Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor

Jared Hennen, Kwang Ho Hur, Siddarth Reddy Karuka, G. W.Gant Luxton, Joachim D. Mueller

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)28-41
Number of pages14
JournalMethods
Volume157
DOIs
StatePublished - Mar 15 2019

Bibliographical note

Funding Information:
We thank Ang Li and Patrick Willey for excellent technical assistance. This work was supported by the National Institutes of Health ( R01 GM064589 ).

Publisher Copyright:
© 2018 Elsevier Inc.

Fingerprint

Dive into the research topics of 'Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor'. Together they form a unique fingerprint.

Cite this