Monomeric Fluorescent Protein Research Articles

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications. Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system (1,2). T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells (3,4). T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity (5). The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression (6,7). The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites (8). Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications. Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process (9,10). We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization (11). Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody (8,9), which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.

The mFruit Collection of Monomeric Fluorescent Proteins

Featured Article: Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 2004:22;1567–72.2 Fluorescent proteins, starting with the green fluorescent protein (GFP)3 from Aequorea victoria, have revolutionized our ability to noninvasively study living systems (1). Practically every cell biologist has used fluorescent proteins to tag a favorite protein and watch its dynamic localization in living cells. For almost 10 years after the gene encoding GFP was cloned, researchers were limited to using only about half of the visible spectrum with these probes and were able to image only 2 different colors simultaneously, at best. In 1999, Mikhail Matz and colleagues at the Russian Academy of Science discovered the first known red fluorescent protein, in Discosoma sp. coral; that protein would become known as DsRed (2). Hopes were high that the wavelength barrier had been broken, and many researchers jumped at the chance to try this new color. Unfortunately, it soon …

New Far-Red Fluorescent Proteins for Non-Invasive Imaging of Stem Cell Differentiation

Non-invasive high-resolution imaging of cell populations in animal disease models would greatly facilitate the development of cell-based therapies. Optical imaging of fluorescent proteins (FPs) that can be excited by red light in the “optical window” above 600 nm, such as the monomeric far-red FP mNeptune, is one potential method for tracking implanted cells. However the utility of red-absorbing FPs for tracking the fates of implanted cells has not yet been explored. Here we report three new red-absorbing monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has an additionally red-shifted absorbance spectrum. We show that mCardinal can be used to non-invasively visualize muscle regeneration from myoblasts and stem cells in a mouse injury model with high anatomical detail. Correction et al.Biophysical JournalApril 16, 2013In Brief2013. New Far-Red Fluorescent Proteins for Non-Invasive Imaging of Stem Cell Differentiation. Jun Chu, Russell D. Haynes, Stephane Y. Corbel, Helen M. Blau. Biophys. J. 104(2)s: 342a. Full-Text PDF Open Archive

Developing Photoactivatable Fluorescent Proteins for Diffraction-Limited and Superresolution Imaging

Photoactivatable fluorescent proteins (PAFPs) are molecules that switch to a new fluorescent state in response to specific light activation, and play vital roles in super-resolution imaging. There are three classes of PAFPs: dark-to-bright photoactivators (PAFPs), irreversible photoconverters (PCFPs), and reversible highlighters (RSFP). However, compared to traditional fluorescent proteins (such as GFP or RFP), only limited PAFPs are available for super-resolution microscopy.Previously, we developed several novel PAFPs, mGeos, with various switching rates, photon numbers and brightness1. And based on the crystal structure of green state mEos2, we evolved two truly monomeric and bright RSFPs, mEos3.1 and mEos3.2, with the good photochemical properties including rapid maturation rate, high photon budget and extremely high labeling density2. Here we present new generation of mGeos2 which are true monomeric PAFPs. These novel fluorescent proteins are suitable for both single color and dual color PALM superresolution imaging, and have a broad brand of applications in traditional fluorescence microscopy such as dynamic tracking and pulse chase labeling of proteins.1. Chang H, Zhang M, Ji W, Chen J, Zhang Y, Liu B, Lu J, Zhang J, Xu P, Xu T, A new series of reversibly switchable fluorescent proteins with beneficial properties for various applications, Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):4455-60.2. Zhang M, Chang H, Zhang Y, Yu J, Wu L, Ji W, Chen J, Liu B, Lu J, Liu Y, Zhang J, Xu P, Xu T. Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods. 2012 May 13;9(7):727-9.

Fluorescence Anisotropy in a Protein: DNA System Undergoing Inducible Assembly

Fluorescence anisotropy may be used as an indicator of homo-FRET making it an attractive reported of oligomer formation and aggregation. A model system was created consisting of a monomeric teal fluorescent protein peptide nucleic acid (mTFP-PNA) fusion. The mTFP-PNA monomer units produced by expressed protein ligation (EPL). An mTFP with a C-terminal thioester group was expressed with a modified intein and subsequently conjugated to a PNA with an N-terminal thiol group. This generates a native peptide bond at the ligation site. Addition of a complementary template DNA to solutions of mTFP-PNA monomer units resulted in template directed induced assembly. Assembly was confirmed by SDS-PAGE, mass spectrometry, and size exclusion HPLC.Fluorescence anisotropy was monitored over the course of a mTFP-PNA:DNA titration. Template coding for dimer formation was studied. The anisotropy showed a decreasing trend related to homo-FRET in the assembled forms. A maximal reduction (41%) was observed at a DNA to mTFP-PNA ratio of 1:2. Anisotropy, then increased steadily up to a 1:1 ratio.This study demonstrates an inducibly assembled homo-FRET system using expressed protein ligation which may be extended to study oligomerization and cluster formation in living systems.

Differential Oligomeric Nature of mEos2 and mEos3.2 Fluorescent Proteins is Consequential to Diffusion and Confinement of Membrane Probes

Lipid-mediated membrane heterogeneity is proposed to be an important organizing principle in mammalian cells. using super-resolution fluorescence localization imaging we track the diffusion of a large panel of fluorescent membrane probes ranging in size, mode of membrane anchoring, and putative phase-association. Our expressed probes include fluorescently-tagged palmitoylated or non-palmitoylated versions of transmembrane domains including linker of activated T-cell and haemagglutinin, and fluorescent protein anchored by GPI, palmitate-myristoyl moieties, or a geranyl-geranyl moiety. The recent advent of photoconvertible fluorescent protein mEos3.2, a “truly monomeric” mutant of its mEos2 predecessor1, has enabled us to compare directly monomeric and oligomeric versions of the same probes. Our results indicate that in some cases rates of diffusion are more than two fold lower in the mEos2 probes when compared to their mEos3.2 counterparts. Many mEos2-labeled probes also differ from mEos3.2-labeled probes in their exponents of anomalous diffusion. We attribute changes in mobility to the proteins' differential propensity to oligomerize, and are using the clustering effect of mEos2 to investigate whether phase heterogeneity influences cluster mobility.1 Zhang, M. et al. Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods 9, 727-729. (2012).

Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.

Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.

R26R-GR: A Cre-Activable Dual Fluorescent Protein Reporter Mouse

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

Caveolin-1 Mediates the Internalization of Functional Insulin Receptors in Beta-Cells

Type 2 diabetes is associated with defects in insulin receptor (InsR) signalling, including in beta-cells. Internalization of the activated InsR complex is necessary to initiate signalling. Mechanisms of InsR internalization in beta-cells remain unclear, although Caveolin-1 (CAV1) has been implicated in other cell types. We labelled InsR with monomeric fluorescent proteins in a non-critical linker region between the furin-like region and the transmembrane domain. Total internal reflection microscopy in living MIN6 beta-cells revealed that CAV1 is recruited to InsR membrane domains right before receptor internalization. These results were confirmed by super-resolution stimulated emission depletion microscopy, which showed a CAV1 protein coat around membrane nanodomains containing InsR. To investigate if CAV1 is essential for InsR internalization, we compared the distribution of InsR in cells expressing CAV1 mutant proteins. MIN6 cells over-expressing dominant negative CAV1-Y14F showed defects of InsR internalization, with virtually complete accumulation plasma membrane of InsR. Overexpression of CAV1 in MIN6 cells leads to an increased Erk1/2 phosphorylation upon insulin treatment compared to empty-vector controls. Erk1/2 signalling is further increased in cells overexpressing the dominant-positive CAV1-Y14D mutant. Interestingly, Akt phosphorylation was not affected by CAV1 overexpression. These data strongly suggest that CAV1-mediated internalization of the InsR is required for Erk1/2 signalling but Akt signalling is mostly induced by insulin receptors localized at the plasma membrane. Together, our data give new insights on the mechanisms in InsR signalling in the beta-cell.

Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP+RFP+ puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.

Improper tagging of the non-essential small capsid protein VP26 impairs nuclear capsid egress of herpes simplex virus.

To analyze the subcellular trafficking of herpesvirus capsids, the small capsid protein has been labeled with different fluorescent proteins. Here, we analyzed the infectivity of several HSV1(17+) strains in which the N-terminal region of the non-essential small capsid protein VP26 had been tagged at different positions. While some variants replicated with similar kinetics as their parental wild type strain, others were not infectious at all. Improper tagging resulted in the aggregation of VP26 in the nucleus, prevented efficient nuclear egress of viral capsids, and thus virion formation. Correlative fluorescence and electron microscopy showed that these aggregates had sequestered several other viral proteins, but often did not contain viral capsids. The propensity for aggregate formation was influenced by the type of the fluorescent protein domain, the position of the inserted tag, the cell type, and the progression of infection. Among the tags that we have tested, mRFPVP26 had the lowest tendency to induce nuclear aggregates, and showed the least reduction in replication when compared to wild type. Our data suggest that bona fide monomeric fluorescent protein tags have less impact on proper assembly of HSV1 capsids and nuclear capsid egress than tags that tend to dimerize. Small chemical compounds capable of inducing aggregate formation of VP26 may lead to new antiviral drugs against HSV infections.

A FRET-Facilitated Photoswitching Using an Orange Fluorescent Protein with the Fast Photoconversion Kinetics

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via Förster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells.

Easy preparation of a large-size random gene mutagenesis library in Escherichia coli

A simple and fast protocol for the preparation of a large-size mutant library for directed evolution in Escherichia coli was developed based on the DNA multimers generated by prolonged overlap extension polymerase chain reaction (POE–PCR). This protocol comprised the following: (i) a linear DNA mutant library was generated by error-prone PCR or shuffling, and a linear vector backbone was prepared by regular PCR; (ii) the DNA multimers were generated based on these two DNA templates by POE–PCR; and (iii) the one restriction enzyme-digested DNA multimers were ligated to circular plasmids, followed by transformation to E. coli. Because the ligation efficiency of one DNA fragment was several orders of magnitude higher than that of two DNA fragments for typical mutant library construction, it was very easy to generate a mutant library with a size of more than 107 protein mutants per 50μl of the POE–PCR product. Via this method, four new fluorescent protein mutants were obtained based on monomeric cherry fluorescent protein. This new protocol was simple and fast because it did not require labor-intensive optimizations in restriction enzyme digestion and ligation, did not involve special plasmid design, and enabled constructing a large-size mutant library for directed enzyme evolution within 1day.

Rational design of true monomeric and bright photoactivatable fluorescent proteins

Monomeric (m)Eos2 is an engineered photoactivatable fluorescent protein widely used for super-resolution microscopy. We show that mEos2 forms oligomers at high concentrations and forms aggregates when labeling membrane proteins, limiting its application as a fusion partner. We solved the crystal structure of tetrameric mEos2 and rationally designed improved versions, mEos3.1 and mEos3.2, that are truly monomeric, are brighter, mature faster and exhibit higher photon budget and label density.

Enhanced detection efficiency of genetically encoded tag allows the visualization of monomeric proteins by electron microscopy

A cadmium-binding, genetically encoded protein tag, consisting of three repeats of metallothionein (3MT), can be used in electron microscopy for the visualization of multimeric- but not monomeric-tagged proteins due to insufficient electron density in monomeric proteins. Here, we present a technique for detecting monomeric 3MT-tagged green fluorescent protein (GFP-3MT) using a platinum compound to intensify the electron density. Substitution of cadmium by platinum as a result of incubating purified cadmium-binding 3MT-tagged GFP (GFP-Cd-3MT) with cis-diamminedichloroplatinum(II) (cisDDP) was assessed by a UV absorption band centered at 284nm thereby indicating platinum-sulfhydryl bonds. The incubation time and the concentration of cisDDP to reach maximal absorption were 2h and 36-fold molar equivalent of cisDDP, respectively. GFP-Pt-3MT isolated by gel filtration chromatography contained 29 platinum atoms per single GFP-3MT molecule. Electron-dense particles were observed in a GFP-Pt-3MT sample by electron microscopy without negative staining. Further image processing and image analysis demonstrated that particles with higher density relative to their surroundings were detectable in both GFP-Cd-3MT and GFP-Pt-3MT samples. These results demonstrate that replacement of cadmium with platinum, together with proper image analyses, improve detection efficiency and enable the visualization of 3MT-tagged monomeric protein by electron microscopy.

Segregation of molecules at cell division reveals native protein localization

We introduce a non-intrusive method exploiting post-division single-cell variability to validate protein localization. The results show that Clp proteases, widely reported to form biologically relevant foci, are in fact uniformly distributed inside Escherichia coli cells, and that many commonly used fluorescent proteins (FPs) cause severe mislocalization when fused to homo-oligomers. Re-tagging five other reportedly foci-forming proteins with the most monomeric FP tested suggests the foci were caused by the FPs.

Assessing the Tendency of Fluorescent Proteins to Oligomerize Under Physiologic Conditions

Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein (EGFP) or monomeric green fluorescent protein (mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize.