Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

Liang, Lifang; Xing, Da; Chen, Tongsheng; Pei, Yanxi

doi:10.1117/12.760226

“…The mean observed ~7 mer cluster diameter from Slimfield data is ~30 nm, which, assuming a spherical packing geometry, suggests a subunit diameter for single Mig1-GFP molecules of ~30/7 1/3 ≈ 15.6 nm, consistent with that predicted from the earlier hydrodynamic expectations. Using Stokes law this estimated hydrodynamic radius indicates an effective viscosity for the cytoplasm and nucleoplasm as low as 2-3cP, compatible with earlier live cell estimates on mammalian cells using fluorescence correlation spectroscopy (FCS) ( Liang et al, 2009 ).…”

Section: Discussionsupporting

confidence: 84%

Transcription factor clusters regulate genes in eukaryotic cells

Wollman

¹

,

Shashkova

²

,

Hedlund

³

et al. 2017

eLife

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Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

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“…The mean observed ~7-mer cluster diameter from Slimfield data is ~30nm, which, assuming a spherical packing geometry, suggests a subunit diameter for single Mig1-GFP molecules of ~30/7 1/3 ≈ 15.6nm, consistent with that predicted from the earlier hydrodynamic expectations. Using Stokes law this estimated hydrodynamic radius indicates an effective viscosity for the cytoplasm and nucleoplasm as low as 2-3cP, compatible with earlier live cell estimates on mammalian cells using fluorescence correlation spectroscopy (FCS) (53).…”

Section: Discussionsupporting

confidence: 84%

Transcription factor clusters regulate genes in eukaryotic cells

Wollman

¹

,

Shashkova

²

,

Hedlund

³

et al. 2017

Preprint

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Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.not peer-reviewed)

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“…In addition, there are lipids diffusing more slowly in intact cells, which cannot be studied with standard NMR probes. Slower moving lipid species may correspond to lipids located in different cellular environments (for example, the viscosity and molecular crowding is different in nucleus and cytoplasm 44–46 ) or even in lipid rafts, where the movement of lipids is highly restricted 47 . This approach opens up new opportunities for investigating lipid behaviour in intact cells.…”

Section: Resultsmentioning

confidence: 99%

Monitoring apoptosis in intact cells by high‐resolution magic angle spinning ¹H NMR spectroscopy

Wylot

¹

,

Whittaker

²

,

Wren

³

et al. 2021

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Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in 1H NMR‐observable resonances associated with lipids stored in lipid droplets. However, standard one‐dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis‐based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high‐resolution magic angle spinning (HRMAS) 1H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution‐state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS 1H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR‐based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with 1H NMR spectroscopy‐derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.

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Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

Cited by 4 publications

References 2 publications

Transcription factor clusters regulate genes in eukaryotic cells

Transcription factor clusters regulate genes in eukaryotic cells

Transcription factor clusters regulate genes in eukaryotic cells

Monitoring apoptosis in intact cells by high‐resolution magic angle spinning ¹H NMR spectroscopy

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Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

Cited by 4 publications

References 2 publications

Transcription factor clusters regulate genes in eukaryotic cells

Transcription factor clusters regulate genes in eukaryotic cells

Transcription factor clusters regulate genes in eukaryotic cells

Monitoring apoptosis in intact cells by high‐resolution magic angle spinning 1H NMR spectroscopy

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Product

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Monitoring apoptosis in intact cells by high‐resolution magic angle spinning ¹H NMR spectroscopy