In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.
Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell–cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow–green split-mNeonGreen21–10/11 that improves the ratio of complemented signal to the background of FP1–10-expressing cells compared to the commonly used split GFP1–10/11; as well as a 10-fold brighter red-colored split-sfCherry21–10/11. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.
A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. CRISPR/Cas9 | GFP library | genome engineering M ore than a decade after the completion of the Human Genome Project (1), over 30% of human genes still lack clear functional annotation (2, 3). Functional tagging is a powerful strategy to characterize the cellular role of proteins. In particular, tags allow access to two key features of protein function: localization (using fluorescent tags) and interaction partners (using epitope tags and immunoprecipitation). Hence, by tagging proteins in a systematic manner, a comprehensive functional description of an organism's proteome can be achieved. The power of systematic tagging approaches is best illustrated by studies conducted in the budding yeast Saccharomyces cerevisiae (4). In particular, a genome-wide collection of GFP-tagged yeast strains enabled the systematic study of protein localization in live cells (5), whereas libraries of strains expressing TAP epitope-fusion proteins paved the way for the large-scale isolation and proteomic analysis of protein complexes (6, 7). One of the great advantages of yeast genetics (especially in S. cerevisiae) is the efficiency and relative simplicity of PCR-based homologous recombination (8). As a result, functional tags can be easily inserted in a gene locus of interest, preserving endogenous expression levels and minimizing genomic disruption. Together, these genome-wide tagged libraries helped provide a comprehensive snapshot of the yeast protein landscape under near-native conditions (4, 5, 9-11).The development of clustered regularly interspersed short palindromic repeat associated protein 9 (CRISPR/Cas9)-based methods has profoundly transformed our ability to dir...
SIGNIFICANCE STATEMENTThe function of a large fraction of the human proteome still remains poorly characterized. Tagging proteins with a functional sequence is a powerful way to access function, and inserting tags at endogenous genomic loci allows the preservation of a near-native cellular background. To characterize the cellular role of human proteins in a systematic manner and in a native context, we developed a method for tagging endogenous human proteins with GFP that is both rapid and readily applicable at a genome-wide scale. Our approach allows studying both localization and interaction partners of the protein target. Our results pave the way for the large-scale generation of endogenously tagged human cell lines for a systematic functional interrogation of the human proteome.
Epidermal growth factor (EGF) family members, including epiregulin (EP), play a fundamental role in epithelial tissues; however, their roles in immune responses and the physiological role of EP remain to be elucidated. The skin has a versatile system of immune surveillance. Biologically active IL-1␣ is released to extracellular space upon damage from keratinocytes and is a major player in skin inflammation. Here, we show that EP is expressed not only in keratinocytes but also in tissue-resident macrophages, and that EP-deficient (EP ؊/؊ ) mice develop chronic dermatitis. Wound healing in the skin in EP ؊/؊ mice was not impaired in vivo, nor was the growth rate of keratinocytes from EP ؊/؊ mice different from that of WT mice in vitro. Of interest is that in WT keratinocytes, both IL-1␣ and the secreted form of EP induced down-regulation of IL-18 mRNA expression, which overexpression in the epidermis was reported to induce skin inflammation in mice, whereas the downregulation of IL-18 induced by IL-1␣ was impaired in EP ؊/؊ keratinocytes. Although bone marrow transfer experiments indicated that EP deficiency in non-bone-marrow-derived cells is essential for the development of dermatitis, production of proinflammatory cytokines by EP ؊/؊ macrophages in response to Toll-like receptor agonists was much lower, compared with WT macrophages, whose dysfunction in EP ؊/؊ macrophages was not compensated by the addition of the secreted form of EP. These findings, taken together, suggested that EP plays a critical role in immune͞inflammatory-related responses of keratinocytes and macrophages at the barrier from the outside milieu and that the secreted and membranebound forms of EP have distinct functions.T he system of epidermal growth factor (EGF) superfamily (1) and EGF receptor (EGFR) family, including EGFR (ErbB1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4) (2), play a fundamental role in epithelial tissues. EGF family members, including EGF, transforming growth factor-␣, amphiregulin, heparinbinding EGF (HB-EGF), epiregulin (EP), and other members, regulate these receptors by inducing their homo-and͞or heterooligomerization (2, 3). EGF family members vary in their ability to activate distinct ErbB heterodimers, and this mechanism may, in part, account for the differences in their bioactivities (4-7).The membrane-anchored precursor of the EGF family is enzymatically processed externally to release a mature soluble form that acts as autocrine and͞or paracrine growth factor (8, 9), whereas some members of the EGF family act in the membrane-anchored form (8, 10). A bioactive transmembrane precursor, pro-HB-EGF, was suggested to induce growth inhibition or apoptosis rather than the proliferative response induced by soluble HB-EGF (10). EP (11-17) acts as an autocrine growth factor in normal human keratinocytes in vitro (15); however, its physiological role in vivo still remains to be elucidated.Keratinocytes in the epidermis play critical roles in the cutaneous immune-related responses (18) and contain biologically active ...
Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact.To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen2 1-10/11 that improves the ratio of complemented signal to the background of FP 1-10 -expressing cells compared to the commonly used split GFP 1-10/11 ; as well as a 10-fold brighter red-colored split-sfCherry2 1-10/11 . Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry2 11 and GFP 11 , revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes. 5,6 , identification of cell contacts and synapses 7,8 , as well as scaffolding protein assembly 3, 9, 10 . Recently, they have also enabled the generation of large-scale human cell line libraries with fluorescently tagged endogenous proteins through CRISPR/Cas9-based gene editing 11 .So far, the most commonly used self-complementing split FP was GFP 1-10D7/11M3 OPT (which we refers to as GFP 1-10/11 ), engineered from super-folder GFP (sfGFP) 12 . With the splitting point between the tenth and eleventh β-strands, the resulting GFP 11 fragment is a 16-amino acid (a.a.) short peptide. The corresponding GFP 1-10 fragment remains almost non-fluorescent until complementation, making GFP 1-10/11 well suited for protein labeling by fusing GFP 11 to the target protein and over-expressing GFP 1-10 in the corresponding subcellular compartments. However, there lacks a second, orthogonal split FP system with comparable complementation performance for multicolor imaging and multiplexed scaffolding of protein assembly. Previously, a sfCherry 1-10/11 system 3 was derived from super-folder Cherry, an mCherry variant optimized for folding efficiency 13 . However, its overall fluorescent brightness is substantially weaker than an intact sfCherry fusion, potentially due to its limited complementation efficiency 3 . Although two-color imaging with sfCherry 1-10/11 and GFP 1-10/11 has been done using tandem sfCherry 11 to amplify the sfCherry signal for over-expressed targets, it is still too dim to detect most endogenous proteins.In this paper, we report a screening strategy for the direct engineering of self-complementing split FPs. Using this strategy, we have generated a yellow-green-colored mNeonGreen2 1-10/11 (mNG2) that has an improved ratio of complemented signal to the background of FP 1-10 -expressing cells as compared to GFP 1-10/11 , as well as a red-colored sfCherry2 1-...
Highlights d Nanopores of Drosophila olfactory sensillum are modified cuticular envelope d Endocytic membrane structures are associated with the site of nanopore formation d Gore-tex/Osiris23 endosomal protein is required for nanopore formation d gore-tex/Osiris23 mutants showed greatly reduced olfactory response
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.