Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light

Gurskaya, Nadya G.; Verkhusha, Vladislav V.; Shcheglov, Alex S.; Staroverov, Dmitry B; Chepurnykh, T. V.; Fradkov, Arkady F.; Lukyanov, Sergey; Lukyanov, Konstantin A.

doi:10.1038/nbt1191

Cited by 664 publications

(545 citation statements)

References 32 publications

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“…This inherent intracellular inertness, which gives rise to the simple diffusion properties, makes mEos2 and similar GFP variants [Dendra2 (25), PA-GFP (26), Dronpa (27), etc.] ideal fusion partners when tracking other proteins for which deviations from simple Brownian diffusion is of biological interest, such as RelA.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule investigations of the stringent response machinery in living bacterial cells

English

Hauryliuk

Sanamrad

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

262

294

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The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism. cytosolic diffusion | single particle tracking | photoactivated localization microscopy | stroboscopic illumination

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Section: Resultsmentioning

confidence: 99%

Single-molecule investigations of the stringent response machinery in living bacterial cells

English

Hauryliuk

Sanamrad

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

262

294

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“…To follow the retrograde movement of dendritically localized CREB2, we tagged CREB2 at its N terminus with the photoconvertible fluorescent protein Dendra, which converts from green to red with brief UV irradiation (27). After photoconverting a small (Ϸ20 m) dendritic region (40 to shows that equal amounts of GST-CREB2 were used in each pull-down assay (note that GST alone runs at a lower MW and is not shown).…”

Section: Nuclear Import and Retrograde Transport Of Creb2 Is Mediated Bymentioning

confidence: 99%

Importin-mediated retrograde transport of CREB2 from distal processes to the nucleus in neurons

Lai¹,

Zhao²,

Ch’ng³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

104

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Signals received at distal synapses of neurons must be conveyed to the nucleus to initiate the changes in transcription that underlie long-lasting synaptic plasticity. The presence of importin nuclear transporters and of select transcription factors at synapses raises the possibility that importins directly transport transcription factors from synapse to nucleus to modulate gene expression. Here, we show that cyclic AMP response element binding protein 2 (CREB2)/activating transcription factor 4 (ATF4), a transcriptional repressor that modulates long-term synaptic plasticity and memory, localizes to distal dendrites of rodent hippocampal neurons and neurites of Aplysia sensory neurons (SNs) and binds to specific importin ␣ isoforms. Binding of CREB2 to importin ␣ is required for its transport from distal dendrites to the soma and for its translocation into the nucleus. CREB2 accumulates in the nucleus during long-term depression (LTD) but not long-term potentiation of rodent hippocampal synapses, and during LTD but not long-term facilitation (LTF) of Aplysia sensory-motor synapses. Time-lapse microscopy of CREB2 tagged with a photoconvertible fluorescent protein further reveals retrograde transport of CREB2 from distal neurites to the nucleus of Aplysia SN during phenylalaninemethionine-arginine-phenylalanine-amide (FMRFamide)-induced LTD. Together, our findings indicate that CREB2 is a novel cargo of importin ␣ that translocates from distal synaptic sites to the nucleus after stimuli that induce LTD of neuronal synapses.Aplysia ͉ hippocampus ͉ transcription ͉ long-term depression ͉ dendra S ynaptic plasticity is critical to cognition and memory formation. Long-lasting forms of synaptic plasticity require both transcription and translation (1-3), and specific patterns and strengths of synaptic stimulation trigger changes in neuronal gene expression (4). However, relatively little is known about how signals are transported from synapse to nucleus to regulate transcription. Many types of synaptic stimulation induce depolarization and electrochemical signaling to rapidly alter transcription in the nucleus (5). A slower, more persistent mechanism of signaling to the nucleus involves the transport of soluble molecules from stimulated synapses to the nucleus (6, 7). Recent studies have indicated that this type of transport may involve the active nuclear import pathway (8-10).In the classical nuclear import pathway, proteins bearing nuclear localization signals (NLSs) bind an adaptor protein of the importin ␣ family, which in turn binds the importin ␤1 nuclear transporter. Importin ␤1 mediates facilitated translocation of this heterotrimeric complex across the nuclear pore. In neurons, importins may also function to carry signals from distal neuronal processes to the soma and into the nucleus (8-11). This finding raises a critical question: what are the synaptically localized cargoes of importins that translocate to the nucleus to alter transcription during synaptic plasticity?One attractive class of potential importin...

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“…Cells were transfected with a Dendra-MKKS expression vector constructed with the vector pDendra-2 (Evrogen, Moscow, Russia) encoding the greento-red photoactivatable fluorescent protein of the octocoral Dendronephthya (Gurskaya et al, 2006). Preconversion green signals of Dendra were collected by excitation with a 488-nm Ar laser set at 1% power, and postconversion red signals were collected by excitation with a 543-nm He-Ne laser at 50% power by using a FV1000 laser scanning microscope with a UApo 40ϫ/1.15 NA 340 water immersion objective (Olympus) at 37°C in a system controlled by Fluoview software version 1.6 (Olympus).…”

Section: Photoconversion Analysismentioning

confidence: 99%

“…Photoconversion is a variation of the photoactivation method (Patterson and Lippincott-Schwartz, 2002), which allows the tracking of movement by fluorescently labeled molecules after pulse activation. MKKS tagged with Dendra, a green-to-red photoactivatable fluorescent protein (Gurskaya et al, 2006), was expressed in HEK293 cells. The green Dendra-MKKS protein concentrated at the centrosome was photoconverted to red by a pulse of laser beam.…”

mentioning

confidence: 99%

MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination

Hirayama

Yamazaki²,

Kitamura³

et al. 2008

MBoC

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McKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin-proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases. INTRODUCTIONMcKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease predominantly characterized by developmental anomalies, including vaginal atresia with hydrometrocolpos, polydactyly, and congenital heart defects (McKusick et al., 1964). The MKKS gene encodes a 570-amino acid polypeptide with weak but significant similarity to group II chaperonins . Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder characterized by obesity, retinal dystrophy, polydactyly, mental retardation, renal malformation, and hypogenitalism (Beales et al., 1999), and thus MKKS is also called BBS6. The MKKS mRNA is widely expressed in various tissues, including those affected by MKKS and BBS diseases . Although more than 10 mutations in the MKKS gene have been identified in patients (Beales et al., 2001;Slavotinek et al., 2002), little is known about how they cause MKKS and BBS.Twelve of the genes that are responsible for BBS (BBS1-12) have been identified so far, and the products of several of these genes have been suggested to be involved in intraflagellar transport in cilia and intracellular trafficking in the cytosol Badano et al., 2005;Beales, 2005;Blacque and Leroux, 2006). Cargo-carrying motors play crucial roles in intraflagellar and intracellular transport systems by bidirectionally moving on microtubules, and BBS4 interacts with the dynactin complex, which mediates interactions between cargoes and the dynein motor (Kim et al., 2004). Retrograde transport of melanosomes in zebrafish is slowed by knockdown of BBS2 or BBS4-8 (Yen et al., 2006), whereas mutations in Caenorhabditis elegans BBS7 and BBS8 cause delays in intraflagellar transport driven by kinesin motors (Ou et al., 2005). The Chlamydomonas homologue of BBS5 is essential for flagellum formation (Li et al., ...

show abstract

Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light

Cited by 664 publications

References 32 publications

Single-molecule investigations of the stringent response machinery in living bacterial cells

Single-molecule investigations of the stringent response machinery in living bacterial cells

Importin-mediated retrograde transport of CREB2 from distal processes to the nucleus in neurons

MKKS Is a Centrosome-shuttling Protein Degraded by Disease-causing Mutations via CHIP-mediated Ubiquitination

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