswal, J.K. ∙ Simon, S.M. Imaging single events at the cell membrane Nat. Chem. Biol. 2007; 3:92-98 Crossref Scopus (113) PubMed Google Scholar
  10. [11]11. Greaves, J. ∙ Duncan, R.R. ... Use of ANF-EGFP for the visualization of secretory vesicles in bovine adrenal chromaffin cells Ann. N. Y. Acad. Sci. 2002; 971:275-276 Crossref Scopus (3) PubMed Google Scholar
  11. [12]12. Axelrod, D. ∙ Burghardt, T.P. ∙ Thompson, N.L. Total internal reflection fluorescence Annu. Rev. Biophys. Bioeng. 1984; 13:247-268 Crossref Scopus (581) PubMed Google Scholar
  12. [13]13. Bal, M. ∙ Zaika, O. ... Calmodulin binding to M-type K+ channels assayed by TIRF/FRET in living cells J. Physiol. 2008; 586:2307-2320 Crossref Scopus (38) PubMed Google Scholar
  13. [14]14. Fowler, C.E. ∙ Aryal, P. ... Evidence for association of GABAB receptors with Kir3 channels and regulators of G protein signaling (RGS4) proteins J. Physiol. 2007; 580:51-65 Crossref Scopus (96) PubMed Google Scholar
  14. [15]15. Khakh, B.S. ∙ Fisher, J.A. ... An Ångstrom scale interaction between plasma membrane ATP-gated P2X2 and α4β2 nicotinic channels measured with fluorescence resonance energy transfer and total internal reflection fluorescence microscopy J. Neurosci. 2005; 25:6911-6920 Crossref Scopus (87) PubMed Google Scholar
  15. [16]16. Riven, I. ∙ Iwanir, S. ∙ Reuveny, E. GIRK channel activation involves a local rearrangement of a preformed G protein channel complex Neuron. 2006; 51:561-573 Full Text Full Text (PDF) Scopus (96) PubMed Google Scholar
  16. [17]17. Dovas, A. ∙ Gevrey, J.C. ... Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages J. Cell Sci. 2009; 122:3873-3882 Crossref Scopus (86) PubMed Google Scholar
  17. [18]18. Beemiller, P. ∙ Hoppe, A.D. ∙ Swanson, J.A. A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during Fcγ receptor-mediated phagocytosis PLoS Biol. 2006; 4:e162 Crossref Scopus (107) PubMed Google Scholar
  18. [19]19. Hoppe, A. ∙ Christensen, K. ∙ Swanson, J.A. Fluorescence resonance energy transfer-based stoichiometry in living cells Biophys. J. 2002; 83:3652-3664 Full Text Full Text (PDF) Scopus (299) PubMed Google Scholar
  19. [20]20. Mattheyses, A.L. ∙ Axelrod, D. Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence J. Biomed. Opt. 2006; 11:014006 Crossref Scopus (105) Google Scholar
  20. [21]21. Oheim, M. ∙ Loerke, D. ... Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999; 354:307-318 Crossref Scopus (55) PubMed Google Scholar
  21. [22]22. van Manen, H.J. ∙ Verkuijlen, P. ... Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy Biophys. J. 2008; 94:L67-L69 Full Text Full Text (PDF) Scopus (117) PubMed Google Scholar
  22. [23]23. Axelrod, D. Total internal reflection fluorescence microscopy Methods Cell Biol. 1989; 30:245-270 Crossref Scopus (268) PubMed Google Scholar
  23. [24]24. Axelrod, D.D. Total internal reflection fluorescence microscopy in cell biology Traffic. 2001; 2:764-774 Crossref Scopus (783) PubMed Google Scholar
  24. [25]25. Allersma, M.W. ∙ Wang, L. ... Visualization of regulated exocytosis with a granule-membrane probe using total internal reflection microscopy Mol. Biol. Cell. 2004; 15:4658-4668 Crossref Scopus (111) PubMed Google Scholar
  25. [26]26. Degtyar, V.E. ∙ Allersma, M.W. ... Increased motion and travel, rather than stable docking, characterize the last moments before secretory granule fusion Proc. Natl. Acad. Sci. USA. 2007; 104:15929-15934 Crossref Scopus (53) PubMed Google Scholar
  26. [27]27. Becherer, U. ∙ Pasche, M. ... Quantifying exocytosis by combination of membrane capacitance measurements and total internal reflection fluorescence microscopy in chromaffin cells PLoS ONE. 2007; 2:e505 Crossref Scopus (35) PubMed Google Scholar
  27. [29]29. Wiegand, U.K. ∙ Don-Wauchope, A. ... Exocytosis studies in a chromaffin cell-free system: imaging of single-vesicle exocytosis in a chromaffin cell-free system using total internal reflection fluorescence microscopy Ann. N. Y. Acad. Sci. 2002; 971:257-261 Crossref Scopus (9) PubMed Google Scholar
  28. [30]30. Zenisek, D. ∙ Steyer, J.A. ∙ Almers, W. Transport, capture and exocytosis of single synaptic vesicles at active zones Nature. 2000; 406:849-854 Crossref Scopus (383) PubMed Google Scholar
  29. [31]31. Zerial, M. ∙ McBride, H. Rab proteins as membrane organizers Nat. Rev. Mol. Cell Biol. 2001; 2:107-117 Crossref Scopus (2810) PubMed Google Scholar
  30. [32]32. Novick, P. ∙ Medkova, M. ... Interactions between Rabs, tethers, SNAREs and their regulators in exocytosis Biochem. Soc. Trans. 2006; 34:683-686 Crossref Scopus (83) PubMed Google Scholar
  31. [33]33. Grosshans, B.L. ∙ Ortiz, D. ∙ Novick, P. Rabs and their effectors: achieving specificity in membrane traffic Proc. Natl. Acad. Sci. USA. 2006; 103:11821-11827 Crossref Scopus (839) PubMed Google Scholar
  32. [34]34. Kasai, K. ∙ Ohara-Imaizumi, M. ... Rab27A mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation J. Clin. Invest. 2005; 115:388-396 Crossref Scopus (147) PubMed Google Scholar
  33. [35]35. Fukuda, M. Versatile role of Rab27 in membrane trafficking: focus on the Rab27 effector families J. Biochem. 2005; 137:9-16 Crossref Scopus (193) PubMed Google Scholar
  34. [36]36. Merrins, M.J. ∙ Stuenkel, E.L. Kinetics of Rab27A-dependent actions on vesicle docking and priming in pancreatic β-cells J. Physiol. 2008; 586:5367-5381 Crossref Scopus (39) PubMed Google Scholar
  35. [37]37. Fukuda, M. Slp4-A/granuphilin-A inhibits dense-core vesicle exocytosis through interaction with the GDP-bound form of Rab27A in PC12 cells J. Biol. Chem. 2003; 278:15390-15396 Crossref Scopus (59) PubMed Google Scholar
  36. [38]38. Yi, Z. ∙ Yokota, H. ... The Rab27A/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules Mol. Cell. Biol. 2002; 22:1858-1867 Crossref Scopus (195) PubMed Google Scholar
  37. [39]39. Allersma, M.W. ∙ Bittner, M.A. ... Motion matters: secretory granule motion adjacent to the plasma membrane and exocytosis Mol. Biol. Cell. 2006; 17:2424-2438 Crossref Scopus (58) PubMed Google Scholar
  38. [40]40. Kozuka, J. ∙ Yokota, H. ... Dynamic polymorphism of single actin molecules in the actin filament Nat. Chem. Biol. 2006; 2:83-86 Crossref Scopus (79) PubMed Google Scholar
  39. [41]41. Margeat, E. ∙ Kapanidis, A.N. ... Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes Biophys. J. 2006; 90:1419-1431 Full Text Full Text (PDF) Scopus (113) PubMed Google Scholar
  40. [42]42. Tateyama, M. ∙ Abe, H. ... Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1α Nat. Struct. Mol. Biol. 2004; 11:637-642 Crossref Scopus (157) PubMed Google Scholar
  41. [43]43. Gomi, H. ∙ Mizutani, S. ... Granuphilin molecularly docks insulin granules to the fusion machinery J. Cell Biol. 2005; 171:99-109 Crossref Scopus (148) PubMed Google Scholar
  42. [44]44. Torii, S. ∙ Takeuchi, T. ... Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1A J. Biol. Chem. 2004; 279:22532-22538 Crossref Scopus (80) PubMed Google Scholar
  43. [45]45. Karatekin, E. ∙ Tran, V.S. ... A 20-nm step toward the cell membrane preceding exocytosis may correspond to docking of tethered granules Biophys. J. 2008; 94:2891-2905 Full Text Full Text (PDF) Scopus (30) PubMed Google Scholar
  44. [46]46. Neher, E. A comparison between exocytic control mechanisms in adrenal chromaffin cells and a glutamatergic synapse Pflugers Arch. 2006; 453:261-268 Crossref Scopus (84) PubMed Google Scholar

Cited by

Sorted by citations

Authors
Lam, A. D., Ismail, S., Wu, R., Yizhar, O., Passmore, D. R., Ernst, S. A., & Stuenkel, E. L.
Affiliations
  • University of Michigan (US)
  • Stanford University (US)
Corresponding author
Stuenkel, Edward L.
ORCID
Venue
Biophysical Journal
Publisher
Elsevier BV
Published
2010-08
Volume
99
Issue
4
Pages
1311-1320
ISSN
0006-3495 (ISSN-L)
DOI
10.1016/j.bpj.2010.06.014
PMC
PMC2920663
OpenAlex
W2027175865
Cited by
23
References
0
Citations by year
2025: 1 · 2024: 1 · 2023: 2 · 2022: 3 · 2021: 1 · 2019: 2 · 2018: 1 · 2017: 1 · 2016: 1 · 2014: 1 · 2013: 4 · 2012: 3
FWCI
1.26
Type
journal article
Language
en
Open access
Licence
Locations
  • Biophysical Journal (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institute of Diabetes and Digestive and Kidney Diseases (DK020572, P30DK034933, R56DK077050, DK077050, P60DK020572, B30 DK-34933, R01DK077050, P30DK020572)
  • National Institute of Neurological Disorders and Stroke (R01NS053978, NS053978, F31NS053263)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Health Sciences › Medicine › Surgery › Pancreatic function and diabetes
Keywords
insulin secretory granules, Rab27a, exocytosis, membrane fusion, protein-protein interaction, plasma membrane, live-cell imaging
MeSH terms
rab27 GTP-Binding Proteins, Insulin Secretion, Animals, Calibration, Cell Line, Fluorescent Dyes, Insulin, Microscopy, Fluorescence, Protein Binding, Solubility, Transfection, rab GTP-Binding Proteins, Secretory Vesicles, Protein Interaction Mapping, Fluorescence Resonance Energy Transfer, Vesicular Transport Proteins, Mice

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