A photostable monomeric superfolder green fluorescent protein

Valbuena, Fernando M.; Fitzgerald, Ivy; Strack, Rita; Andruska, Neal; Smith, Luke; Glick, Benjamin S.

doi:10.1111/tra.12737

Cited by 34 publications

(44 citation statements)

References 47 publications

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“…To generate the Flp-In 293 T-REx cell line stably expressing GalNAc-T2-GFP, a synthetic gBlock (IDT) encoding a mammalian codon-optimized GalNAc-T2-GFP gene was fused to the msGFP2 gene ( Valbuena et al. , 2020 ), and this construct was inserted into pcDNA5/FRT/TO (Thermo Fisher; V652020) to generate pcDNA5-GalNAc-T2-GFP.…”

Section: Methodsmentioning

confidence: 99%

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

Casler

Zajac

Valbuena

et al. 2020

MBoC

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Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms. [Media: see text] [Media: see text] [Media: see text]

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Section: Methodsmentioning

confidence: 99%

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

Casler

Zajac

Valbuena

et al. 2020

MBoC

Self Cite

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“… References cited in this file: Guntas et al, 2015 ; Munjal et al, 2015 ; Valbuena et al, 2020 ; Wagner and Glotzer, 2016 ; Wenzl et al, 2010 . …”

Section: Additional Filesmentioning

confidence: 99%

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos

Rich

Fehon

Glotzer

2020

eLife

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Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Here, using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, we show that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. Our results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and reveal that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.

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“…To generate the Flp-In 293 T-REx cell line stably expressing GalNAc-T2-GFP, a synthetic gBlock (IDT) encoding a mammalian codon-optimized GalNAc-T2-GFP gene was fused to the msGFP2 gene (Valbuena et al, 2020), and this construct was inserted into pcDNA5/FRT/TO (Thermo Fisher; V652020) to generate pcDNA5-GalNAc-T2-GFP. This plasmid was transfected into cells together with the Flprecombinase expression vector pOG44 (Thermo Fisher; V600520).…”

Section: Mammalian Cell Culture Engineering and Microscopymentioning

confidence: 99%

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

Casler

Zajac

Valbuena

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block exit of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid transport out of the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

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A photostable monomeric superfolder green fluorescent protein

Cited by 34 publications

References 47 publications

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

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