Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Abstract

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-...

Memory sequencing reveals heritable single cell gene expression programs associated with distinct cellular behaviors

“…To create the NGFR-mNG2(11) WM989-A6-G3 cell line, we used the split mNeonGreen2(mNG2) system described in Ref. (Feng et al, 2017) . In brief, we first transduced WM989 A6-G3 with 10/11ths of mNG2, which is non-fluorescent without the remaining 1/11th of the protein.…”

Kinase-mediated RAS signaling via membraneless cytoplasmic protein granules

“…To uncover the connection between EML4-ALK granules and RAS activation, we created a library of gene-edited Beas2B cell lines by introducing a split mNeonGreen21-10/11 tag (mNG2) at the endogenous locus of canonical adaptor and effector proteins in the RTK/RAS/MAPK signaling pathway, including GRB2, GAB1, SOS1, RAS GTPases (H/N/K isoforms) and RAF (A/B/C isoforms) [21]. This suite of isogenic cell lines avoids potential biases that can arise when overexpressing labeled proteins or fixing and permeabilizing cells for immunofluorescence.…”

Bright split red fluorescent proteins with enhanced complementation efficiency for the tagging of endogenous proteins and visualization of synapses

“…Previously, we have shown that split GFP 1-10/11 has nearly identical overall brightness as its fulllength counterpart, whereas both split mNeonGreen2 1-10/11 (mNG2 1-10/11 ) and split sfCherry2 1-10/11 are substantially dimmer (9). This sub-optimal performance of mNG2 1-10/11 and sfCherry2 1-10/11 could be attributed to either (a) the lower molecular brightness of complemented split FPs or (b) incomplete complementation between the FP 1-10 and FP 11 fragments.…”

“…Self-associating split fluorescent proteins (FPs) are a powerful tool for protein labeling and livecell imaging. In this system, the eleventh β-strand of FP (FP 11 , 16 amino acids) is separated out from the remainder of FP (FP [1][2][3][4][5][6][7][8][9][10] ) and is genetically fused to the protein of interest (POI).…”

“…While all these applications have focused on a single, green-colored channel, expanding the color palette will greatly benefit the investigation of more complex biological systems by enabling multicolor imaging. For this purpose, we have recently developed a red-colored split sfCherry 1-10/11 (3) and then a brighter split sfCherry2 1-10/11 (9), enabling dual-color endogenous labeling in human cells using orthogonal FP 11 tags (9) and visualization of Listeria protein secretion in infection (10). However, unlike split GFP 1-10/11 , which is as bright as its full-length, split sfCherry2 1-10/11 produces substantially lower overall fluorescence signal than its full-length counterpart.…”