Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples

Goedhart, Joachim; Vermeer, Joop E. M.; Adjobo‐Hermans, Merel J. W.; Weeren, Laura van; Gadella, Theodorus W. J.

doi:10.1371/journal.pone.0001011

Cited by 80 publications

(105 citation statements)

References 50 publications

(71 reference statements)

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“…The efficiency of energy transfer between the donor and acceptor is a measure for the intermolecular average distance. For eGFP and mCherry, the R 0 is 5.4 nm (Goedhart et al 2007); a similar R 0 is calculated for eGFP and FM 5-95.…”

Section: Microscopysupporting

confidence: 65%

Positive control of cell division: FtsZ is recruited by SsgB during sporulation ofStreptomyces

Willemse¹,

Borst²,

Waal³

et al. 2011

Genes Dev.

168

201

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In bacteria that divide by binary fission, cell division starts with the polymerization of the tubulin homolog FtsZ at mid-cell to form a cell division scaffold (the Z ring), followed by recruitment of the other divisome components. The current view of bacterial cell division control starts from the principle of negative checkpoints that prevent incorrect Z-ring positioning. Here we provide evidence of positive control of cell division during sporulation of Streptomyces, via the direct recruitment of FtsZ by the membrane-associated divisome component SsgB. In vitro studies demonstrated that SsgB promotes the polymerization of FtsZ. The interactions are shown in vivo by time-lapse imaging and Fö rster resonance energy transfer and fluorescence lifetime imaging microscopy (FRET-FLIM), and are corroborated independently via two-hybrid studies. As determined by fluorescence recovery after photobleaching (FRAP), the turnover of FtsZ protofilaments increased strongly at the time of Z-ring formation. The surprising positive control of Z-ring formation by SsgB implies the evolution of an entirely new way of Z-ring control, which may be explained by the absence of a mid-cell reference point in the long multinucleoid hyphae. In turn, the localization of SsgB is mediated through the orthologous SsgA, and premature expression of the latter is sufficient to directly activate multiple Z-ring formation and hyperdivision at early stages of the Streptomyces cell cycle.

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Section: Microscopysupporting

confidence: 65%

Positive control of cell division: FtsZ is recruited by SsgB during sporulation ofStreptomyces

Willemse¹,

Borst²,

Waal³

et al. 2011

Genes Dev.

168

201

View full text Add to dashboard Cite

show abstract

“…For mammalian constructs, JKD, BIB, SCR, and SHR coding sequences (CDSs) were amplified using primers described in Supplemental Table 1 and subcloned by ligation into mammalian vector containing mTurquoise, SYFP2, or mCherry under the constitutive cytomegalovirus promoter (Kremers et al, 2006;Goedhart et al, 2007Goedhart et al, , 2010. The resulting clones are listed in Supplemental Table 2.…”

Section: Constructs For Cells and Plant Transformationmentioning

confidence: 99%

Arabidopsis BIRD Zinc Finger Proteins Jointly Stabilize Tissue Boundaries by Confining the Cell Fate Regulator SHORT-ROOT and Contributing to Fate Specification

et al. 2015

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Plant cells cannot rearrange their positions; therefore, sharp tissue boundaries must be accurately programmed. Movement of the cell fate regulator SHORT-ROOT from the stele to the ground tissue has been associated with transferring positional information across tissue boundaries. The zinc finger BIRD protein JACKDAW has been shown to constrain SHORT-ROOT movement to a single layer, and other BIRD family proteins were postulated to counteract JACKDAW's role in restricting SHORT-ROOT action range. Here, we report that regulation of SHORT-ROOT movement requires additional BIRD proteins whose action is critical for the establishment and maintenance of the boundary between stele and ground tissue. We show that BIRD proteins act in concert and not in opposition. The exploitation of asymmetric redundancies allows the separation of two BIRD functions: constraining SHORT-ROOT spread through nuclear retention and transcriptional regulation of key downstream SHORT-ROOT targets, including SCARECROW and CYCLIND6. Our data indicate that BIRD proteins promote formative divisions and tissue specification in the Arabidopsis thaliana root meristem ground tissue by tethering and regulating transcriptional competence of SHORT-ROOT complexes. As a result, a tissue boundary is not "locked in" after initial patterning like in many animal systems, but possesses considerable developmental plasticity due to continuous reliance on mobile transcription factors.

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“…Subsequently the YFP/CFP ratio was calculated as a measure of kinase activity. FLIM experiments and subsequent image analysis were performed as described (Goedhart et al, 2007). A 442 nm helium-cadmium laser (125 mW, MellesGriot, Carlsbad, CA) was intensity modulated at a frequency of 75.1 MHz.…”

Section: Live Cell Microscopymentioning

confidence: 99%

PKCγ mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling

Verbeek

Goedhart

Bruinsma

et al. 2008

Journal of Cell Science

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Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disorder caused by mutations in the neuronal-specific protein kinase C gamma (PKCγ) gene. Since most mutations causing SCA14 are located in the PKCγ C1B regulatory subdomain, we investigated the impact of three C1B mutations on the intracellular kinetics, protein conformation and kinase activity of PKCγ in living cells. SCA14 mutant PKCγ proteins showed enhanced phorbol-ester-induced kinetics when compared with wild-type PKCγ. The mutations led to a decrease in intramolecular FRET of PKCγ, suggesting that they `open' PKCγ protein conformation leading to unmasking of the phorbol ester binding site in the C1 domain. Surprisingly, SCA14 mutant PKCγ showed reduced kinase activity as measured by phosphorylation of PKC reporter MyrPalm-CKAR, as well as downstream components of the MAPK signaling pathway. Together, these results show that SCA14 mutations located in the C1B subdomain `open' PKCγ protein conformation leading to increased C1 domain accessibility, but inefficient activation of downstream signaling pathways.

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Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples

Cited by 80 publications

References 50 publications

Positive control of cell division: FtsZ is recruited by SsgB during sporulation ofStreptomyces

Positive control of cell division: FtsZ is recruited by SsgB during sporulation ofStreptomyces

Arabidopsis BIRD Zinc Finger Proteins Jointly Stabilize Tissue Boundaries by Confining the Cell Fate Regulator SHORT-ROOT and Contributing to Fate Specification

PKCγ mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling

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