The Fluorescent Protein Color Palette

Olenych, Scott G.; Claxton, Nathan S.; Ottenberg, Gregory; Davidson, Michael W.

doi:10.1002/0471143030.cb2105s33

Cited by 36 publications

(40 citation statements)

References 158 publications

(289 reference statements)

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“…For many experimental applications, these oligomeric tendencies can be alleviated by mutagenesis of the dimer interface, at least when examined using biochemical techniques such as size-exclusion chromatography (26) and analytical ultracentrifugation (23). Although most of the FPs used in this study (mEGFP, mNeGr, mCit, tagRFPt, and mApple) were previously found to be monomeric in solution, the FPs may be prone to oligomerization artifacts when a high local concentration is generated by fusion to oligomeric proteins (24). For example, tagRFP and EGFP were susceptible to oligomerization when fused to the cytoplasmic face of a resident endoplasmic reticulum membrane protein (27).…”

Section: Discussionmentioning

confidence: 99%

Influence of Fluorescent Tag on the Motility Properties of Kinesin-1 in Single-Molecule Assays

Norris

Nunez

Verhey

2015

Biophysical Journal

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Molecular motors such as kinesin and dynein use the energy derived from ATP hydrolysis to walk processively along microtubule tracks and transport various cargoes inside the cell. Recent advancements in fluorescent protein (FP) research enable motors to be fluorescently labeled such that single molecules can be visualized inside cells in multiple colors. The performance of these fluorescent tags can vary depending on their spectral properties and a natural tendency for oligomerization. Here we present a survey of different fluorescent tags fused to kinesin-1 and studied by single-molecule motility assays of mammalian cell lysates. We tested eight different FP tags and found that seven of them display sufficient fluorescence intensity and photostability to visualize motility events. Although none of the FP tags interfere with the enzymatic properties of the motor, four of the tags (EGFP, monomeric EGFP, tagRFPt, and mApple) cause aberrantly long motor run lengths. This behavior is unlikely to be due to electrostatic interactions and is probably caused by tag-dependent oligomerization events that appear to be facilitated by fusion to the dimeric kinesin-1. We also compared the single-molecule performance of various fluorescent SNAP and HALO ligands. We found that although both green and red SNAP ligands provide sufficient fluorescent signal, only the tetramethyl rhodamine (TMR) HALO ligand provides sufficient signal for detection in these assays. This study will serve as a valuable reference for choosing fluorescent labels for single-molecule motility assays.

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Section: Discussionmentioning

confidence: 99%

Influence of Fluorescent Tag on the Motility Properties of Kinesin-1 in Single-Molecule Assays

Norris

Nunez

Verhey

2015

Biophysical Journal

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“…Finally, for VLP-forming proteins, the tag must not interfere with the capacity of the viral protein or proteins to self-assemble. eGFP was therefore chosen as the fluorescent tag because of its enhanced fluorescent properties, photostability, and relatively small size, as well as the fact that it is a widely used research tool (45)(46)(47). For the eGFP-labeled VLPs described in this report, fluorescence was stable even after particle purification (Fig.…”

Section: Discussionmentioning

confidence: 99%

Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP)

Young¹,

Arthus-Cartier²,

Yam³

et al. 2015

The FASEB Journal

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Medicago, Inc. has developed an efficient virus-like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza-based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA-based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5-eGFP in N. benthamiana produced brightly fluorescent ∼160 nm particles resembling H5-VLPs. H5-eGFP-VLPs elicited anti-H5 serologic responses in mice comparable to those elicited by H5-VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti-GFP IgG response (mean optical density = 2.52 ± 0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5-eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5-eGFP-VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5-eGFP-protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines.

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“…Before transfection, growth media was replaced by Optimem media without FBS (Gibco) and 8 µg of plasmid DNA (mCherry-Mito-7 was a gift from Michael Davidson (Addgene plasmid # 55102) [22] was transfected using 20 µL of lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) per condition. After 5 h of transfection, the media was replaced by complete media.…”

Section: Mcherry-mito7 Stable Transfection In A549 Cellsmentioning

confidence: 99%

Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer

Rodilla

Korrodi‐Gregório

Hernando

et al. 2017

Biochemical Pharmacology

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Current pharmacological treatments for lung cancer show very poor clinical outcomes, therefore, the development of novel anticancer agents with different mechanisms of action is urgently needed. Cancer cells have a reversed pH gradient compared to normal cells, which favors cancer progression by promoting proliferation, metabolic adaptation and evasion of apoptosis. In this regard, the use of ionophores to modulate intracellular pH appears as a promising new therapeutic strategy. Indeed, there is a growing body of evidence supporting ionophores as a novel antitumor drugs. Despite this, little is known about the implications of pH deregulation and homeostasis imbalance triggered by ionophores at the cellular level. In this work, we deeply analyze for the first time the anticancer effects of tambjamine analogues, a group of highly effective anion selective ionophores, at the cellular and molecular level. First, their effects on cell viability were determined in several lung cancer cell lines and patient-derived cancer stem cells, demonstrating their potent cytotoxic effects. Then, we have characterized the induced lysosomal deacidification, as well as, the massive cytoplasmic vacuolization observed after treatment with these compounds, which is consistent with mitochondrial swelling. Finally, the activation of several proteins involved in stress response, autophagy and apoptosis was also detected, although necrosis was the main mechanism of cell death induced.Altogether, these evidences suggest that tambjamine analogues provoke an imbalance in cellular ion homeostasis that triggers mitochondrial dysfunction and lysosomal deacidification leading to a potent cytotoxic effect through necrosis in lung cancer cell lines and cancer stem cells.

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The Fluorescent Protein Color Palette

Cited by 36 publications

References 158 publications

Influence of Fluorescent Tag on the Motility Properties of Kinesin-1 in Single-Molecule Assays

Influence of Fluorescent Tag on the Motility Properties of Kinesin-1 in Single-Molecule Assays

Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP)

Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer

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