RESUMO
Cell migration in confined 3D tissue microenvironments is critical for both normal physiological functions and dissemination of tumor cells. We discovered a cytoskeletal structure that prevents damage to the nucleus during migration in confined microenvironments. The formin-family actin filament nucleator FMN2 associates with and generates a perinuclear actin/focal adhesion (FA) system that is distinct from previously characterized actin/FA structures. This system controls nuclear shape and positioning in cells migrating on 2D surfaces. In confined 3D microenvironments, FMN2 promotes cell survival by limiting nuclear envelope damage and DNA double-strand breaks. We found that FMN2 is upregulated in human melanomas and showed that disruption of FMN2 in mouse melanoma cells inhibits their extravasation and metastasis to the lung. Our results indicate a critical role for FMN2 in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage to promote cell survival during confined migration and thus promote cancer metastasis.
Assuntos
Núcleo Celular/metabolismo , Adesões Focais , Neoplasias Pulmonares/secundário , Melanoma/patologia , Proteínas dos Microfilamentos/metabolismo , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Actinas/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos/citologia , Matriz Extracelular/metabolismo , Feminino , Forminas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido NervosoRESUMO
Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple "cryptic leading edges" within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as "cell polarization barriers," decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.
Assuntos
Polaridade Celular , Matriz Extracelular/patologia , Adesões Focais , Metástase Neoplásica , Neoplasias/patologia , Fibras de Estresse/patologia , HumanosRESUMO
Inferring the organization of fluorescently labeled nanosized structures from single molecule localization microscopy (SMLM) data, typically obscured by stochastic noise and background, remains challenging. To overcome this, we developed a method to extract high-resolution ordered features from SMLM data that requires only a low fraction of targets to be localized with high precision. First, experimentally measured localizations are analyzed to produce relative position distributions (RPDs). Next, model RPDs are constructed using hypotheses of how the molecule is organized. Finally, a statistical comparison is used to select the most likely model. This approach allows pattern recognition at sub-1% detection efficiencies for target molecules, in large and heterogeneous samples and in 2D and 3D data sets. As a proof-of-concept, we infer ultrastructure of Nup107 within the nuclear pore, DNA origami structures, and α-actinin-2 within the cardiomyocyte Z-disc and assess the quality of images of centrioles to improve the averaged single-particle reconstruction.
Assuntos
DNA , Imagem Individual de MoléculaRESUMO
The advent of fluorescent proteins (FPs) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight ß-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.
Assuntos
Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Proteínas Recombinantes de Fusão/análise , Espectrometria de Fluorescência/métodos , Fluorescência , Células HeLa , HumanosRESUMO
Nanoclustering is an emerging organizational principle for membrane-associated proteins. The functional consequences of nanoclustering for receptor signaling remain largely unknown. Here, we applied quantitative multi-channel high- and super-resolution imaging to analyze the endothelial cell surface receptor CD36, the clustering of which upon binding to multivalent ligands, such as the anti-angiogenic factor thrombospondin-1 (TSP-1), is thought to be crucial for signaling. We found that a substantial fraction of unligated CD36 exists in nanoclusters, which not only promote TSP-1 binding but are also enriched with the downstream effector Fyn. Exposure to multivalent ligands (TSP-1 or anti-CD36 IgM) that result in larger and denser CD36 clusters activates Fyn. Conversely, pharmacological perturbations that prevent the enhancement of CD36 clustering by TSP-1 abrogate Fyn activation. In both cases, there is no detectable change in Fyn enrichment at CD36 nanoclusters. These observations reveal a crucial role for the basal organization of a receptor into nanoclusters that are enriched with the signal-transducing downstream effectors of that receptor, such that enhancement of clustering by multivalent ligands is necessary and sufficient to activate the downstream effector without the need for its de novo recruitment.
Assuntos
Antígenos CD36/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Transdução de Sinais , Actinas/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Células Endoteliais/metabolismo , Ativação Enzimática , Humanos , Ligantes , Microvasos/citologia , Modelos Biológicos , Ligação Proteica , Trombospondina 1/metabolismoRESUMO
Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.
Assuntos
Proteínas Luminescentes/metabolismo , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cricetulus , Fluorescência , Células HeLa , Humanos , Proteínas Luminescentes/genética , Imagem Molecular/métodos , Dados de Sequência Molecular , Tetróxido de Ósmio/química , Fotoquímica/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.
Assuntos
Proteínas de Fluorescência Verde/química , Raios Infravermelhos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , DNA/química , Biologia do Desenvolvimento , Drosophila melanogaster , Corantes Fluorescentes/química , Células HeLa , Histidina/química , Humanos , Proteínas Luminescentes/química , Camundongos , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Plasmídeos/metabolismo , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Transfecção , Peixe-ZebraRESUMO
Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be â¼97 nm in length and oriented at â¼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.
Assuntos
Adesões Focais/metabolismo , Nanoestruturas , Talina/fisiologia , Humanos , Microscopia de FluorescênciaRESUMO
A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.
Assuntos
Diferenciação Celular , Diagnóstico por Imagem/métodos , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Animais , Cristalografia por Raios X , Biblioteca Gênica , Células HeLa , Hemoglobinas/química , Humanos , Ligação de Hidrogênio , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Células Musculares/metabolismo , Músculo Esquelético/patologia , Músculos/patologia , Mutagênese , Mioblastos/metabolismo , Mioglobina/química , Células NIH 3T3 , Regeneração , Células-Tronco/citologia , Proteína Vermelha FluorescenteRESUMO
Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.
Assuntos
Algoritmos , Aumento da Imagem/instrumentação , Microscopia de Vídeo/instrumentação , Imagem Molecular/instrumentação , Nanotecnologia/instrumentação , Reconhecimento Automatizado de Padrão/métodos , Semicondutores , Desenho de Equipamento , Análise de Falha de Equipamento , Processamento de Sinais Assistido por Computador/instrumentaçãoRESUMO
We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.
Assuntos
Cordados/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Animais , Dados de Sequência Molecular , Processos EstocásticosRESUMO
Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.
Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Vinculina/metabolismo , Animais , Colágeno/genética , Matriz Extracelular/genética , Fibroblastos/citologia , Camundongos , Camundongos Knockout , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Pseudópodes/genética , Pseudópodes/metabolismo , Vinculina/genéticaRESUMO
Mutations and alterations in caveolin-1 expression levels have been linked to a number of human diseases. How misregulation of caveolin-1 contributes to disease is not fully understood, but has been proposed to involve the intracellular accumulation of mutant forms of the protein. To better understand the molecular basis for trafficking defects that trap caveolin-1 intracellularly, we compared the properties of a GFP-tagged version of caveolin-1 P132L, a mutant form of caveolin-1 previously linked to breast cancer, with wild-type caveolin-1. Unexpectedly, wild-type caveolin-1-GFP also accumulated intracellularly, leading us to examine the mechanisms underlying the abnormal localization of the wild type and mutant protein in more detail. We show that both the nature of the tag and cellular context impact the subcellular distribution of caveolin-1, demonstrate that even the wild-type form of caveolin-1 can function as a dominant negative under some conditions, and identify specific conformation changes associated with incorrectly targeted forms of the protein. In addition, we find intracellular caveolin-1 is phosphorylated on Tyr14, but phosphorylation is not required for mistrafficking of the protein. These findings identify novel properties of mistargeted forms of caveolin-1 and raise the possibility that common trafficking defects underlie diseases associated with overexpression and mutations in caveolin-1.
Assuntos
Caveolina 1/metabolismo , Mutação de Sentido Incorreto , Fenótipo , Animais , Neoplasias da Mama/genética , Células COS , Caveolina 1/química , Caveolina 1/genética , Chlorocebus aethiops , Feminino , Células HeLa , Humanos , Fosforilação , Conformação Proteica , Transporte ProteicoRESUMO
A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Sequência de Bases , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína Vermelha FluorescenteRESUMO
BACKGROUND AIMS: Pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) represent an unlimited source for the treatment of various neurological disorders. NPCs are usually derived from PSCs through the formation of embryoid body (EB), an aggregate structure mimicking embryonic development. This study investigated the effect of labeling multicellular EB-NPC aggregates with micron-sized particles of iron oxide (MPIO) for cell tracking using magnetic resonance imaging (MRI). METHODS: Intact and dissociated EB-NPC aggregates were labeled with various concentrations of MPIOs (0, 2.5, 5 and 10 µg Fe/mL). The labeled cells were analyzed by fluorescent imaging, flow cytometry and in vitro MRI for labeling efficiency and detectability. Moreover, the biological effects of intracellular MPIO on cell viability, cytotoxicity, proliferation and neural differentiation were evaluated. RESULTS: Intact EB-NPC aggregates showed higher cell proliferation and viability compared with the dissociated cells. Despite diffusion limitation at low MPIO concentration, higher concentration of MPIO (i.e., 10 µg Fe/mL) was able to label EB-NPC aggregates at similar efficiency to the single cells. In vitro MRI showed concentration-dependent MPIO detection in EB-NPCs over 2.0-2.6 population doublings. More important, MPIO incorporation did not affect the proliferation and neural differentiation of EB-NPCs. CONCLUSIONS: Multicellular EB-NPC aggregates can be efficiently labeled and tracked with MPIO while maintaining cell proliferation, phenotype and neural differentiation potential. This study demonstrated the feasibility of labeling EB-NPC aggregates with MPIO for cellular monitoring of in vitro cultures and in vivo transplantation.
Assuntos
Células-Tronco Embrionárias/citologia , Compostos Férricos/farmacologia , Nanopartículas de Magnetita/administração & dosagem , Células-Tronco Mesenquimais/citologia , Células-Tronco Neurais/citologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Rastreamento de Células/métodos , Células-Tronco Embrionárias/efeitos dos fármacos , Imageamento por Ressonância Magnética/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Tamanho da Partícula , Coloração e Rotulagem/métodosRESUMO
Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling but is independent of all activity by Toll-like receptors and does not require the presence of the adaptor protein Card9. We further demonstrate that reactive oxygen species generation by NADPH oxidase is required for LC3 recruitment to the fungal phagosome. These observations directly link LC3 to the inflammatory pathway against C. albicans in macrophages.
Assuntos
Fungos/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Candida albicans/imunologia , Linhagem Celular , Interleucina-1beta/biossíntese , Interleucina-6/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/imunologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Modelos Biológicos , NADPH Oxidases/metabolismo , Fagossomos/imunologia , Fagossomos/microbiologia , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteoglicanas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Quinase Syk , Fator de Necrose Tumoral alfa/biossíntese , beta-Glucanas/metabolismoRESUMO
We developed a coating method to produce functionalized small quantum dots (sQDs), about 9â nm in diameter, that were stable for over a month. We made sQDs in four emission wavelengths, from 527 to 655â nm and with different functional groups. AMPA receptors on live neurons were labeled with sQDs and postsynaptic density proteins were visualized with super-resolution microscopy. Their diffusion behavior indicates that sQDs access the synaptic clefts significantly more often than commercial QDs.
Assuntos
Corantes Fluorescentes/análise , Neurônios/citologia , Pontos Quânticos/análise , Receptores de AMPA/análise , Animais , Células Cultivadas , Microscopia de Fluorescência , Imagem Óptica , RatosRESUMO
BACKGROUND AIMS: Human mesenchymal stem cells (hMSCs) have gained interest for treatment of stroke injury. Using in vitro culture, the purpose of this study was to investigate the long-term detectability of hMSCs by magnetic resonance imaging (MRI) after transfection with a superparamagnetic iron oxide (SPIO) and evaluate the effects of SPIO on cellular activity, particularly under an ischemic environment. METHODS: hMSCs were exposed to low doses of SPIOs. After a short incubation period, cells were cultured for additional 1, 7 and 14 d to evaluate proliferation, colony formation and multilinear potential. Labeled cells were imaged and evaluated in agarose to quantify R2 and R2∗ contrast at each time point. Cells were placed in a low-oxygen, low-serum environment and tested for cytotoxicity. In addition, labeled cells were transplanted into an ischemic stroke model and evaluated with ex vivo MRI and histology. RESULTS: Cellular events such as proliferation and differentiation were not affected at any of the exposures tested when cultured for 14 d. The low iron exposure and short incubation time are sufficient for detectability with MRI. However, the higher iron dosage results in higher calcification and cytotoxicity under in vitro ischemic conditions. Transplantation of the hMSCs labeled with an initial exposure of 22.4 µg of Fe showed excellent retention of contrast in stroke-induced rats. CONCLUSIONS: Although SPIO labeling is stable for long-term MRI detection and has limited effects on the multilineage potential of hMSCs, high-dose SPIO labeling may affect hMSC survival under serum and oxygen withdrawal.
Assuntos
Óxido Ferroso-Férrico , Espectroscopia de Ressonância Magnética , Células-Tronco Mesenquimais/diagnóstico por imagem , Acidente Vascular Cerebral/terapia , Animais , Diferenciação Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos , Meios de Contraste/química , Óxido Ferroso-Férrico/química , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Radiografia , Ratos , Acidente Vascular Cerebral/diagnósticoRESUMO
The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy.