English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Background  Fluorescent proteins are powerful molecular biology tools that have been used to study the subcellular dynamics of proteins within live cells for well over a decade. Two fluorescent proteins commonly used to enable dual protein labelling are GFP (green) and mCherry (red). Sporulation in the Gram positive bacterium  Bacillus subtilis  has been studied for many years as a paradigm for understanding the molecular basis for differential gene expression. As sporulation initiates, cells undergo an asymmetric division leading to differential gene expression in the small prespore and large mother cell compartments. Use of two fluorescent protein reporters permits time resolved examination of differential gene expression either in the same compartments or between compartments. Due to the spectral properties of GFP and mCherry, they are considered an ideal combination for co-localisation and co-expression experiments. They can also be used in combination with fluorescent DNA stains such as DAPI to correlate protein localisation patterns with the developmental stage of sporulation which can be linked to well characterised changes in DNA staining patterns.    Findings  While observing the recruitment of the transcription machinery into the forespore of sporulating  Bacillus subtilis , we noticed the occurrence of stage-specific fluorescence intensity differences between GFP and mCherry. During vegetative growth and the initial stages of sporulation, fluorescence from both GFP and mCherry fusions behaved similarly. During stage II-III of sporulation we found that mCherry fluorescence was considerably diminished, whilst GFP signals remained clearly visible. This fluorescence pattern reversed during the final stage of sporulation with strong mCherry and low GFP fluorescence. These trends were observed in reciprocal tagging experiments indicating a direct effect of sporulation on fluorescent protein fluorophores.    Conclusions  Great care should be taken when interpreting the results of protein localisation and quantitative gene expression patterns using fluorescent proteins in experiments involving intracellular physiological change. We believe changes in the subcellular environment of the sporulating cell leads to conditions that differently alter the spectral properties of GFP and mCherry making an accurate interpretation of expression profiles technically challenging.

Stage-specific fluorescence intensity of GFP and mCherry during sporulation In Bacillus Subtilis