DOCSLIB.ORG

Characterization and Development of Photoactivatable Fluorescent Proteins for Single-Molecule–Based Superresolution Imaging

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Characterization and Development of Photoactivatable Fluorescent Proteins for Single-Molecule–Based Superresolution Imaging Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging Siyuan Wanga, Jeffrey R. Moffitta, Graham T. Dempseya, X. Sunney Xiea, and Xiaowei Zhuanga,b,c,1 Departments of aChemistry and Chemical Biology and bPhysics, Harvard University, cHoward Hughes Medical Institute, Cambridge, MA 02138 Contributed by Xiaowei Zhuang, April 10, 2014 (sent for review February 7, 2014) Photoactivatable fluorescent proteins (PAFPs) have been widely photons, a higher photon budget leads to higher localization used for superresolution imaging based on the switching and precision and hence higher image resolution (7, 9). (ii) The localization of single molecules. Several properties of PAFPs second property is the on–off switching rate ratio (on–off ratio), strongly influence the quality of the superresolution images. defined as the ratio between the on-switching (activation) and These properties include (i) the number of photons emitted per off-switching or photobleaching rates under the illumination of switching cycle, which affects the localization precision of individ- the imaging light only (7). Even in the absence of activation light, ual molecules; (ii) the ratio of the on- and off-switching rate con- the imaging light itself can also switch on the PAFPs, albeit at stants, which limits the achievable localization density; (iii) the a low rate. Thus, the ratio between the on-switching and off- dimerization tendency, which could cause undesired aggregation switching rates under this condition determines the lower bound of target proteins; and (iv) the signaling efficiency, which de- of the fraction of PAFP molecules in the on-state at any given termines the fraction of target–PAFP fusion proteins that is de- time. The presence of activation light would increase this frac- tectable in a cell. Here, we evaluated these properties for 12 tion. When the product of this fraction and the density of fluo- rescent labels reaches approximately one fluorophore per commonly used PAFPs fused to both bacterial target proteins, diffraction-limited volume, it becomes difficult to resolve and H-NS, HU, and Tar, and mammalian target proteins, Zyxin and precisely localize the activated fluorophores. Hence the on–off Vimentin. Notably, none of the existing PAFPs provided optimal ratio limits the density of fluorescent labels that can be localized, performance in all four criteria, particularly in the signaling effi- which in turn affects the effective image resolution based on the ciency and dimerization tendency. The PAFPs with low dimeriza- Nyquist sampling theorem (10). (iii) The third property is the tion tendencies exhibited low signaling efficiencies, whereas dimerization tendency. Many PAFPs have a weak tendency to mMaple showed the highest signaling efficiency but also a high form dimers; this could even be true for the PAFPs that are dimerization tendency. To address this limitation, we engineered reported as being monomeric. When these proteins are fused to two new PAFPs based on mMaple, which we termed mMaple2 and target proteins that also tend to polymerize, they may cause mMaple3. These proteins exhibited substantially reduced or unde- undesired aggregation of the target proteins and distort the na- tectable dimerization tendencies compared with mMaple but main- tive distribution of the protein of interest. (iv) The fourth tained the high signaling efficiency of mMaple. In the meantime, property is the signaling efficiency, defined as the ratio between these proteins provided photon numbers and on–off switching rate the number of detectable PAFP-fusion molecules per cell and ratios that are comparable to the best achieved values among PAFPs. the expression level of the fusion protein. Fluorescent proteins do not necessarily fold with 100% efficiency. Among the folded STORM | PALM | fPALM | photoconvertible | photoswitchable molecules, not all of them will become mature at the time of imaging. Among the matured PAFP molecules, only a subset can hotoactivated localization microscopy, stochastic optical re- be photoactivated and imaged. Because of these deficiencies, the construction microscopy, and related imaging methods take number of fusion molecules detected could be substantially P lower than the expression level of the fusion protein. PAFPs with advantage of photoswitching and imaging of single molecules to higher signaling efficiencies will lead to higher localization circumvent the diffraction limit of spatial resolution in light – densities for a given target protein, which will in turn increase the microscopy (1 3). In these methods, only a subset of the fluo- effective image resolution. rescent labels in the sample is switched on at any given time such In this work, we measured the above properties of 12 commonly that the positions of individual fluorophores can be localized used PAFPs, including PAGFP (11), Dendra2 (12, 13), mEos2 from their images with high precision. Iteration of this process allows numerous fluorescent labels to be localized and an image Significance with sub–diffraction-limit resolution to be reconstructed from the fluorophore localizations. Fluorescent proteins that can be activated from dark to fluorescent or converted from one color Photoactivatable fluorescent proteins (PAFPs) are important probes for superresolution fluorescence microscopy, which allows the to another are widely used for such imaging approaches (4, 5). – Although photoactivatable fluorescent proteins (PAFPs) are spatial organization of proteins in living cells to be probed with sub generally dimmer than photoswitchable dyes (6, 7) and hence diffraction-limit resolution. Here, we compare four properties of give lower image resolution, the ease and high specificity of la- PAFPs that are critical for superresolution imaging and report two beling protein targets in living cells with fluorescent proteins new PAFPs that exhibit excellent performance in all four properties. makes PAFPs highly appealing probes for imaging the dynamics Author contributions: S.W., J.R.M., and X.Z. designed research; S.W., J.R.M., and G.T.D. of cellular structures (4, 8). performed research; S.W. and J.R.M. contributed new reagents/analytic tools; S.W., J.R.M., For single-molecule–based superresolution imaging methods, X.S.X., and X.Z. analyzed and interpreted data; and S.W., J.R.M., G.T.D., X.S.X., and X.Z. several properties of PAFPs are particularly important for the wrote the paper. image quality. Here, we focus on four such key properties. (i) Conflict of interest statement: A US provisional patent application has been filed for the The first property is the photon budget, defined as the average new fluorescent proteins developed in this work. number of photons emitted in each switching event. Given that 1To whom correspondence should be addressed. E-mail: [email protected]. the error of localizing an individual fluorophore approximately This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. scales with the inverse square root of the number of detected 1073/pnas.1406593111/-/DCSupplemental. 8452–8457 | PNAS | June 10, 2014 | vol. 111 | no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1406593111 Downloaded by guest on September 28, 2021 (14), mEos3.2 (15), tdEos (16), mKikGR (17), PAmCherry (18), and switching off (or photobleaching) the PAFPs in the presence PAtagRFP (19), mMaple (20), PSCFP2 (13, 21), Dronpa (22), and of imaging light only. By definition, the on-switching rate is the mGeosM (23). From this screen, we found that none of these increment in probability of the on-switching events per unit time. PAFPs was simultaneously optimal in all four criteria described To measure this quantity, we imaged the Zyxin-PAFP–express- above. For example, PAtagRFP and mEos3.2 exhibited the ing cells in the superresolution mode for a short period without highest photon budgets among PAFPs, excellent on–off ratios, and any activation light (with imaging light only). The samples were undetectable dimerization tendencies, but showed poor signaling then imaged to completion with an additional activation light at efficiencies. Alternatively, mMaple provided excellent signaling 405 nm. The ratio of the total number of activation events ac- efficiency and on–off ratio with a photon budget nearly equal to cumulated by a certain time during the period without activation those of PAtagRFP and mEos3.2, but had a substantial di- light over the total number of activation events by the end of the merization tendency. To address this limitation, we developed two imaging process was determined. The slope of this cumulative new PAFPs based on mMaple that exhibited substantially reduced activation probability against time then gave the on-switching or undetectable dimerization tendencies while maintaining the rate (Fig. 2 A and B, and Table S1). The off-switching rate was high signaling efficiency, high photon budget, and low on–off ratio determined from the inverse of the mean lifetime of the on-state of mMaple. These PAFPs will substantially facilitate super- of each PAFP in the presence of the imaging light (Fig. 2 C and resolution imaging of cellular structures. D, and Table S1). The ratios between the on- and off-switching rates were determined for all 12 PAFPs (Table 1). Because both Results on- and off-switching rates scale linearly with the illumination in- Photon Budget of Photoactivatable Fluorescent Proteins. tensity, the on–off ratio should be independent of the imaging light To evalu- − intensity. The on–off ratios were generally very small (10 5 to ate the properties of PAFPs under conditions similar to typical − − superresolution imaging experiments, we fused each PAFP to 10 6) except for PAGFP, Dronpa, and mGeosM, which were ∼10 3. various target proteins and expressed these fusion proteins in From the superresolution images of Zyxin, it is evident that the either mammalian cells or bacteria. To measure the photon bud- PAFP with a smaller on–off ratio gave images with a higher lo- get, we fused the PAFPs to the mammalian focal adhesion protein calization density and hence higher image quality (Fig. 2 E and F).
Load more

Recommended publications
  • Desmoplakin Assembly Dynamics in Four Dimensions
    JCB: ARTICLE Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin Lisa M. Godsel,1 Sherry N. Hsieh,1 Evangeline V. Amargo,1 Amanda E. Bass,1 Lauren T. Pascoe-McGillicuddy,1,4 Arthur C. Huen,1 Meghan E. Thorne,1 Claire A. Gaudry,1 Jung K. Park,1 Kyunghee Myung,3 Robert D. Goldman,3,4 Teng-Leong Chew,3 and Kathleen J. Green1,2 1Department of Pathology, 2Department of Dermatology, 3Department of Cell and Molecular Biology, and 4The R.H. Lurie Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611 he intermediate filament (IF)–binding protein des- sensitive translocation of cytoplasmic particles to matur- moplakin (DP) is essential for desmosome function ing borders, with kinetics ranging from 0.002 to 0.04 T and tissue integrity, but its role in junction assembly ␮m/s. DP mutants that abrogate or enhance association Downloaded from is poorly understood. Using time-lapse imaging, we show with IFs exhibit delayed incorporation into junctions, al- that cell–cell contact triggers three temporally overlap- tering particle trajectory or increasing particle pause ping phases of DP-GFP dynamics: (1) the de novo ap- times, respectively. Our data are consistent with the idea pearance of punctate fluorescence at new contact zones that DP assembles into nascent junctions from both diffus- after as little as 3 min; (2) the coalescence of DP and the ible and particulate pools in a temporally overlapping jcb.rupress.org armadillo protein plakophilin 2 into discrete cytoplasmic series of events triggered by cell–cell contact and regu- particles after as little as 15 min; and (3) the cytochalasin- lated by actin and DP–IF interactions.
    [Show full text]
  • And G- Actin Regulates Cell Migration Pavan Vedula1, Satoshi Kurosaka2, Brittany Mactaggart1, Qin Ni3, Garegin Papoian4, Yi Jiang5, Dawei W Dong1,6, Anna Kashina1*
    RESEARCH ARTICLE Different translation dynamics of b- and g- actin regulates cell migration Pavan Vedula1, Satoshi Kurosaka2, Brittany MacTaggart1, Qin Ni3, Garegin Papoian4, Yi Jiang5, Dawei W Dong1,6, Anna Kashina1* 1Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States; 2Institute of Advanced Technology, Kindai University, Kainan, Wakayama, Japan; 3Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, United States; 4Department of Chemistry, University of Maryland, College Park, United States; 5Department of Mathematics and Statistics, Georgia State University, Atlanta, United States; 6Institute for Biomedical Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States Abstract b- and g-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and mesenchymal cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequences; however, it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in b- and g- cytoplasmic actins’ intracellular functions, using stable polyclonal populations of immortalized mouse embryonic fibroblasts with exogenously expressed actin isoforms and their ‘codon- switched’ variants. When targeted to the cell periphery using b-actin 30UTR; b-actin and g-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single- molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in b- and g-actins’ translation elongation rates in cells, leading to changes in their dynamics at focal adhesions, impairments in actin bundle *For correspondence: formation, and reduced cell anchoring to the substrate during migration.
    [Show full text]
  • Apkc-Mediated Displacement and Actomyosin-Mediated Retention
    RESEARCH ARTICLE aPKC-mediated displacement and actomyosin-mediated retention polarize Miranda in Drosophila neuroblasts Matthew Robert Hannaford, Anne Ramat, Nicolas Loyer, Jens Januschke* Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dundee, United Kingdom Abstract Cell fate assignment in the nervous system of vertebrates and invertebrates often hinges on the unequal distribution of molecules during progenitor cell division. We address asymmetric fate determinant localization in the developing Drosophila nervous system, specifically the control of the polarized distribution of the cell fate adapter protein Miranda. We reveal a step- wise polarization of Miranda in larval neuroblasts and find that Miranda’s dynamics and cortical association are differently regulated between interphase and mitosis. In interphase, Miranda binds to the plasma membrane. Then, before nuclear envelope breakdown, Miranda is phosphorylated by aPKC and displaced into the cytoplasm. This clearance is necessary for the subsequent establishment of asymmetric Miranda localization. After nuclear envelope breakdown, actomyosin activity is required to maintain Miranda asymmetry. Therefore, phosphorylation by aPKC and differential binding to the actomyosin network are required at distinct phases of the cell cycle to polarize fate determinant localization in neuroblasts. DOI: https://doi.org/10.7554/eLife.29939.001 Introduction *For correspondence: The development of the central nervous system depends on asymmetric cell divisions for the bal- [email protected] anced production of progenitor and differentiating cells. During vertebrate and invertebrate neuro- Competing interests: The genesis, cell fates can be established through the asymmetric inheritance of cortical domains or fate authors declare that no determinants during asymmetric division of progenitor cells (Alexandre et al., 2010; Doe, 2008; competing interests exist.
    [Show full text]
  • Regulation of Keratin Filament Network Dynamics
    Regulation of keratin filament network dynamics Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom Biologe Marcin Maciej Moch aus Dzierżoniów (früher Reichenbach, NS), Polen Berichter: Universitätsprofessor Dr. med. Rudolf E. Leube Universitätsprofessor Dr. phil. nat. Gabriele Pradel Tag der mündlichen Prüfung: 19. Juni 2015 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. This work was performed at the Institute for Molecular and Cellular Anatomy at University Hospital RWTH Aachen by the mentorship of Prof. Dr. med. Rudolf E. Leube. It was exclusively performed by myself, unless otherwise stated in the text. 1. Reviewer: Univ.-Prof. Dr. med. Rudolf E. Leube 2. Reviewer: Univ.-Prof. Dr. phil. nat. Gabriele Pradel Ulm, 15.02.2015 2 Publications Publications Measuring the regulation of keratin filament network dynamics. Moch M, and Herberich G, Aach T, Leube RE, Windoffer R. 2013. Proc Natl Acad Sci U S A. 110:10664-10669. Intermediate filaments and the regulation of focal adhesion. Leube RE, Moch M, Windoffer R. 2015. Current Opinion in Cell Biology. 32:13–20. "Panta rhei": Perpetual cycling of the keratin cytoskeleton. Leube RE, Moch M, Kölsch A, Windoffer R. 2011. Bioarchitecture. 1:39-44. Intracellular motility of intermediate filaments. Leube RE, Moch M, Windoffer R. Under review in: The Cytoskeleton. Editors: Pollard T., Dutcher S., Goldman R. Cold Springer Harbor Laboratory Press, Cold Spring Harbor. Multidimensional monitoring of keratin filaments in cultured cells and in tissues. Schwarz N, and Moch M, Windoffer R, Leube RE.
    [Show full text]
  • Genetically Encoded Biosensors Based on Fluorescent Proteins
    sensors Review Genetically Encoded Biosensors Based on Fluorescent Proteins Hyunbin Kim 1,2,†, Jeongmin Ju 1,2,†, Hae Nim Lee 1,3,†, Hyeyeon Chun 1,† and Jihye Seong 1,2,3,* 1 Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea; [email protected] (H.K.); [email protected] (J.J.); [email protected] (H.N.L.); [email protected] (H.C.) 2 Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Korea 3 Department of Converging Science and Technology, Kyung Hee University, Seoul 02453, Korea * Correspondence: [email protected]; Tel.: +82-2-958-5904 † These authors contributed equally to this work. Abstract: Genetically encoded biosensors based on fluorescent proteins (FPs) allow for the real-time monitoring of molecular dynamics in space and time, which are crucial for the proper functioning and regulation of complex cellular processes. Depending on the types of molecular events to be monitored, different sensing strategies need to be applied for the best design of FP-based biosensors. Here, we review genetically encoded biosensors based on FPs with various sensing strategies, for example, translocation, fluorescence resonance energy transfer (FRET), reconstitution of split FP, pH sensitivity, maturation speed, and so on. We introduce general principles of each sensing strategy and discuss critical factors to be considered if available, then provide representative examples of these FP-based biosensors. These will help in designing the best sensing strategy for the successful development of new genetically encoded biosensors based on FPs. Keywords: fluorescent protein; genetically encoded biosensor; FRET; ddFP; BiFC; split FP; circular permutation; fluorescent timer Citation: Kim, H.; Ju, J.; Lee, H.N.; Chun, H.; Seong, J.
    [Show full text]
  • Phototransformable Fluorescent Proteins: Future Challenges. Virgile Adam, Romain Berardozzi, Martin Byrdin, Dominique Bourgeois
    Phototransformable fluorescent proteins: Future challenges. Virgile Adam, Romain Berardozzi, Martin Byrdin, Dominique Bourgeois To cite this version: Virgile Adam, Romain Berardozzi, Martin Byrdin, Dominique Bourgeois. Phototransformable fluores- cent proteins: Future challenges.. Current Opinion in Chemical Biology, Elsevier, 2014, 20, pp.92-102. hal-01093360 HAL Id: hal-01093360 https://hal.univ-grenoble-alpes.fr/hal-01093360 Submitted on 19 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. *Manuscript Click here to view linked References Phototransformable fluorescent proteins 19/05/2014 Phototransformable fluorescent proteins: future challenges Virgile Adam*,1,2,3, Romain Berardozzi1,2,3, Martin Byrdin1,2,3 and Dominique Bourgeois*,1,2,3 1Université Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38000 Grenoble, France ; 2CNRS, IBS, F-38000 Grenoble, France ; 3CEA, DSV, IBS, F-38000 Grenoble, France *Authors for correspondence: [email protected], Tel: +33 (0)4 57 42 85 67 [email protected], Tel: +33 (0)4 57 42 86 44
    [Show full text]
  • Individual and Combined Use of Ultraviolet (UV) Light with Hydrogen Peroxide, Peracetic Acid, and Ferrate(VI) for Wastewater Disinfection
    Individual and combined use of ultraviolet (UV) light with hydrogen peroxide, peracetic acid, and ferrate(VI) for wastewater disinfection by Natalie Linklater A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree Doctor of Philosophy in Environmental Engineering Carleton University Ottawa, Ontario © 2017 Natalie Linklater Abstract This work explores the use of chlorine-free chemicals hydrogen peroxide (HP), peracetic acid (PAA), and ferrate(VI) as well as physical disinfection method, ultraviolet light (UV), alone and combination for wastewater disinfection. To evaluate treatments the culturability of indicator-organism Escherichia coli was compared to that of spore- forming Bacillus subtilis and C. perfringens. Remaining culturable and viable E. coli were studied using a method combining propidium monoazide (PMA) and quantitative polymerase chain reaction (qPCR). Changes to culturability were monitored for up to 48 hours after treatment to evaluate regrowth, reactivation and resuscitation levels. In Chapter 4 culturable and viable E. coli were studied following disinfection with sodium hypochlorite (NaClO), HP, PAA, ferrate(VI), and ultraviolet light (UV). All methods reduced culturable E. coli to below 2 log CFU/mL but 3-6 log CCE/mL of E. coli remained viable following chemical treatments. The largest reduction of viable cells was achieved with Ferrate(VI) followed by PAA, NaClO, and HP. In Chapters 5 and 6 the use of UV and ferrate(VI) both alone and in combination was studied. The combination of 40 mJ/cm2 UV and 9 mg/L ferrate(VI) provided an additional 80% reduction in coliform bacteria compared to the use of UV alone.
    [Show full text]
  • Formin-2 Drives Polymerisation of Actin Filaments Enabling
    RESEARCH ADVANCE Formin-2 drives polymerisation of actin filaments enabling segregation of apicoplasts and cytokinesis in Plasmodium falciparum Johannes Felix Stortz1, Mario Del Rosario1, Mirko Singer2, Jonathan M Wilkes1, Markus Meissner1,2*, Sujaan Das1,2* 1Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity & Inflammation, University of Glasgow, Glasgow, United Kingdom; 2Faculty of Veterinary Medicine, Experimental Parasitology, Ludwig Maximilian University, Munich, Germany Abstract In addition to its role in erythrocyte invasion, Plasmodium falciparum actin is implicated in endocytosis, cytokinesis and inheritance of the chloroplast-like organelle called the apicoplast. Previously, the inability to visualise filamentous actin (F-actin) dynamics had restricted the characterisation of both F-actin and actin regulatory proteins, a limitation we recently overcame for Toxoplasma (Periz et al, 2017). Here, we have expressed and validated actin-binding chromobodies as F-actin-sensors in Plasmodium falciparum and characterised in-vivo actin dynamics. F-actin could be chemically modulated, and genetically disrupted upon conditionally deleting actin-1. In a comparative approach, we demonstrate that Formin-2, a predicted nucleator of F-actin, is responsible for apicoplast inheritance in both Plasmodium and Toxoplasma, and additionally mediates efficient cytokinesis in Plasmodium. Finally, time-averaged local intensity *For correspondence: measurements of F-actin in Toxoplasma conditional mutants revealed molecular determinants of [email protected]. spatiotemporally regulated F-actin flow. Together, our data indicate that Formin-2 is the primary uni-muenchen.de (MM); F-actin nucleator during apicomplexan intracellular growth, mediating multiple essential functions. [email protected] DOI: https://doi.org/10.7554/eLife.49030.001 muenchen.de (SD) Competing interests: The authors declare that no competing interests exist.
    [Show full text]
  • EOS, an Ikaros Family Zinc Finger Transcription Factor, Interacts with the HTLV-1 Oncoprotein Tax and Is Downregulated in Periph
    Naito et al. Virology Journal (2019) 16:160 https://doi.org/10.1186/s12985-019-1270-1 RESEARCH Open Access EOS, an Ikaros family zinc finger transcription factor, interacts with the HTLV-1 oncoprotein Tax and is downregulated in peripheral blood mononuclear cells of HTLV-1-infected individuals, irrespective of clinical statuses Tadasuke Naito1, Hiroshi Ushirogawa1, Takuya Fukushima2, Yuetsu Tanaka3 and Mineki Saito1* Abstract Background: EOS plays an important role in maintaining the suppressive function of regulatory T cells (Tregs), and induces a regulated transformation of Tregs into T helper-like cells, which are capable of secreting proinflammatory cytokines in response to specific inflammatory signals. Meanwhile, significant reduction in Treg activity along with production of proinflammatory cytokines has been reported in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Methods: In this study, to examine whether there is an alteration in EOS expression in peripheral blood mononuclear cells (PBMCs) derived from HTLV-1-infected individuals especially HAM/TSP, we investigated the expression of HTLV-1 tax genotype, proviral load (PVL), and the mRNA expression of tax, HBZ and EOS in HTLV-1 infected individuals including adult T-cell leukemia/lymphoma (ATL), HAM/TSP, or asymptomatic carriers. The expression levels of EOS mRNA and protein in various HTLV-1-infected or uninfected human T-cell lines were also investigated. Results: EOS was highly expressed at the protein level in most HTLV-1 infected T-cell lines, and was augmented after the HTLV-1 regulatory factor Tax was induced in a Tax-inducible JPX-9 cell line. Immunoprecipitation experiments demonstrated a physical interaction between EOS and the viral regulatory protein Tax, but not HBZ.
    [Show full text]
  • Advances in Fluorescent Protein Technology
    Commentary 4247 Advances in fluorescent protein technology Nathan C. Shaner1, George H. Patterson2 and Michael W. Davidson3 1The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA 2Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development (NICHD), Bethesda, MD 20892, USA 3National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, Tallahassee, FL 32310, USA e-mails: [email protected]; [email protected]; [email protected] Accepted 31 October 2007 Journal of Cell Science 120, 4247-4260 Published by The Company of Biologists 2007 doi:10.1242/jcs.005801 Summary Current fluorescent protein (FP) development strategies monomeric and fast-maturing red FP has yielded a host of are focused on fine-tuning the photophysical properties of excellent candidates, although none is yet optimal for all blue to yellow variants derived from the Aequorea victoria applications. Meanwhile, photoactivatable FPs are jellyfish green fluorescent protein (GFP) and on the emerging as a powerful class of probes for intracellular development of monomeric FPs from other organisms that dynamics and, unexpectedly, as useful tools for the emit in the yellow-orange to far-red regions of the visible development of superresolution microscopy applications. light spectrum. Progress toward these goals has been substantial, and near-infrared emitting FPs may loom over the horizon. The latest efforts in jellyfish variants have Key words: Fluorescent proteins, Mutagenesis, Optical highlighters, resulted in new and improved monomeric BFP, CFP, GFP Photoactivation, Photoconversion, Photoswitching, Live-cell and YFP variants, and the relentless search for a bright, imaging Introduction fusions.
    [Show full text]
  • Internet-Based Education on the Structure, Function, and Imaging Of
    The Fluorescent Protein Color Palette Scott G. Olenych, Nathan S. Claxton, Gregory K. Ottenberg, and Michael W. Davidson* National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, The Florida State University, Tallahassee, Florida 32310-3706 ABSTRACT Advances in fluorescent protein development over the past 10 years have led to fine-tuning of the Aequorea victoria jellyfish color palette in the emission color range from blue to yellow, while a significant amount of progress has been achieved with reef coral species in the generation of monomeric fluorescent proteins emitting in the orange to far-red spectral regions. It is not inconceivable that near-infrared fluorescent proteins loom on the horizon. Expansion of the fluorescent protein family to include optical highlighters and FRET biosensors further arms this ubiquitous class of fluorophores with biological probes capable of photoactivation, photoconversion, and detection of molecular interactions beyond the resolution limits of optical microscopy. The success of these endeavors certainly suggests that almost any biological parameter can be investigated using the appropriate fluorescent protein-based application. Keywords: GFP, fluorescent proteins, optical highlighters, biosensors, photoconversion, photoactivation, chromophore, fluorophore, FRET, FRAP, microscopy, confocal INTRODUCTION Although the first report of fluorescence emission in the bioluminescent hydrozoan jellyfish species Aequorea victoria was recorded in the mid-twentieth century (Davenport and Nichol, 1955) and a protein extract was independently demonstrated by two investigators to be responsible for this “green” fluorescence in the 1960s and 1970s (Shimomura, et al., 1962; Morin and Hastings, 1971), it took another 20 years and a number of significant advances in the technology of molecular and cellular biology to witness the elucidation of the primary amino acid structure (Prasher, et al., 1992).
    [Show full text]
  • Fluorescent Proteins and Two-Color PALM Methods
    UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Use of a Novel Two Color PALM Method to Examine Structural Properties of Drp1 Helical Rings during Mammalian Mitochondrial Fission In Situ. Permalink https://escholarship.org/uc/item/2wq8b3vm Author Rosenbloom, Alyssa Publication Date 2012 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Use of a Novel Two Color PALM Method to Examine Structural Properties of Drp1 Helical Rings during Mammalian Mitochondrial Fission In Situ. by Alyssa Blair Rosenbloom A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Carlos Bustamante, Chair Professor Jasper Rine Professor Jan Liphardt Professor Jodi Nunnari Fall 2012 Use of a Novel Two Color PALM Method to Examine Structural Properties of Drp1 Helical Rings during Mammalian Mitochondrial Fission In Situ. Copyright ©2012 by Alyssa Blair Rosenbloom Abstract Use of a Novel Two Color PALM Method to Examine Structural Properties of Drp1 Helical Rings during Mammalian Mitochondrial Fission In Situ. by Alyssa Blair Rosenbloom Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Carlos Bustamante, Chair Visual observation is a powerful way to examine a biological structure. Since the development of the basic microscope, scientists have attempted to get a closer look at smaller and smaller biological systems and molecules. Until recently, the greatest technical challenge to visualization with visible light has been the diffraction limit. With the advent of photoactivatable light microscopy (PALM), the diffraction limit barrier has been circumvented, allowing for improved resolution by an order of magnitude and a much closer observation of cell organelles and individual proteins.
    [Show full text]