Live Cell Imaging in Fission Yeast

Mulvihill, Daniel P.

doi:10.1101/pdb.top090621

Cited by 11 publications

(4 citation statements)

References 65 publications

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“…Samples were taken for imaging and protein analysis 2 h after 10 µg/ml induction or 5 h after 20 µg/ml induction. For cell imaging, cells were mounted on LB-agarose pads under coverslips, as described previously and imaged as described below 26 . For protein analysis, cells were pelleted at 16, 100 × RCF, resuspended in an equilibrated volume of 1 × SDS-PAGE loading, and boiled for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage

Clancy

Ramadurai

Needham

et al. 2023

Sci Rep

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Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5–14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54–1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.

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Section: Methodsmentioning

confidence: 99%

Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage

Clancy

Ramadurai

Needham

et al. 2023

Sci Rep

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“…Use appropriate light source and filter combinations for fluorescent protein(s)/dye(s) being used 6 .…”

Section: Protocolmentioning

confidence: 99%

Optimised protocol for production and analysis of recombinant protein-filled vesicles fromE. coli.

Streather

Baker

Eastwood

et al. 2023

Preprint

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We have recently developed and described an innovative system that exports diverse recombinant proteins in membrane bound vesicles from E. coli. These recombinant vesicles compartmentalise proteins within a micro-environment that enables production of otherwise challenging insoluble, toxic, or disulphide-bond containing proteins from bacteria. The release of vesicle-packaged proteins supports isolation from the culture and allows long-term storage of active protein. This technology results in high yields of vesicle-packaged, functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine. In this article, and associated video, we provide a detailed protocol of the method, and highlight key steps in the methodology to maximise recombinant protein filled vesicle production.

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“…Materials) and filter combinations for the fluorescent protein(s)/dye(s) being used6 .3. Use a high magnification (i.e., 100x or 150x) and high numerical aperture (i.e., NA ≥1.4) lens for imaging the microbial cells and vesicles.4.…”

mentioning

confidence: 99%

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from <em>E. coli</em>

Streather

Baker

Eastwood

et al. 2023

JoVE

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This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression.These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfidebond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

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Live Cell Imaging in Fission Yeast

Cited by 11 publications

References 65 publications

Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage

Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage

Optimised protocol for production and analysis of recombinant protein-filled vesicles fromE. coli.

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from <em>E. coli</em>

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