دورية أكاديمية
Photobleaching step analysis for robust determination of protein complex stoichiometries.
| العنوان: | Photobleaching step analysis for robust determination of protein complex stoichiometries. |
|---|---|
| المؤلفون: | Hummert J; Institute of Physical Chemistry, Heidelberg University, D-69120 Heidelberg, Germany.; Institute of Cardiovascular Sciences, College of Medical and Dental Sciences & School of Chemistry, University of Birmingham, Birmingham, B152TT UK.; Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, Birmingham, B15 2TT UK., Yserentant K; Institute of Physical Chemistry, Heidelberg University, D-69120 Heidelberg, Germany.; Faculty of Biosciences, Heidelberg University, D-69120 Heidelberg, Germany.; Institute of Cardiovascular Sciences, College of Medical and Dental Sciences & School of Chemistry, University of Birmingham, Birmingham, B152TT UK.; Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, Birmingham, B15 2TT UK., Fink T; Institute of Physical Chemistry, Heidelberg University, D-69120 Heidelberg, Germany., Euchner J; Institute of Physical Chemistry, Heidelberg University, D-69120 Heidelberg, Germany.; Institute of Cardiovascular Sciences, College of Medical and Dental Sciences & School of Chemistry, University of Birmingham, Birmingham, B152TT UK.; Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, Birmingham, B15 2TT UK., Ho YX; Institute of Cardiovascular Sciences, College of Medical and Dental Sciences & School of Chemistry, University of Birmingham, Birmingham, B152TT UK.; Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, Birmingham, B15 2TT UK., Tashev SA; Institute of Cardiovascular Sciences, College of Medical and Dental Sciences & School of Chemistry, University of Birmingham, Birmingham, B152TT UK.; Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, Birmingham, B15 2TT UK., Herten DP; Institute of Physical Chemistry, Heidelberg University, D-69120 Heidelberg, Germany.; Institute of Cardiovascular Sciences, College of Medical and Dental Sciences & School of Chemistry, University of Birmingham, Birmingham, B152TT UK.; Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, Birmingham, B15 2TT UK. |
| المصدر: | Molecular biology of the cell [Mol Biol Cell] 2021 Nov 01; Vol. 32 (21), pp. ar35. Date of Electronic Publication: 2021 Sep 29. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: American Society for Cell Biology Country of Publication: United States NLM ID: 9201390 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1939-4586 (Electronic) Linking ISSN: 10591524 NLM ISO Abbreviation: Mol Biol Cell Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Bethesda, MD : American Society for Cell Biology, c1992- |
| مواضيع طبية MeSH: | Microscopy, Fluorescence/*methods , Photobleaching/*drug effects, Algorithms ; DNA ; Fluorescence ; Fluorescent Dyes |
| مستخلص: | The counting of discrete photobleaching steps in fluorescence microscopy is ideally suited to study protein complex stoichiometry in situ. The counting range of photobleaching step analysis has been significantly improved with more-sophisticated algorithms for step detection, albeit at an increasing computational cost and with the necessity for high-quality data. Here, we address concerns regarding robustness, automation, and experimental validation, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labeling strategies with respect to their molecular brightness, photostability, and photoblinking. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we validate the developed methods with experimental data acquired on DNA origami labeled with defined fluorophore numbers, demonstrating counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis by counting labeled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated manner. |
| References: | Mol Syst Biol. 2013;9:648. (PMID: 23511206) Science. 2005 Oct 14;310(5746):310-4. (PMID: 16224022) Nat Methods. 2016 May;13(5):439-42. (PMID: 27018580) Phys Chem Chem Phys. 2017 Mar 29;19(13):8962-8969. (PMID: 28300271) Nat Methods. 2007 Apr;4(4):319-21. (PMID: 17369835) Angew Chem Int Ed Engl. 2020 Jan 7;59(2):804-810. (PMID: 31638314) Phys Chem Chem Phys. 2010 Sep 21;12(35):10295-300. (PMID: 20603676) Sci Rep. 2018 Jan 12;8(1):667. (PMID: 29330459) J Mol Biol. 1990 Nov 5;216(1):49-68. (PMID: 2146398) Nat Methods. 2012 Jul;9(7):671-5. (PMID: 22930834) Sci Rep. 2015 Mar 23;5:9372. (PMID: 25797490) Biophys J. 2008 Mar 1;94(5):1826-35. (PMID: 17921203) J Chem Phys. 2020 Jan 14;152(2):024110. (PMID: 31941327) Angew Chem Int Ed Engl. 2008;47(29):5465-9. (PMID: 18601270) FEBS Lett. 2018 Feb;592(4):475-488. (PMID: 29119545) Methods Appl Fluoresc. 2019 Jan 15;7(1):012003. (PMID: 30524087) Annu Rev Phys Chem. 2012;63:595-617. (PMID: 22404588) Biochemistry. 2021 Aug 24;60(33):2560-2575. (PMID: 34339177) Mol Biol Cell. 2016 Nov 7;27(22):3601-3615. (PMID: 27654946) ACS Nano. 2019 Oct 22;13(10):11955-11966. (PMID: 31513377) Chembiochem. 2007 Jun 18;8(9):994-9. (PMID: 17503420) J Am Chem Soc. 2019 May 1;141(17):6976-6985. (PMID: 30950273) Proc Natl Acad Sci U S A. 2013 Oct 1;110(40):16015-20. (PMID: 24043832) Nat Methods. 2015 Mar;12(3):244-50, 3 p following 250. (PMID: 25599551) Nat Methods. 2019 Oct;16(10):1045-1053. (PMID: 31562488) Science. 2015 Dec 11;350(6266):aaa2245. (PMID: 26659058) Biophys J. 1995 Jun;68(6):2588-600. (PMID: 7647262) Sci Rep. 2019 Oct 23;9(1):15238. (PMID: 31645577) Nat Methods. 2012 Jun 28;9(7):676-82. (PMID: 22743772) J Biol Methods. 2014;1(2):. (PMID: 25606571) Chembiochem. 2021 Dec 2;22(23):3283-3291. (PMID: 34296494) Cell. 2013 Dec 5;155(6):1233-43. (PMID: 24315095) Cell Chem Biol. 2019 Apr 18;26(4):584-592.e6. (PMID: 30745239) J Cell Biol. 2009 Oct 5;187(1):81-9. (PMID: 19805630) Chemphyschem. 2014 Mar 17;15(4):600-5. (PMID: 24481650) Bioinformatics. 2014 Aug 15;30(16):2389-90. (PMID: 24771516) Nat Methods. 2018 May;15(5):367-369. (PMID: 29630062) Nature. 2006 Sep 21;443(7109):355-8. (PMID: 16971952) Proc Natl Acad Sci U S A. 2012 Oct 23;109(43):17436-41. (PMID: 23045631) Proc Natl Acad Sci U S A. 2015 Jan 13;112(2):E110-8. (PMID: 25535361) Nat Protoc. 2014;9(6):1367-91. (PMID: 24833175) Elife. 2015 Feb 25;4:. (PMID: 25714924) Angew Chem Int Ed Engl. 2015 Oct 5;54(41):12049-52. (PMID: 26289028) Biophys J. 2015 Dec 1;109(11):2352-62. (PMID: 26636946) Int J Biochem Cell Biol. 2021 Jun;135:105978. (PMID: 33865985) Nat Methods. 2013 May;10(5):407-9. (PMID: 23524392) ACS Nano. 2012 Jul 24;6(7):6364-9. (PMID: 22703450) |
| المشرفين على المادة: | 0 (Fluorescent Dyes) 9007-49-2 (DNA) |
| تواريخ الأحداث: | Date Created: 20210929 Date Completed: 20220321 Latest Revision: 20240404 |
| رمز التحديث: | 20240404 |
| مُعرف محوري في PubMed: | PMC8693960 |
| DOI: | 10.1091/mbc.E20-09-0568 |
| PMID: | 34586828 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1939-4586 |
|---|---|
| DOI: | 10.1091/mbc.E20-09-0568 |