Fluorescence fluctuation spectroscopy reveals differential SUN protein oligomerization in living cells

Hennen, Jared; Saunders, Cosmo A.; Mueller, Joachim D.; Luxton, G. W. Gant

doi:10.1091/mbc.e17-04-0233

Cited by 40 publications

(74 citation statements)

References 47 publications

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“…To overcome this challenge we replaced ACF with MSQ analysis and more recently with the improved tsMSQ algorithm [14,18]. Both methods have proven to be robust for measuring homotypic protein interactions within the NE of living cells [16,25]. To extend crosscorrelation analysis to DC FFS data obtained in the NE, we generalize single-color to dual-color tsMSQ.…”

Section: Resultsmentioning

confidence: 99%

“…To begin to test this model of LINC complex assembly in the NE of living cells we first performed measurements on cells coexpressing mCherry-SR-KASH2 with SS-EGFP-SUN2 595-731 . The SUN domain (SUN2 595-731 ) contains the KASH binding sites and has previously been shown to remain monomeric using SC tsMSQ [16]. Thus, the SUN domain permits us to directly test for the presence of monomermonomer interactions with nesprin-2.…”

Section: Resultsmentioning

confidence: 99%

“…The generation of SS-EGFP, SS-EGFP-torsinA NTD-2xLeu , SS-EGFP-SUN2 595-731 , and SS-EGFP-SUN2 261-731 constructs were described previously [14,16]. The generation of SS-mCherry-KDEL, the SUN domain-linked SS-EGFP-SUN2 595-731 -mCherry (SS-EGFP-SL-mCherry), and mCherry-SR-KASH2 constructs are described in Text S6 of the Supporting Materials and Methods.…”

Section: Dna Constructsmentioning

confidence: 99%

“…Although the NE has been identified as a critical signaling node of the cell [15], a mechanistic understanding of how these hetero-protein complexes assemble remains limited due to the lack of quantitative biophysical techniques suitable for use in the NE. To address this challenge, we explored the use of single-color (SC) FFS for characterizing homo-protein association in the NE of living cells [14,16]. We found that slow undulations of the nuclear membranes give rise to local volume fluctuations that are not properly accounted for by conventional FFS methods [14].…”

Section: Introductionmentioning

confidence: 99%

“…Thus, the homo-trimerization of the SUN2 luminal domain enables the recruitment of 3 nesprin-2 luminal domains resulting in the assembly of a SUN2nesprin-2 hetero-hexamer. We recently succeeded in directly measuring the homo-trimerization of the EGFP-tagged luminal domain of SUN2 in the NE of living cells by SC FFS [16]. Here we utilize DC tsMSQ to directly observe the heterotypic interactions formed between the EGFPtagged SUN2 luminal domain and an mCherry-tagged construct that encodes the last three spectrin-like repeats (SRs), the transmembrane domain, and the luminal KASH peptide of nesprin-2 in the NE.…”

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: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Dna Constructsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Identifying hetero-protein complexes in the nuclear envelope

Hennen

Hur

Kohler

et al. 2019

Preprint

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Dynamic regulation of LINC complex composition and function across tissues and contexts

King

2023

FEBS Letters

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The concept of mechanotransduction to the nucleus through a direct force transmission mechanism has fascinated cell biologists for decades. Central to such a mechanism is the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which spans the nuclear envelope to couple the cytoplasmic cytoskeleton to the nuclear lamina. In reality, there is not one LINC complex identity, but instead a family of protein configurations of varied composition that exert both shared and unique functions. Regulated expression of LINC complex components, splice variants, and mechanoresponsive protein turnover mechanisms together shape the complement of LINC complex forms present in a given cell type. Disrupting specific gene(s) encoding LINC complex components therefore gives rise to a range of organismal defects. Moreover, evidence suggests that the mechanical environment remodels LINC complexes, providing a feedback mechanism by which cellular context influences the integration of the nucleus into the cytoskeleton. In particular, evidence for crosstalk between the nuclear and cytoplasmic intermediate filament networks communicated through the LINC complex represents an emerging theme in this active area of ongoing investigation.

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Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy

Hennen

Angert

Hur

et al. 2018

Methods in Molecular Biology

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Linkers of nucleoskeleton and cytoskeleton (LINC) complexes are conserved nuclear envelope (NE) spanning molecular bridges which mechanically integrate the nucleus with the cytoskeleton and mediate force transmission into the nucleoplasm. Despite their critical roles in fundamental cellular processes such as meiotic chromosome and nuclear positioning, the mechanism of LINC complex assembly in cells remains unclear. To begin to address this deficit, we recently developed z-scan fluorescence fluctuation spectroscopy (FFS) and brightness analysis as a method for quantifying the oligomeric states of fluorescent protein-tagged NE proteins including nesprins and SUN proteins. Since the homo-oligomerization of SUN2 is critical for its ability to interact with nesprins within the perinuclear space, the knowledge obtained through quantitative brightness experiments reveals important insights into the in vivo mechanisms of LINC complex assembly. Here we describe the procedure we use to determine the brightness of proteins in the NE of living cells. In addition to the measurement procedure, we discuss the instrumentation requirements and present the results of applying this procedure to measure the brightness of nesprin-2 and SUN2.

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Fluorescence fluctuation spectroscopy reveals differential SUN protein oligomerization in living cells

Cited by 40 publications

References 47 publications

Identifying hetero-protein complexes in the nuclear envelope

Identifying hetero-protein complexes in the nuclear envelope

Dynamic regulation of LINC complex composition and function across tissues and contexts

Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy

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