Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor.
Journal
Methods (San Diego, Calif.)
ISSN: 1095-9130
Titre abrégé: Methods
Pays: United States
ID NLM: 9426302
Informations de publication
Date de publication:
15 03 2019
15 03 2019
Historique:
received:
14
07
2018
revised:
14
09
2018
accepted:
25
09
2018
pubmed:
1
10
2018
medline:
22
11
2019
entrez:
1
10
2018
Statut:
ppublish
Résumé
Analysis of fluorescence fluctuation experiments by the mean-segmented Q (MSQ) method was recently used to successfully characterize the oligomeric state and mobility of proteins within the nuclear envelope (NE) of living cells. However, two significant shortcomings of MSQ were recognized. Non-ideal detector behavior due to dead-time and afterpulsing as well as the lack of error analysis currently limit the potential of MSQ. This paper presents time-shifted MSQ (tsMSQ), a new formulation of MSQ that is robust with respect to dead-time and afterpulsing. In addition, a protocol for performing error analysis on tsMSQ data is introduced to assess the quality of fit models and estimate the uncertainties of fit parameters. Together, these developments significantly simplify and improve the analysis of fluorescence fluctuation data taken within the NE. To demonstrate these new developments, tsMSQ was used to characterize the oligomeric state and mobility of the luminal domains of two inner nuclear membrane SUN proteins. The results for the luminal domain of SUN2 obtained through tsMSQ without correction for non-ideal detector effects agree with a recent study that was conducted using the original MSQ formulation. Finally, tsMSQ was applied to characterize the oligomeric state and mobility of the luminal domain of the germline-restricted SUN3.
Identifiants
pubmed: 30268407
pii: S1046-2023(18)30135-X
doi: 10.1016/j.ymeth.2018.09.008
pmc: PMC6401277
mid: NIHMS999076
pii:
doi:
Substances chimiques
Membrane Proteins
0
Nuclear Proteins
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
28-41Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM064589
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM129374
Pays : United States
Informations de copyright
Copyright © 2018 Elsevier Inc. All rights reserved.
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