[4] Comparison of enhanced green fluorescent protein and its destabilized form as transcription reporters

Zhao, Xiaoning; Duong, Tommy; Huang, Chiao-Chian; Kain, Steven R.; Li, Xianoiang

doi:10.1016/s0076-6879(99)02006-6

Cited by 11 publications

(6 citation statements)

References 9 publications

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“…In this case, a smaller increase in fluorescence occurred immediately after the shift, but the promoter activity remained fairly constant, with a small decrease in rate. This may be a combined result of continued high rates of transcription during the transition between methanol-and succinate-growth modes, consistent with the results from flux analysis (Pedraza & van Oudenaarden, 2005), and of negligible degradation of GFPuv over time due to an in vivo half-life that is >24 h (Leveau & Lindow, 2001; Andersen et al , 1998; Zhao et al , 1999). …”

Section: Resultssupporting

confidence: 71%

Population heterogeneity in Methylobacterium extorquens AM1

Strovas

Lidstrom

2009

Microbiology

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Heterogeneity of cells within exponentially growing populations was addressed in a bacterium, the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methanol-inducible promoter (P mxaF ) and gfp uv was used with flow cytometry to analyse the distribution of gene expression in populations grown on either succinate or methanol, correlated with forward scatter as a measure of cell size. These cell populations were found to consist of three major subpopulations defined by cells that were actively growing and dividing, newly divided, and non-dividing. Through the use of flow cytometry, it was demonstrated that a significant percentage of the total population did not respond to carbon shift. In addition, these experiments demonstrated that a small subset of the total population was significantly brighter than the rest of the population and dominated fluorimetry data. These results were corroborated with a continuous flow-through system and laser scanning microscopy, confirming that subpopulations, not discernible in the population average, dominate population response. These results demonstrate that the combination of flow cytometry and microscopic single-cell analysis can be effectively used to determine the dynamics of subpopulations in population response. In addition, they support the concept that physiological diversity in isogenic populations can poise some proportion of the population to respond appropriately to changing conditions.

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Section: Resultssupporting

confidence: 71%

Population heterogeneity in Methylobacterium extorquens AM1

Strovas

Lidstrom

2009

Microbiology

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“…It is conceivable that nuclear import of newly synthesized transcription factor occurs at the same rate, but due to the experimental limitation of GFP maturation is picked up with a delay. Possibly, with a more sensitive microscopy set up providing higher temporal resolution, and by using faster maturing and/or faster decaying GFP variants, such as TurboGFP (Evdokimov., 2006 ) or labile GFPs (Deichsel., 1999 ; Zhao., 1999 ), it could be demonstrated that the cells actually respond and reach higher nuclear transcription factor concentrations much quicker.…”

Section: Discussionmentioning

confidence: 99%

Nucleo‐cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulase and xylanase gene expression in Trichoderma reesei

Lichius

Seidl‐Seiboth

Schink

et al. 2014

Molecular Microbiology

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Trichoderma reesei is a model for investigating the regulation of (hemi-)cellulase gene expression. Cellulases are formed adaptively, and the transcriptional activator XYR1 and the carbon catabolite repressor CRE1 are main regulators of their expression. We quantified the nucleo-cytoplasmic shuttling dynamics of GFP-fusion proteins of both transcription factors under cellulase and xylanase inducing conditions, and correlated their nuclear presence/absence with transcriptional changes. We also compared their subcellular localization in conidial germlings and mature hyphae. We show that cellulase gene expression requires de novo biosynthesis of XYR1 and its simultaneous nuclear import, whereas carbon catabolite repression is regulated through preformed CRE1 imported from the cytoplasmic pool. Termination of induction immediately stopped cellulase gene transcription and was accompanied by rapid nuclear degradation of XYR1. In contrast, nuclear CRE1 rapidly decreased upon glucose depletion, and became recycled into the cytoplasm. In mature hyphae, nuclei containing activated XYR1 were concentrated in the colony center, indicating that this is the main region of XYR1 synthesis and cellulase transcription. CRE1 was found to be evenly distributed throughout the entire mycelium. Taken together, our data revealed novel aspects of the dynamic shuttling and spatial bias of the major regulator of (hemi-)cellulase gene expression, XYR1, in T. reesei.

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“…This degron is directly fused to intrabodies that prevent misfolding of their target antigen [13, 14]. The degron used in this study is from mouse Ornithine Decarboxylase (ODC), a short-lived protein containing a C-terminal PEST degron that has been shown to heterologously reduce the half-life of GFP transcription reporters [15–17]. …”

Section: Introductionmentioning

confidence: 99%

Bifunctional Anti-Non-Amyloid Component α-Synuclein Nanobodies Are Protective In Situ

et al. 2016

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Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson’s disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts. We compared this anti-NAC candidate to NbSyn87, which binds to the C-terminus of α-Syn. NbSyn87PEST degrades α-Syn as well or better than VH14PEST. However, while both candidates reduced toxicity, VH14PEST appears more effective in both proteostatic stress and toxicity assays. These results show that the approach of reducing intracellular monomeric targets with novel antibody engineering technology should allow in vivo modulation of proteostatic pathologies.

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[4] Comparison of enhanced green fluorescent protein and its destabilized form as transcription reporters

Cited by 11 publications

References 9 publications

Population heterogeneity in Methylobacterium extorquens AM1

Population heterogeneity in Methylobacterium extorquens AM1

Nucleo‐cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulase and xylanase gene expression in Trichoderma reesei

Bifunctional Anti-Non-Amyloid Component α-Synuclein Nanobodies Are Protective In Situ

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