Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

Zhou, Shuqin; Feng, Siyu; Brown, David

doi:10.1371/journal.pone.0242592

Cited by 12 publications

(10 citation statements)

References 18 publications

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“…This challenge could be overcome by inserting multiple repeats of the mNG211 sequence to increase fluorescent signal as has been demonstrated for split-GFP (He et al, 2019;Hefel and Smolikove, 2019;Kamiyama et al, 2016Kamiyama et al, , 2021Noma et al, 2017). Additionally, a third generation split-mNG system was recently developed and reported to have improved spectral properties (Zhou et al, 2020), which may extend the use of split-FP labeling to low or moderately expressed proteins.…”

Section: Discussionmentioning

confidence: 99%

Tissue-specific and endogenous protein labeling with split fluorescent proteins

Ligunas,

Paniagua,

LaBelle

et al. 2024

Preprint

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The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11(split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10is expressed under a tissue-specific promoter using standard transgenesis while mNG211is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genestubb4bandkrt8in various tissues. We also demonstrate that by anchoring the mNG21-10component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein function. Our approach should be broadly useful for a wide range of applications.

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Section: Discussionmentioning

confidence: 99%

Tissue-specific and endogenous protein labeling with split fluorescent proteins

Ligunas,

Paniagua,

LaBelle

et al. 2024

Preprint

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“…N-terminally His-tagged GRK2 / pFastBac was previously described 132 . FLAG-SMO-nanoluc-IRES-mNG3k/pEF5-FRT-hygro was constructed by fusing in tandem: 1) a full-length FLAG-tagged SMO construct containing the CRD, 7TM domain, pCT, and dCT, and with nanoluciferase (nanoluc) fused at the C-terminus; the native SMO signal sequence was replaced by an N-terminal FLAG tag and an HA signal sequence 38 ; 2) an IRES element; and 3) a mNeonGreen3k fragment (mNG3k) 133 . The mNG3k was included to enable potential mNeonGreen labeling of endogenous NIH3T3 proteins that had been tagged with an mNG11 helix, via mNeonGreen complementation 133 ; however, we ultimately did not pursue this strategy in our experiments, and so this feature of our expression vector was not used.…”

Section: Methodsmentioning

confidence: 99%

GRK2 Kinases in the Primary Cilium Initiate SMOOTHENED-PKA Signaling in the Hedgehog Cascade

Walker

Zhang

Steiner

et al. 2023

Preprint

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During Hedgehog (Hh) signal transduction in development, homeostasis, and cancer, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by directly binding the PKA catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh signal transduction in the vertebrate primary cilium. Hh pathway activation triggers rapid GRK2 relocalization from the base to the shaft of the cilium, leading to SMO phosphorylation and ultimately formation of SMO / PKA-C complexes in this compartment. In vitro reconstitution experiments reveal that GRK2 phosphorylation is sufficient to trigger direct binding of the SMO active conformation to PKA-C, without participation from additional proteins. Lastly, the SMO-GRK2-PKA communication pathway operates during Hh signaling in a range of cellular and in vivo models. Our work highlights GRK2 phosphorylation of ciliary SMO as a key initiating event for the intracellular steps of the Hh cascade, enabling a deeper understanding of how Hh signals are transduced intracellularly in tissues and organs to orchestrate proliferative and differentiative decisions. More broadly, our study hints at an expanded role for GRKs in enabling direct GPCR interactions with a diverse array of intracellular effectors.

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“…The recently developed self-complementing split fluorescent proteins can be used as tools for large-scale endogenous tagging ( Feng et al, 2017 ; Feng et al, 2019 ; Kamiyama et al, 2016 ; Tamura et al, 2021 ; Zhou et al, 2020 ). In the split mNeonGreen system, the mNeonGreen protein is expressed as two separate fragments: a large fragment composed of the first ten beta-strands of mNeonGreen (mNG2 1-10 ) and a short fragment corresponding to the eleventh beta strand (mNG2 11 ; Feng et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Husser,

Pham,

Law

et al. 2024

eLife

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Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

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Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

Cited by 12 publications

References 18 publications

Tissue-specific and endogenous protein labeling with split fluorescent proteins

Tissue-specific and endogenous protein labeling with split fluorescent proteins

GRK2 Kinases in the Primary Cilium Initiate SMOOTHENED-PKA Signaling in the Hedgehog Cascade

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

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