A palette of fluorescent proteins optimized for diverse cellular environments

Costantini, Lindsey M.; Baloban, Mikhail; Markwardt, Michele L.; Rizzo, Mark A.; Guo, Feng; Verkhusha, Vladislav V.; Snapp, Erik L.

doi:10.1038/ncomms8670

Cited by 215 publications

(225 citation statements)

References 60 publications

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“…To address this issue, we developed improved reagents for in vivo imaging of the ER. First, we utilized a mutant GFP, termed Superfolder GFP (sfGFP) (Aronson et al, 2011;Costantini et al, 2015;Pédelacq et al, 2006), that has been found to fold properly in oxidizing environments such as the ER lumen. This protein traffics to the ER because of addition of the Drosophila BiP signal sequence and is retained by addition of the HDEL ER retention signal from calreticulin (BiP-sfGFP-HDEL).…”

Section: Resultsmentioning

confidence: 99%

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster

Summerville

Faust

Fan

et al. 2016

Journal of Cell Science

107

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Hereditary spastic paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate endoplasmic reticulum (ER) morphology. Atlastin 1 (ATL1, also known as SPG3A) encodes an ER membrane fusion GTPase and reticulon 2 (RTN2, also known as SPG12) helps shape ER tube formation. Here, we use a new fluorescent ER marker to show that the ER within wild-type Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse upon loss of the atlastin 1 ortholog atl. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similar to other HSP proteins, Atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a crucial role for ER in neuronal function, and identify mechanistic links between ER morphology, neuronal function, BMP signaling and adult behavior.

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Section: Resultsmentioning

confidence: 99%

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster

Summerville

Faust

Fan

et al. 2016

Journal of Cell Science

107

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“…We examined RFPs, including: mCherry, TagRFP, mKate2, mRuby2, and Fusion Red (12,13), in a live cell assay (14) that assesses the propensity of FPs to oligomerize. Importantly, reportedly monomeric TagRFP, mRuby2, and mKate2 all formed inappropriate oligomers, as did mCherry, but to a much more limited degree (15,16).…”

Section: The Oligomeric Tendency Of Fluorescent Proteinsmentioning

confidence: 98%

“…Either modification can induce gross misfolding and produce a population of nonfluorescent misfolded FPs (15,20). To protect against these cellular environments, we recommend selecting FPs with low pK a values and no or few residues subject to posttranslational modifications, i.e., cysteines and N-linked glycosylation consensus sequences (N-X-S/T).…”

Section: The Impact Of the Cellular Environment On Fluorescent Proteinsmentioning

confidence: 99%

“…For expressing proteins in oxidizing cellular compartments (secretory pathway, inner membrane space of mitochondria, and periplasm of Gram-negative bacteria), we recommend the "mox" FPs we recently developed, including moxsynGFP, moxCerulean3, moxVenus, and moxBFP (15). These genuinely monomeric FPs lack cysteines and, thus, cannot form disulfide bonds.…”

Section: The Impact Of the Cellular Environment On Fluorescent Proteinsmentioning

confidence: 99%

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Going Viral with Fluorescent Proteins

Costantini

Snapp

2015

J Virol

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Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses. Statements such as "my fluorescent virus can barely replicate" and "I cannot see my fluorescent protein fusion" are, unfortunately, not uncommon complaints about fusion proteins made with fluorescent proteins (FPs). In some cases, FPs are inappropriate for the system or question. Other times, FPs are incorporated in a haphazard manner. Finally, FPs may not perform as advertised or the investigator may not read the "fine print." In this review, we describe how to avoid common FP problems and how to select appropriate FPs for FP fusions.With multiple fluorescent labeling strategies available, why use FP tags? Generally, FPs have many desirable characteristics. They are genetically encoded, have sufficiently short sequences for incorporation into many viral genomes, and enable site-specific labeling of a viral or cellular protein of interest. Dye-labeling techniques often randomly label proteins and require extracellular delivery, and dye-labeled proteins cannot be delivered into subcellular organelles, such as the endoplasmic reticulum. Difficulties frequently arise when investigators expect FPs to be inert, well behaved in all environments, and provide a bright signal. While few, if any, FPs satisfy every item on a wish list, FPs are undeniably powerful cell biology tools. For a detailed list of virus-relevant methods that exploit FPs, see reference 1 (especially Tables 1 and 2). For basic considerations for developing FP fusion proteins, see reference 2. PHYSICAL PROPERTIES OF FLUORESCENT PROTEINSFirst, let us consider the general properties of FPs. They have short primary sequences but fold into proteins that are not small (ϳ5 nm in diameter) (3). FPs are evolved as soluble cytoplasmic proteins, with only the environmental considerations related to the pH-neutral cytoplasm as a selective pressure. Taken together, these properties suggest significant concerns when using FPs, such as the potential for steric hindrance in fusion proteins and for the performance of FPs in subcellular compartments. The photophysical characteristics of FPs range tremendously in brightness and photostability. Unless the investigator is studying isolated FPs or using advanced microscopy techniques, including total internal-reflection fluorescence (TIRF) (4) and photoactivated localization microscopy (PALM) (5), it is difficult to detect the signal of a single FP in the presence of the cellular autofluorescence background (2). The photophysical pr...

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“…1,2 Various fluorescent labeling methods for proteins have been developed, with those based on fluorescent proteins being most widely used due to their simplicity and specificity, where target proteins are fused with fluorescent proteins by introducing genes encoding the appropriate fusion proteins into cells. [3][4][5][6][7][8] Concomitant with the dissemination of fluorescent protein-based labeling methods, new labeling methods based on chemical biology have been proposed, such as tag-probe labeling technologies, in which synthetic fluorescent probes are attached to the target proteins carrying genetically encoded tags through specific interactions between the fluorescent probes and the tags. [9][10][11][12] These chemical biology-based approaches allow for the labeling of proteins with synthetic fluorophores bearing various properties and controlling the timing of the labeling, thus broadening their applications in protein analysis.…”

Section: Introductionmentioning

confidence: 99%

Labeling of Cytoskeletal Proteins in Living Cells Using Biotin Ligase Carrying a Fluorescent Protein

et al. 2017

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Labeling with fluorescent proteins is now widely exploited for elucidating the functions and roles of target proteins in living cells. Previously, we developed a protein labeling method by combining a fluorescent protein with a biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). By taking advantage of this unique property, a target protein carrying BCCP in living cells can be labeled through biotinylation with BPL carrying a fluorescent protein. In the present work, to demonstrate the utility and performance of this labeling system in more detail, the cytoskeletal proteins β-actin and α-tubulin were selected as target proteins and labeled in living cells. With this approach, we succeeded in fluorescent imaging of actin filaments and microtubules in living cells, and shows the advantages of our approach over the conventional labeling methods with fluorescent proteins.

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A palette of fluorescent proteins optimized for diverse cellular environments

Cited by 215 publications

References 60 publications

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster

Going Viral with Fluorescent Proteins

Labeling of Cytoskeletal Proteins in Living Cells Using Biotin Ligase Carrying a Fluorescent Protein

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