Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics

Jameson, David M.; Ross, Justin A.; Albanesi, Joseph P.

doi:10.1007/s12551-009-0013-8

Cited by 55 publications

(51 citation statements)

References 103 publications

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“…The data and fit closely match ( Fig. S8A-C), with the recovered HSP brightness values   , gr bb  b centered near (1,0) in a brightness plot, which is consistent with the EGFP species ( Fig. S8D-E).…”

Section: Resultssupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

“…Fluorescence fluctuation spectroscopy (FFS) refers to a collection of related biophysical techniques that exploit the stochastic intensity of fluorescently labeled biomolecules passing through a small observation volume created by confocal or two-photon microscopy [1]. The primary parameters accessible by FFS are the concentration, mobility, and oligomeric state of the labeled biomolecule [1]. While the original analysis of FFS results was based on the autocorrelation function (ACF), many other methods have been introduced over the years, each with its own strengths and weaknesses [2][3][4][5].…”

Section: Introductionmentioning

confidence: 99%

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Identifying hetero-protein complexes in the nuclear envelope

Hennen

Hur

Kohler

et al. 2019

Preprint

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The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ~40 nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes which are mediated by protein complexes within this subcellular compartment. While fluorescence fluctuation spectroscopy (FFS) is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional FFS methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted meansegmented Q (tsMSQ) analysis and applied it to successfully characterize protein homooligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton (LINC) complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE hetero-protein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies hetero-protein complexes and their stoichiometry in the NE by accounting for spectral crosstalk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the LINC complex, a hetero-protein complex composed of Klarsicht/ANC-1/SYNE homology (KASH) and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the KASH protein nesprin-2 to form a hetero-complex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE heteroprotein complexes. Statement of SignificanceProtein complexes found within the nuclear envelope (NE) play a vital role in regulating cellular functions ranging from gene expression to cellular movement. However, the assembly state of these complexes within their native environment remains poorly understood, which is compounded by a general lack of fluorescence techniques suitable for quantifying the oligomeric state of NE protein complexes. This study aims at addressing this issue by introducing dual-color time-shifted mean-segmented Q analysis as a fluorescence fluctuation method specifically designed to identify the average oligomeric state of hetero-protein complexes within the NE of living cells.

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Section: Resultssupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identifying hetero-protein complexes in the nuclear envelope

Hennen

Hur

Kohler

et al. 2019

Preprint

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show abstract

“…For this purpose, different methodologies and techniques have been developed over the past few decades to describe membrane protein dynamics. These methodologies include fluorescence recovery after photobleaching (FRAP), single‐particle tracking (SPT), single molecule tracking, and different fluorescence fluctuation spectroscopy such as fluorescence correlation spectroscopy (FCS), stimulated emission depletion (STED)–FCS, image correlation spectroscopy, and raster image correlation spectroscopy (RICS) (Alenghat & Golan, ; Briddon, Kilpatrick, & Hill, ; Jameson, Ross, & Albanesi, ; Kusumi, Tsunoyama, Hirosawa, Kasai, & Fujiwara, ; Owen, Williamson, Rentero, & Gaus, ).…”

Section: Introductionmentioning

confidence: 99%

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

Planes

Vanderheyden²,

Gratton

et al. 2019

Microscopy Res & Technique

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The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G‐protein‐coupled receptors namely Angiotensin II type 1 (AT1) and Endothelin 1 type A (ETA) receptors and their corresponding receptor–ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT1 and ETA receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local diffusivity and the confinement zone of AT1 receptors were reduced by the binding of its antagonist losartan, and the long‐range diffusion with the local diffusivity coefficient of ETA receptors was reduced upon exposure to its antagonist BQ123. To the best of our knowledge, this is the first study addressing the protein diffusion laws of these two receptors on living cells using total internal reflection fluorescence microscopy and iMSD.

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“…Fluorescent correlation spectroscopy, a single molecule sensitive fluorescence-based technique (Elson and Magde 1974;Jameson, Ross et al 2009) was used to measure aK d s.…”

Section: Introductionmentioning

confidence: 99%

Dynamic changes in Sox2 spatio-temporal expression direct the second cell fate decision throughFgf4/Fgfr2signaling in preimplantation mouse embryos

Mistri

Arindrarto

et al. 2016

Preprint

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Oct4 and Sox2 regulate the expression of target genes such as Nanog, Fgf4 and Utf1, by binding to their respective regulatory motifs. Their functional cooperation is reflected in their ability to heterodimerise on adjacent cis regulatory elements, the composite Sox/Oct motif. Given that Oct4 and Sox2 regulate many developmental genes, a quantitative analysis of their synergistic action on different Sox/Oct motifs would yield valuable insights into the mechanisms of early embryonic development. In this study, we measured binding affinities of Oct4 and Sox2 to different Sox/Oct motifs using fluorescence correlation spectroscopy (FCS). We found that the synergistic binding interaction is driven mainly by the level of Sox2 in the case of the Fgf4 Sox/Oct motif. Taking into account Sox2 expression levels fluctuate more than Oct4, our finding provides an explanation on how Sox2 controls the segregation of the epiblast (EPI) and primitive endoderm (PE) populations within the inner cell mass (ICM) of the developing rodent blastocyst.

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Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics

Cited by 55 publications

References 103 publications

Identifying hetero-protein complexes in the nuclear envelope

Identifying hetero-protein complexes in the nuclear envelope

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

Dynamic changes in Sox2 spatio-temporal expression direct the second cell fate decision throughFgf4/Fgfr2signaling in preimplantation mouse embryos

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