A monomeric StayGold fluorescent protein

Ivorra-Molla, Esther; Akhuli, Dipayan; McAndrew, Martin B. L.; Scott, William; Kumar, Lokesh; Palani, Saravanan; Mishima, Masanori; Crow, Allister; Balasubramanian, Mohan K.

doi:10.1038/s41587-023-02018-w

Cited by 25 publications

(14 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These results support that the scFv-FPs containing newer generation monomeric FPs are more suitable for translation analysis, as they are less likely to form aggregates compared to sfGFP which is able to form weak dimers 42,47 .…”

Section: Resultssupporting

confidence: 71%

“…Although few aggregates were also present in cells expressing scFv-msGFP2 with the SunTagRLuc-MS2 reporter (Figure S3d), this occurred with a significantly lower frequency (~55% of cells without aggregates compared to ~15% using scFv-sfGFP) and smaller aggregate size, suggesting that msGFP2 is less prone to aggregate formation. These results support that the scFv-FPs containing newer generation monomeric FPs are more suitable for translation analysis, as they are less likely to form aggregates compared to sfGFP which is able to form weak dimers 42,47 .…”

Section: New Scfv-fp Prevents Aggregate Formationsupporting

confidence: 71%

“…Although the sfGFP (weak dimer) was the standard FP for fusion with the scFv in mammalian cell culture, we found that it might present aggregation issue in Drosophila. Moreover, During the revision process of our manuscript, three monomeric versions of StayGold proteins were published [62][63][64] , and future optimization should consider testing these FPs for imaging translation, taking into account the specific imaging setup 65 . Local translation of specific mRNAs is an important biological process to control gene expression in space and time.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Boosting the toolbox for live imaging of translation

Bellec

Chen²,

Dhayni

et al. 2023

Preprint

View full text Add to dashboard Cite

Live imaging of translation based on a tag recognition by a single chain antibody is a powerful technique to assess the fine tuning of translation regulation in living cells. However, especially in a multicellular organism, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different Green Fluorescent Protein variants fused to the single chain fragment variable (scFv) and uncover photobleaching, aggregation and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. Finally, we introduced a new translation imaging method, based on a nanobody/tag system, named ALFA_array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species.

show abstract

Section: Resultssupporting

confidence: 71%

Section: New Scfv-fp Prevents Aggregate Formationsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Boosting the toolbox for live imaging of translation

Bellec

Chen²,

Dhayni

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Investigating the extremely slow motion of single-molecule kinesin-9A requires a more photostable uorescent label than mEGFP. Thus, for the TtK9A uorescent tag, mStayGold, a monomeric, highly photostable, and bright green uorescent protein derived from StayGold [28] with the E138D mutation [29] , was used; whereas for TtK9B1, mEGFP was used (Fig. 1c).…”

Section: Resultsmentioning

confidence: 99%

“…AviHis refers to the Avi-tag (GLNDIFEAQKIEWHE), which is biotinylated by BirA in Escherichia coli [46] and 6× His-tag. The sequence of mStayGold, a monomeric green uorescent protein derived from StayGold [28] with the E138D mutation [29] , was used. All the constructs were veri ed using DNA sequencing.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Characterizing the in vitro motor properties of two kinesin-9 family members from Tetrahymena

Ishii,

Yamagishi,

Yajima

2024

Preprint

View full text Add to dashboard Cite

The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates, is a common feature of the ciliary kinesin-9 family.

show abstract