1
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Verma S, Moreno IY, Gesteira TF, Coulson-Thomas VJ. Toxicity of nuclear-localized GFP in reporter mice. Sci Rep 2024; 14:24642. [PMID: 39428407 PMCID: PMC11491490 DOI: 10.1038/s41598-024-75741-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2024] [Accepted: 10/08/2024] [Indexed: 10/22/2024] Open
Abstract
Various techniques using fluorescent reporter probes have been developed, such as GFP transgenic mouse lines that are used to detect spatial-temporal expression levels of genes. Although GFP expression is largely considered non-toxic, recent reports have indicated that under certain conditions GFP can display cellular toxicity. We hereby report the nuclear toxicity of H2B-GFP using a K14 specific Tet-on reporter mouse system. Using this system, GFP accumulates in the nucleus of all K14 expressing cells, such as the ocular surface epithelia and ocular adnexa. Expression of high levels of nuclear GFP during embryonic stages led to an eye open-at-birth (EOB) phenotype and abnormal ocular adnexa development and during adult and aging stages showed notable toxicity to ocular tissues. Other tissues, such as skin, also presented multiple defects associated with H2B-GFP expression. This toxicity was found to be concentration dependent, with homozygous mice presenting extremely high toxicity, while heterozygous mice presented limited toxicity. Upon induction, the accumulation of H2B-GFP in the nucleus of homozygous mice led to apoptosis within 2 weeks. This study therefore shows that although the use of nuclear GFP reporter mice is a valuable tool, at high levels, nuclear GFP can be toxic, leading to cell death and affecting tissue function.
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Affiliation(s)
- Sudhir Verma
- College of Optometry, University of Houston, 4401 Martin Luther King Boulevard, Houston, TX, 77204-2020, USA
- Department of Zoology, Deen Dayal Upadhyaya College, University of Delhi, Delhi, 110078, India
| | - Isabel Y Moreno
- College of Optometry, University of Houston, 4401 Martin Luther King Boulevard, Houston, TX, 77204-2020, USA
| | - Tarsis F Gesteira
- College of Optometry, University of Houston, 4401 Martin Luther King Boulevard, Houston, TX, 77204-2020, USA
| | - Vivien J Coulson-Thomas
- College of Optometry, University of Houston, 4401 Martin Luther King Boulevard, Houston, TX, 77204-2020, USA.
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2
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Hockenberry MA, Daugird TA, Legant WR. Cell dynamics revealed by microscopy advances. Curr Opin Cell Biol 2024; 90:102418. [PMID: 39159598 PMCID: PMC11392612 DOI: 10.1016/j.ceb.2024.102418] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2024] [Revised: 07/23/2024] [Accepted: 07/24/2024] [Indexed: 08/21/2024]
Abstract
Cell biology emerges from spatiotemporally coordinated molecular processes. Recent advances in live-cell microscopy, fueled by a surge in optical, molecular, and computational technologies, have enabled dynamic observations from single molecules to whole organisms. Despite technological leaps, there is still an untapped opportunity to fully leverage their capabilities toward biological insight. We highlight how single-molecule imaging has transformed our understanding of biological processes, with a focus on chromatin organization and transcription in the nucleus. We describe how this was enabled by the close integration of new imaging techniques with analysis tools and discuss the challenges to make a comparable impact at larger scales from organelles to organisms. By highlighting recent successful examples, we describe an outlook of ever-increasing data and the need for seamless integration between dataset visualization and quantification to realize the full potential warranted by advances in new imaging technologies.
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Affiliation(s)
- Max A Hockenberry
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, Chapel Hill, NC, USA
| | - Timothy A Daugird
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Wesley R Legant
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Joint Department of Biomedical Engineering, North Carolina State University, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
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3
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Takeda R, Tsutsumi E, Okatsu K, Fukai S, Takeda K. Structural characterization of green fluorescent protein in the I-state. Sci Rep 2024; 14:22832. [PMID: 39353998 PMCID: PMC11445422 DOI: 10.1038/s41598-024-73696-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2024] [Accepted: 09/19/2024] [Indexed: 10/03/2024] Open
Abstract
Green fluorescent protein (GFP) is widely utilized as a fluorescent tag in biochemical fields. Whereas the intermediate (I) state has been proposed in the photoreaction cycle in addition to the A and B states, until now the structure of I has only been estimated by computational studies. In this paper, we report the crystal structures of the I stabilizing variants of GFP at high resolutions where respective atoms can be observed separately. Comparison with the structures in the other states highlights the structural feature of the I state. The side chain of one of the substituted residues, Val203, adopts the gauche- conformation observed for Thr203 in the A state, which is different from the B state. On the other hand, His148 interacts with the chromophore by ordinary hydrogen bonding with a distance of 2.85 Å, while the weaker interaction by longer distances is observed in the A state. Therefore, it was indicated that it is possible to distinguish three states A, B and I by the two hydrogen bond distances Oγ-Thr203···Oη-chromophore and Nδ1-His148···Oη-chromophore. We discuss the characteristics of the I intermediate of wild-type GFP on the bases of the structure estimated from the variant structures by quantum chemical calculations.
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Affiliation(s)
- Ryota Takeda
- Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan
| | - Erika Tsutsumi
- Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan
| | - Kei Okatsu
- Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan
| | - Shuya Fukai
- Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan
| | - Kazuki Takeda
- Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
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4
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Guerrero JF, Lesko SL, Evans EL, Sherer NM. Studying Retroviral Life Cycles Using Visible Viruses and Live Cell Imaging. Annu Rev Virol 2024; 11:125-146. [PMID: 38876144 PMCID: PMC11697243 DOI: 10.1146/annurev-virology-100422-012608] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/16/2024]
Abstract
Viruses exploit key host cell factors to accomplish each individual stage of the viral replication cycle. To understand viral pathogenesis and speed the development of new antiviral strategies, high-resolution visualization of virus-host interactions is needed to define where and when these events occur within cells. Here, we review state-of-the-art live cell imaging techniques for tracking individual stages of viral life cycles, focusing predominantly on retroviruses and especially human immunodeficiency virus type 1, which is most extensively studied. We describe how visible viruses can be engineered for live cell imaging and how nonmodified viruses can, in some instances, be tracked and studied indirectly using cell biosensor systems. We summarize the ways in which live cell imaging has been used to dissect the retroviral life cycle. Finally, we discuss select challenges for the future including the need for better labeling strategies, increased resolution, and multivariate systems that will allow for the study of full viral replication cycles.
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Affiliation(s)
- Jorge F Guerrero
- McArdle Laboratory for Cancer Research, Department of Oncology, and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA;
| | - Sydney L Lesko
- McArdle Laboratory for Cancer Research, Department of Oncology, and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA;
| | - Edward L Evans
- Current affiliation: Department of Biomedical Engineering and Center for Quantitative Imaging, University of Wisconsin-Madison, Madison, Wisconsin, USA
- McArdle Laboratory for Cancer Research, Department of Oncology, and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA;
| | - Nathan M Sherer
- McArdle Laboratory for Cancer Research, Department of Oncology, and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA;
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5
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Feng B, Hu W, Duan Y, Li Z. A flexible 'plug and play' bicistronic construct and its application in the screening of protein expression system in Escherichia coli. J Microbiol Methods 2024; 221:106928. [PMID: 38583783 DOI: 10.1016/j.mimet.2024.106928] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2024] [Revised: 04/02/2024] [Accepted: 04/04/2024] [Indexed: 04/09/2024]
Abstract
The bicistronic expression system that utilizes fluorescent reporters has been demonstrated to be a straightforward method for detecting recombinant protein expression levels, particularly when compared to polyacrylamide gel electrophoresis and immunoblot analysis, which are tedious and labor-intensive. However, existing bicistronic reporter systems are less capable of quantitative measurement due to the lag in reporter expression and its negative impact on target protein. In this work, a plug and play bicistronic construct using mCherry as reporter was applied in the screening of optimal replicon and promoter for Sortase expression in Escherichia coli (E. coli). The bicistronic construct allowed the reporter gene and target open reading frame (ORF) to be co-transcribed under the same promoter, resulting in a highly positive quantitative correlation between the expression titer of Sortase and the fluorescent intensity (R2 > 0.97). With the correlation model, the titer of target protein can be quantified by noninvasively measuring the fluorescent intensity. On top of this, the expression of reporter has no significant effect on the yield of target protein, thus favoring a plug and play design for removing reporter gene to generate a plain plasmid for industrial use.
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Affiliation(s)
- Binbin Feng
- Microbial and Viral Platform, WuXi Biologics (Hangzhou FTZ) Co., Ltd, #9, 388 of No.16 street, Baiyang Avenue, Hangzhou Free Trade Zone, Hangzhou, Zhejiang Province, China
| | - Weiwei Hu
- Microbial and Viral Platform, WuXi Biologics (Hangzhou FTZ) Co., Ltd, #9, 388 of No.16 street, Baiyang Avenue, Hangzhou Free Trade Zone, Hangzhou, Zhejiang Province, China
| | - Yingyi Duan
- Microbial and Viral Platform, WuXi Biologics (Hangzhou FTZ) Co., Ltd, #9, 388 of No.16 street, Baiyang Avenue, Hangzhou Free Trade Zone, Hangzhou, Zhejiang Province, China.
| | - Zhaopeng Li
- Microbial and Viral Platform, WuXi Biologics (Hangzhou FTZ) Co., Ltd, #9, 388 of No.16 street, Baiyang Avenue, Hangzhou Free Trade Zone, Hangzhou, Zhejiang Province, China.
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6
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Luo Y, Pi S, Liu YJ. Mechanistic Insights into the Bacterial Luciferase-based Bioluminescence Resonance Energy Transfer Luminescence: The Role of Protein Complex Dimer. Chemphyschem 2024; 25:e202300973. [PMID: 38345139 DOI: 10.1002/cphc.202300973] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Revised: 02/11/2024] [Indexed: 02/29/2024]
Abstract
Bacterial bioluminescence holds significant potential in the realm of optical imaging due to the inherent advantages of bioluminescence and ease of operation. However, its practical utility is hindered by its low light intensity. The fusion of bacterial luciferase with a highly fluorescent protein has been demonstrated to significantly enhance autonomous luminescence. Nevertheless, the underlying mechanism behind this enhancement remains unclear, and there is a dearth of research investigating the mechanistic aspects of bioluminescence resonance energy transfer (BRET) luminescence, whether it occurs naturally or can be achieved through experimental means. In this study, we investigated the phenomenon of bacterial luciferase-based BRET luminescence employing a range of computational techniques, including structural modeling, molecular docking, molecular dynamics simulations, as well as combined quantum mechanics and molecular mechanics calculations. The theoretical findings suggest that the BRET luminescence occurs through resonance energy transfer between the excited bioluminophore and the ground chromophore within the protein complex dimer. The proposed mechanism of the protein complex dimer offers a microscopic understanding of the intriguing BRET phenomenon and has the potential to inspire further practical applications in the field of optical imaging.
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Affiliation(s)
- Yanling Luo
- School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin, China
| | - Shuangqi Pi
- Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China
| | - Ya-Jun Liu
- Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China
- Center for Advanced Materials Research, Beijing Normal University, Zhuhai, China
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7
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Sanchez C, Ramirez A, Hodgson L. Unravelling molecular dynamics in living cells: Fluorescent protein biosensors for cell biology. J Microsc 2024. [PMID: 38357769 PMCID: PMC11324865 DOI: 10.1111/jmi.13270] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2023] [Revised: 01/11/2024] [Accepted: 01/22/2024] [Indexed: 02/16/2024]
Abstract
Genetically encoded, fluorescent protein (FP)-based Förster resonance energy transfer (FRET) biosensors are microscopy imaging tools tailored for the precise monitoring and detection of molecular dynamics within subcellular microenvironments. They are characterised by their ability to provide an outstanding combination of spatial and temporal resolutions in live-cell microscopy. In this review, we begin by tracing back on the historical development of genetically encoded FP labelling for detection in live cells, which lead us to the development of early biosensors and finally to the engineering of single-chain FRET-based biosensors that have become the state-of-the-art today. Ultimately, this review delves into the fundamental principles of FRET and the design strategies underpinning FRET-based biosensors, discusses their diverse applications and addresses the distinct challenges associated with their implementation. We place particular emphasis on single-chain FRET biosensors for the Rho family of guanosine triphosphate hydrolases (GTPases), pointing to their historical role in driving our understanding of the molecular dynamics of this important class of signalling proteins and revealing the intricate relationships and regulatory mechanisms that comprise Rho GTPase biology in living cells.
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Affiliation(s)
- Colline Sanchez
- Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA
- Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, USA
| | - Andrea Ramirez
- Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA
- Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, USA
| | - Louis Hodgson
- Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA
- Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, USA
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8
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Voeltz GK, Sawyer EM, Hajnóczky G, Prinz WA. Making the connection: How membrane contact sites have changed our view of organelle biology. Cell 2024; 187:257-270. [PMID: 38242082 PMCID: PMC11830234 DOI: 10.1016/j.cell.2023.11.040] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Revised: 11/13/2023] [Accepted: 11/29/2023] [Indexed: 01/21/2024]
Abstract
The view of organelles and how they operate together has changed dramatically over the last two decades. The textbook view of organelles was that they operated largely independently and were connected by vesicular trafficking and the diffusion of signals through the cytoplasm. We now know that all organelles make functional close contacts with one another, often called membrane contact sites. The study of these sites has moved to center stage in cell biology as it has become clear that they play critical roles in healthy and developing cells and during cell stress and disease states. Contact sites have important roles in intracellular signaling, lipid metabolism, motor-protein-mediated membrane dynamics, organelle division, and organelle biogenesis. Here, we summarize the major conceptual changes that have occurred in cell biology as we have come to appreciate how contact sites integrate the activities of organelles.
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Affiliation(s)
- G K Voeltz
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Boulder, CO 80309, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
| | - E M Sawyer
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Boulder, CO 80309, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
| | - G Hajnóczky
- MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA
| | - W A Prinz
- Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
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9
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Yun WS, Cho H, Jeon SI, Lim DK, Kim K. Fluorescence-Based Mono- and Multimodal Imaging for In Vivo Tracking of Mesenchymal Stem Cells. Biomolecules 2023; 13:1787. [PMID: 38136656 PMCID: PMC10742164 DOI: 10.3390/biom13121787] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2023] [Revised: 12/01/2023] [Accepted: 12/11/2023] [Indexed: 12/24/2023] Open
Abstract
The advancement of stem cell therapy has offered transformative therapeutic outcomes for a wide array of diseases over the past decades. Consequently, stem cell tracking has become significant in revealing the mechanisms of action and ensuring safe and effective treatments. Fluorescence stands out as a promising choice for stem cell tracking due to its myriad advantages, including high resolution, real-time monitoring, and multi-fluorescence detection. Furthermore, combining fluorescence with other tracking modalities-such as bioluminescence imaging (BLI), positron emission tomography (PET), photoacoustic (PA), computed tomography (CT), and magnetic resonance (MR)-can address the limitations of single fluorescence detection. This review initially introduces stem cell tracking using fluorescence imaging, detailing various labeling strategies such as green fluorescence protein (GFP) tagging, fluorescence dye labeling, and nanoparticle uptake. Subsequently, we present several combinations of strategies for efficient and precise detection.
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Affiliation(s)
- Wan Su Yun
- KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Republic of Korea; (W.S.Y.); (D.-K.L.)
| | - Hanhee Cho
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Woman’s University, Seoul 03760, Republic of Korea; (H.C.); (S.I.J.)
| | - Seong Ik Jeon
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Woman’s University, Seoul 03760, Republic of Korea; (H.C.); (S.I.J.)
| | - Dong-Kwon Lim
- KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Republic of Korea; (W.S.Y.); (D.-K.L.)
| | - Kwangmeyung Kim
- Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Woman’s University, Seoul 03760, Republic of Korea; (H.C.); (S.I.J.)
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10
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Lee Y, Kim KM, Nguyen DL, Jannah F, Seong HJ, Kim JM, Kim YP. Cyclized proteins with tags as permeable and stable cargos for delivery into cells and liposomes. Int J Biol Macromol 2023; 252:126520. [PMID: 37625744 DOI: 10.1016/j.ijbiomac.2023.126520] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Revised: 08/18/2023] [Accepted: 08/23/2023] [Indexed: 08/27/2023]
Abstract
Despite the therapeutic potential of recombinant proteins, their cell permeabilities and stabilities remain significant challenges. Here we demonstrate that cyclized recombinant proteins can be used as universal cargos for permeable and stable delivery into cells and polydiacetylene liposomes. Utilizing a split intein-mediated process, cyclized model fluorescent proteins containing short tetraarginine (R4) and hexahistidine (H6) tags were generated without compromising their native protein functions. Strikingly, as compared to linear R4/H6-tagged proteins, the cyclized counterparts have substantially increased permeabilities in both cancer cells and synthetic liposomes, as well as higher resistances to enzymatic degradation in cancer cells. These properties are likely a consequence of structural constraints imposed on the proteins in the presence of short functional peptides. Additionally, photodynamic therapy by cyclized photoprotein-loaded liposomes in cancer cells was significantly improved in comparison to that by their non-cyclized counterparts. These findings suggest that our strategy will be universally applicable to intercellular delivery of proteins and therapeutics.
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Affiliation(s)
- Yeonju Lee
- Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea
| | - Kyung-Min Kim
- Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea
| | - Duc Long Nguyen
- Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea; Research Institute for Convergence of Basic Science, Hanyang University, Seoul 04763, Republic of Korea
| | - Fadilatul Jannah
- Department of Chemical Engineering, Hanyang University, Seoul 04763, Republic of Korea
| | - Hyun-Jung Seong
- Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea
| | - Jong-Man Kim
- Department of Chemical Engineering, Hanyang University, Seoul 04763, Republic of Korea; Institute of Nano Science and Technology, Hanyang University, Seoul 04763, Republic of Korea.
| | - Young-Pil Kim
- Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea; Research Institute for Convergence of Basic Science, Hanyang University, Seoul 04763, Republic of Korea; Institute of Nano Science and Technology, Hanyang University, Seoul 04763, Republic of Korea; Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Republic of Korea; Department of HY-KIST Bio-Convergence, Hanyang University, Seoul 04763, Republic of Korea; Hanyang Institute of Bioscience and Biotechnology, Hanyang University, Seoul 04763, Republic of Korea.
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11
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Takada Y, Fierro L, Sato K, Sanada T, Ishii A, Yamamoto T, Kotani T. Mature mRNA processing that deletes 3' end sequences directs translational activation and embryonic development. SCIENCE ADVANCES 2023; 9:eadg6532. [PMID: 38000026 PMCID: PMC10672166 DOI: 10.1126/sciadv.adg6532] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/13/2023] [Accepted: 10/25/2023] [Indexed: 11/26/2023]
Abstract
Eggs accumulate thousands of translationally repressed mRNAs that are translated into proteins after fertilization to direct diverse developmental processes. However, molecular mechanisms underlying the translation of stored mRNAs after fertilization remain unclear. Here, we report a previously unknown RNA processing of 3' end sequences of mature mRNAs that activates the translation of stored mRNAs. Specifically, 9 to 72 nucleotides at the 3' ends of zebrafish pou5f3 and mouse Pou5f1 mRNAs were deleted in the early stages of development. Reporter assays illustrated the effective translation of the truncated forms of mRNAs. Moreover, promotion and inhibition of the shortening of 3' ends accelerated and attenuated Pou5f3 accumulation, respectively, resulting in defective development. Identification of proteins binding to unprocessed and/or processed mRNAs revealed that mRNA shortening acts as molecular switches. Comprehensive analysis revealed that >250 mRNAs underwent this processing. Therefore, our results provide a molecular principle that triggers the translational activation and directs development.
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Affiliation(s)
- Yuki Takada
- Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan
- Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan
| | - Ludivine Fierro
- Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan
| | - Keisuke Sato
- Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan
| | - Takahiro Sanada
- Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan
| | - Anna Ishii
- Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan
| | - Takehiro Yamamoto
- Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Tomoya Kotani
- Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan
- Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan
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12
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Plante M. Epistemology of synthetic biology: a new theoretical framework based on its potential objects and objectives. Front Bioeng Biotechnol 2023; 11:1266298. [PMID: 38053845 PMCID: PMC10694798 DOI: 10.3389/fbioe.2023.1266298] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2023] [Accepted: 11/07/2023] [Indexed: 12/07/2023] Open
Abstract
Synthetic biology is a new research field which attempts to understand, modify, and create new biological entities by adopting a modular and systemic conception of the living organisms. The development of synthetic biology has generated a pluralism of different approaches, bringing together a set of heterogeneous practices and conceptualizations from various disciplines, which can lead to confusion within the synthetic biology community as well as with other biological disciplines. I present in this manuscript an epistemological analysis of synthetic biology in order to better define this new discipline in terms of objects of study and specific objectives. First, I present and analyze the principal research projects developed at the foundation of synthetic biology, in order to establish an overview of the practices in this new emerging discipline. Then, I analyze an important scientometric study on synthetic biology to complete this overview. Afterwards, considering this analysis, I suggest a three-level classification of the object of study for synthetic biology (which are different kinds of living entities that can be built in the laboratory), based on three successive criteria: structural hierarchy, structural origin, functional origin. Finally, I propose three successively linked objectives in which synthetic biology can contribute (where the achievement of one objective led to the development of the other): interdisciplinarity collaboration (between natural, artificial, and theoretical sciences), knowledge of natural living entities (past, present, future, and alternative), pragmatic definition of the concept of "living" (that can be used by biologists in different contexts). Considering this new theoretical framework, based on its potential objects and objectives, I take the position that synthetic biology has not only the potential to develop its own new approach (which includes methods, objects, and objectives), distinct from other subdisciplines in biology, but also the ability to develop new knowledge on living entities.
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Affiliation(s)
- Mirco Plante
- Collège Montmorency, Laval, QC, Canada
- Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique, Université du Québec, Laval, QC, Canada
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13
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van Schaik J, Li Z, Cheadle J, Crook N. Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for Studying Plant-Microbe Interactions. ACS Synth Biol 2023; 12:3030-3040. [PMID: 37712562 DOI: 10.1021/acssynbio.3c00371] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/16/2023]
Abstract
Sustainably enhancing crop production is a global necessity to meet the escalating demand for staple crops while sustainably managing their associated carbon/nitrogen inputs. Leveraging plant-associated microbiomes is a promising avenue for addressing this demand. However, studying these communities and engineering them for sustainable enhancement of crop production have remained a challenge due to limited genetic tools and methods. In this work, we detail the development of the Maize Root Microbiome ToolKit (MRMTK), a rapid Modular Cloning (MoClo) toolkit that only takes 2.5 h to generate desired constructs (5400 potential plasmids) that replicate and express heterologous genes in Enterobacter ludwigii strain AA4 (Elu), Pseudomonas putida strain AA7 (Ppu), Herbaspirillum robiniae strain AA6 (Hro), Stenotrophomonas maltophilia strain AA1 (Sma), and Brucella pituitosa strain AA2 (Bpi), which comprise a model maize root synthetic community (SynCom). In addition to these genetic tools, we describe a highly efficient transformation protocol (107-109 transformants/μg of DNA) 1 for each of these strains. Utilizing this highly efficient transformation protocol, we identified endogenous Expression Sequences (ES; promoter and ribosomal binding sites) for each strain via genomic promoter trapping. Overall, MRMTK is a scalable and adaptable platform that expands the genetic engineering toolbox while providing a standardized, high-efficiency transformation method across a diverse group of root commensals. These results unlock the ability to elucidate and engineer plant-microbe interactions promoting plant growth for each of the 5 bacterial strains in this study.
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Affiliation(s)
- John van Schaik
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Room 2109, Partners II, 840 Main Campus Drive, Raleigh, North Carolina 27606, United States
| | - Zidan Li
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Room 2109, Partners II, 840 Main Campus Drive, Raleigh, North Carolina 27606, United States
| | - John Cheadle
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Room 2109, Partners II, 840 Main Campus Drive, Raleigh, North Carolina 27606, United States
| | - Nathan Crook
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Room 2109, Partners II, 840 Main Campus Drive, Raleigh, North Carolina 27606, United States
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14
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Wang Y, Zhao Y, Li Y, Zhang K, Fan Y, Li B, Su W, Li S. piggyBac-mediated genomic integration of linear dsDNA-based library for deep mutational scanning in mammalian cells. Cell Mol Life Sci 2023; 80:321. [PMID: 37815732 PMCID: PMC11071730 DOI: 10.1007/s00018-023-04976-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2023] [Revised: 09/15/2023] [Accepted: 09/20/2023] [Indexed: 10/11/2023]
Abstract
Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here, we developed two novel strategies for DMS library construction in mammalian cells, namely 'piggyBac-in vitro ligation' and 'piggyBac-in vitro ligation-PCR'. For the first strategy, we took the 'in vitro ligation' approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore-randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino-acid sequences. And we further identified a novel transcriptional activator peptide with the 'piggyBac-in vitro ligation-PCR' strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future.
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Affiliation(s)
- Yi Wang
- Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
- Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China
- Tianjin's Clinical Research Center for Cancer, Tianjin, 300060, China
| | - Yanjie Zhao
- Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
- Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China
- Tianjin's Clinical Research Center for Cancer, Tianjin, 300060, China
| | - Yifan Li
- Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
- Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China
- Tianjin's Clinical Research Center for Cancer, Tianjin, 300060, China
| | - Kaili Zhang
- Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China
- Key Laboratory of Cancer Prevention and Therapy, Tianjin, China
- Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China
- Tianjin's Clinical Research Center for Cancer, Tianjin, 300060, China
| | - Yan Fan
- School of Medicine, Nankai University, Tianjin, 300071, China
| | - Bo Li
- Beijing Key Laboratory for Separation and Analysis in Biomedicine and Pharmaceuticals, School of Life Science, Beijing Institute of Technology, Beijing, China
- Advanced Research Institute of Multidisciplinary Science, Beijing Institute of Technology, Beijing, 100081, China
| | - Weijun Su
- School of Medicine, Nankai University, Tianjin, 300071, China.
| | - Shuai Li
- Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China.
- Key Laboratory of Cancer Prevention and Therapy, Tianjin, China.
- Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China.
- Tianjin's Clinical Research Center for Cancer, Tianjin, 300060, China.
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15
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Liu JQ, Min D, He RL, Cheng ZH, Wu J, Liu DF. Efficient and precise control of gene expression in Geobacter sulfurreducens through new genetic elements and tools for pollutant conversion. Biotechnol Bioeng 2023; 120:3001-3012. [PMID: 37209207 DOI: 10.1002/bit.28433] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2023] [Revised: 05/05/2023] [Accepted: 05/07/2023] [Indexed: 05/22/2023]
Abstract
Geobacter species, exhibiting exceptional extracellular electron transfer aptitude, hold great potential for applications in pollution remediation, bioenergy production, and natural elemental cycles. Nonetheless, a scarcity of well-characterized genetic elements and gene expression tools constrains the effective and precise fine-tuning of gene expression in Geobacter species, thereby limiting their applications. Here, we examined a suite of genetic elements and developed a new genetic editing tool in Geobacter sulfurreducens to enhance their pollutant conversion capacity. First, the performances of the widely used inducible promoters, constitutive promoters, and ribosomal binding sites (RBSs) elements in G. sulfurreducens were quantitatively evaluated. Also, six native promoters with superior expression levels than constitutive promoters were identified on the genome of G. sulfurreducens. Employing the characterized genetic elements, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was constructed in G. sulfurreducens to achieve the repression of an essential gene-aroK and morphogenic genes-ftsZ and mreB. Finally, applying the engineered strain to the reduction of tungsten trioxide (WO3 ), methyl orange (MO), and Cr(VI), We found that morphological elongation through ftsZ repression amplified the extracellular electron transfer proficiency of G. sulfurreducens and facilitated its contaminant transformation efficiency. These new systems provide rapid, versatile, and scalable tools poised to expedite advancements in Geobacter genomic engineering to favor environmental and other biotechnological applications.
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Affiliation(s)
- Jia-Qi Liu
- CAS Key Laboratory of Urban Pollutant Conversion, Department of Environmental Science and Engineering, University of Science & Technology of China, Hefei, China
| | - Di Min
- CAS Key Laboratory of Urban Pollutant Conversion, Department of Environmental Science and Engineering, University of Science & Technology of China, Hefei, China
| | - Ru-Li He
- CAS Key Laboratory of Urban Pollutant Conversion, Department of Environmental Science and Engineering, University of Science & Technology of China, Hefei, China
| | - Zhou-Hua Cheng
- School of Life Sciences, University of Science & Technology of China, Hefei, China
| | - Jie Wu
- CAS Key Laboratory of Urban Pollutant Conversion, Department of Environmental Science and Engineering, University of Science & Technology of China, Hefei, China
| | - Dong-Feng Liu
- CAS Key Laboratory of Urban Pollutant Conversion, Department of Environmental Science and Engineering, University of Science & Technology of China, Hefei, China
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16
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Hoang MD, Polte I, Frantzmann L, von den Eichen N, Heins AL, Weuster-Botz D. Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor. Microb Cell Fact 2023; 22:153. [PMID: 37574555 PMCID: PMC10424407 DOI: 10.1186/s12934-023-02165-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2023] [Accepted: 08/01/2023] [Indexed: 08/15/2023] Open
Abstract
BACKGROUND The omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood. RESULTS Therefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L-1 demonstrating the consistency of this study's microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process. CONCLUSIONS The here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.
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Affiliation(s)
- Manh Dat Hoang
- Chair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstr. 15, 85748, Garching, Germany
| | - Ingmar Polte
- Chair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstr. 15, 85748, Garching, Germany
| | - Lukas Frantzmann
- Chair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstr. 15, 85748, Garching, Germany
| | - Nikolas von den Eichen
- Chair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstr. 15, 85748, Garching, Germany
| | - Anna-Lena Heins
- Chair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstr. 15, 85748, Garching, Germany.
| | - Dirk Weuster-Botz
- Chair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstr. 15, 85748, Garching, Germany
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17
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Aslopovsky VR, Scherbinin AV, Kleshchina NN, Bochenkova AV. Impact of the Protein Environment on Two-Photon Absorption Cross-Sections of the GFP Chromophore Anion Resolved at the XMCQDPT2 Level of Theory. Int J Mol Sci 2023; 24:11266. [PMID: 37511026 PMCID: PMC10379633 DOI: 10.3390/ijms241411266] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2023] [Revised: 06/16/2023] [Accepted: 07/05/2023] [Indexed: 07/30/2023] Open
Abstract
The search for fluorescent proteins with large two-photon absorption (TPA) cross-sections and improved brightness is required for their efficient use in bioimaging. Here, we explored the impact of a single-point mutation close to the anionic form of the GFP chromophore on its TPA activity. We considered the lowest-energy transition of EGFP and its modification EGFP T203I. We focused on a methodology for obtaining reliable TPA cross-sections for mutated proteins, based on conformational sampling using molecular dynamics simulations and a high-level XMCQDPT2-based QM/MM approach. We also studied the numerical convergence of the sum-over-states formalism and provide direct evidence for the applicability of the two-level model for calculating TPA cross-sections in EGFP. The calculated values were found to be very sensitive to changes in the permanent dipole moments between the ground and excited states and highly tunable by internal electric field of the protein environment. In the case of the GFP chromophore anion, even a single hydrogen bond was shown to be capable of drastically increasing the TPA cross-section. Such high tunability of the nonlinear photophysical properties of the chromophore anions can be used for the rational design of brighter fluorescent proteins for bioimaging using two-photon laser scanning microscopy.
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Affiliation(s)
- Vladislav R Aslopovsky
- Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia
| | - Andrei V Scherbinin
- Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia
| | - Nadezhda N Kleshchina
- Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia
| | - Anastasia V Bochenkova
- Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia
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18
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Zare M, Farhadi A, Zare F, Dehbidi GR, Zarghampoor F, Ahmadi MKB, Behbahani AB. Genetically engineered E. coli invade epithelial cells and transfer their genetic cargo into the cells: an approach to a gene delivery system. Biotechnol Lett 2023:10.1007/s10529-023-03387-7. [PMID: 37166604 DOI: 10.1007/s10529-023-03387-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2022] [Revised: 04/21/2023] [Accepted: 04/27/2023] [Indexed: 05/12/2023]
Abstract
PURPOSE Despite advances in gene therapy, the lack of safe and efficient gene delivery systems limited the clinical effectiveness of gene therapy. Due to the inherent potential of bacteria, they can be considered as a good option for the gene transfer system. This study aimed to create a genetically engineered bacterium capable of entering epithelial cells and transferring its genetic cargo to the cell's cytoplasm, eventually expressing the gene of interest in the cell. METHODS The invasin (inv) gene from Yersinia pseudotuberculosis and the listeriolysin (hlyA) gene from Listeria monocytogenes were isolated by PCR assay and inserted into a pACYCDuet-1 vector. The recombinant plasmid was then transformed into E. coli strain BL21. Subsequently, pEGFP-C1 plasmids containing a CMV promoter were transformed into the engineered bacteria. Finally, the engineered bacteria containing the reporter genes were incubated with the HeLa and LNCaP cell lines. Fluorescence microscopy, flow cytometry, and TEM were used to monitor bacterial entry into the cells and gene expression. We used native E. coli strain BL21 as a control. RESULTS A fluorescence microscope showed that, in contrast to the control group, the manipulated E. coli were able to penetrate the cells and transport the plasmid pEGFP-C1 to the target cells. Flow cytometry also showed fluorescence intensity of 54.7% in HeLa cells and 71% in LNCaP cells, respectively. In addition, electron micrographs revealed the presence of bacteria in the cell endosomes and in the cytoplasm of the cells. CONCLUSION This study shows that genetically engineered E. coli can enter cells, transport cargo into cells, and induce gene expression in the target cell. In addition, flow cytometry shows that the gene transfer efficiency was sufficient for protein expression.
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Affiliation(s)
- Maryam Zare
- Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
- Division of Medical Biotechnology, Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Ali Farhadi
- Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
- Division of Medical Biotechnology, Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Farahnaz Zare
- Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Gholamreza Rafiei Dehbidi
- Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Farzaneh Zarghampoor
- Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mohammad Karimi Baba Ahmadi
- Department of Advanced Medical Technology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Abbas Behzad Behbahani
- Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
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19
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Chen YL, Xie XX, Zhong N, Sun LC, Lin D, Zhang LJ, Weng L, Jin T, Cao MJ. Research Progresses and Applications of Fluorescent Protein Antibodies: A Review Focusing on Nanobodies. Int J Mol Sci 2023; 24:4307. [PMID: 36901737 PMCID: PMC10002328 DOI: 10.3390/ijms24054307] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2023] [Revised: 02/16/2023] [Accepted: 02/18/2023] [Indexed: 02/24/2023] Open
Abstract
Since the discovery of fluorescent proteins (FPs), their rich fluorescence spectra and photochemical properties have promoted widespread biological research applications. FPs can be classified into green fluorescent protein (GFP) and its derivates, red fluorescent protein (RFP) and its derivates, and near-infrared FPs. With the continuous development of FPs, antibodies targeting FPs have emerged. The antibody, a class of immunoglobulin, is the main component of humoral immunity that explicitly recognizes and binds antigens. Monoclonal antibody, originating from a single B cell, has been widely applied in immunoassay, in vitro diagnostics, and drug development. The nanobody is a new type of antibody entirely composed of the variable domain of a heavy-chain antibody. Compared with conventional antibodies, these small and stable nanobodies can be expressed and functional in living cells. In addition, they can easily access grooves, seams, or hidden antigenic epitopes on the surface of the target. This review provides an overview of various FPs, the research progress of their antibodies, particularly nanobodies, and advanced applications of nanobodies targeting FPs. This review will be helpful for further research on nanobodies targeting FPs, making FPs more valuable in biological research.
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Affiliation(s)
- Yu-Lei Chen
- College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
| | - Xin-Xin Xie
- College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
| | - Ning Zhong
- College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
| | - Le-Chang Sun
- College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
| | - Duanquan Lin
- College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
| | - Ling-Jing Zhang
- College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
| | - Ling Weng
- College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
| | - Tengchuan Jin
- Institute of Health and Medicine, Hefei Comprehensive National Science Center, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science & Technology of China, Hefei 230007, China
| | - Min-Jie Cao
- College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China
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20
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Abstract
The genetically encoded fluorescent sensors convert chemical and physical signals into light. They are powerful tools for the visualisation of physiological processes in living cells and freely moving animals. The fluorescent protein is the reporter module of a genetically encoded biosensor. In this study, we first review the history of the fluorescent protein in full emission spectra on a structural basis. Then, we discuss the design of the genetically encoded biosensor. Finally, we briefly review several major types of genetically encoded biosensors that are currently widely used based on their design and molecular targets, which may be useful for the future design of fluorescent biosensors.
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Affiliation(s)
- Minji Wang
- Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, No. 3663 Zhong Shan Road North, Shanghai, 200062, China
| | - Yifan Da
- Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, No. 3663 Zhong Shan Road North, Shanghai, 200062, China
| | - Yang Tian
- Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, No. 3663 Zhong Shan Road North, Shanghai, 200062, China
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21
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Lim B, Kim J, Desai MS, Wu W, Chae I, Lee SW. Elastic Fluorescent Protein-Based Down-Converting Optical Films for Flexible Display. Biomacromolecules 2023; 24:118-131. [PMID: 36507771 DOI: 10.1021/acs.biomac.2c00957] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Protein-based material design provides great advantages to developing smart biomaterials with tunable structures and desired functions. They have been widely used in many biomedical applications including tissue engineering and drug delivery. However, protein-based materials are not yet widely used in optoelectronic materials despite their excellent optical and tunable mechanical properties. Here, we synthesized engineered fluorescent proteins (FPs) fused with elastic protein for the development of optoelectrical down-converting optical filters for flexible display materials. We synthesized sequence-specific FPs to tune blue, green, yellow, and red colors and fused them with elastic protein to tune mechanical properties. We fabricated flexible self-supporting film materials and characterized mechanical properties and down-converting optical properties. We also fabricated a hybrid light-emitting diode (LED) to down convert blue to desired green, red, and white colors. Furthermore, we constructed a flexible white LED using organic LED as a flexible substrate. Our modular synthesis approach of tunable bio-optoelectrical material approaches will be useful to design future biocompatible and flexible display materials and technologies.
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Affiliation(s)
- Butaek Lim
- Department of Bioengineering, University of California, Berkeley, Berkeley, California94720, United States.,Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California94720, United States
| | - Jinyeong Kim
- Samsung Display Co Ltd, 1 Samsung-ro, Giheung-gu, Yongin-si17113, Republic of Korea
| | - Malav S Desai
- Department of Bioengineering, University of California, Berkeley, Berkeley, California94720, United States.,Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California94720, United States
| | - Weiyu Wu
- Department of Bioengineering, University of California, Berkeley, Berkeley, California94720, United States
| | - Inseok Chae
- Department of Bioengineering, University of California, Berkeley, Berkeley, California94720, United States.,Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California94720, United States
| | - Seung-Wuk Lee
- Department of Bioengineering, University of California, Berkeley, Berkeley, California94720, United States.,Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California94720, United States
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22
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Sharma M. Selecting the Fluorescent Protein for In Vivo Imaging Experiments. Methods Mol Biol 2023; 2564:47-52. [PMID: 36107336 DOI: 10.1007/978-1-0716-2667-2_2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Many fluorescent proteins are available nowadays suitable for in vivo imaging experiments. Each fluorescent protein has unique biophysical properties, such as emission and excitation spectra, quantum yield, oligomerization state, pH sensitivity, fluorescence lifetime, and stability within the cellular environment. Even a small variation in fluorescent protein properties might result in significant differences in the experimental outcomes. This chapter discusses the aspects that need to be considered while selecting the fluorescent proteins for in vivo imaging experiment.
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Affiliation(s)
- Mayank Sharma
- Institute of Molecular Plant Biology, ETH Zürich, Zürich, Switzerland.
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23
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Barrantes FJ. Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu. Front Mol Biosci 2022; 9:1014659. [PMID: 36518846 PMCID: PMC9743973 DOI: 10.3389/fmolb.2022.1014659] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2022] [Accepted: 11/01/2022] [Indexed: 05/02/2024] Open
Abstract
Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200 nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4 nm-10 nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.
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Affiliation(s)
- Francisco J. Barrantes
- Biomedical Research Institute (BIOMED), Catholic University of Argentina (UCA)–National Scientific and Technical Research Council (CONICET), Buenos Aires, Argentina
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24
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Zhang Q, Shu F, Chen X, Liu W, Bian Y, Kang H. Construction of nucleus-directed fluorescent reporter systems and its application to verification of heterokaryon formation in Morchella importuna. Front Microbiol 2022; 13:1051013. [PMID: 36478869 PMCID: PMC9720127 DOI: 10.3389/fmicb.2022.1051013] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2022] [Accepted: 11/04/2022] [Indexed: 08/26/2023] Open
Abstract
INTRODUCTION Morchella importuna (M. importuna) is a rare fungus with high nutrition value and distinct flavor. Despite the successful artificial cultivation, its genetic characteristics and biological processes such as life cycle, reproductive system, and trophic mode remain poorly understood. METHODS Considering this, we constructed pEH2B and pMH2B vectors by fusing M. importuna endogenous histone protein H2B with fluorescent proteins eGFP or mCherry, respectively. Based on the constructed pEH2B and pMH2B vectors, nuclear fluorescence localization was performed via Agrobacterium tumefaciens-mediated transformation (ATMT). These two vectors were both driven by two endogenous promoters glyceraldehyde 3-phosphate dehydrogenase (GPD) and ubiquitin (UBI). The vector-based reporter systems were tested by the paired culture of two genetically modified strains pEH2B-labeled M04M24 (24e, MAT1-1-1) and pMH2B-abeled M04M26 (26m, MAT1-2-1). RESULTS The fluorescence observation and molecular identification results indicated the successful hyphal fusion and heterokaryon formation. We found that the expression of the reporter genes was stable, and it did not interfere with the growth of the fungus. DISCUSSION Our constructed nucleus-directed fluorescent systems in M. importuna can be used for monitoring the dynamic development and reproductive processes in living cells and also for monitoring the interaction between morels and plant roots. Therefore, morels exhibit the potential to be a candidate organism used for the research on basic biology and genetics of ascomycetes.
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Affiliation(s)
- Qianqian Zhang
- Institute of Applied Mycology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
- Hubei Hongshan Laboratory, Wuhan, China
| | - Fang Shu
- Institute of Applied Mycology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
- Hubei Hongshan Laboratory, Wuhan, China
| | - Xin Chen
- Institute of Applied Mycology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
- Hubei Hongshan Laboratory, Wuhan, China
| | - Wei Liu
- Institute of Applied Mycology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
- Hubei Hongshan Laboratory, Wuhan, China
| | - Yinbing Bian
- Institute of Applied Mycology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
- Hubei Hongshan Laboratory, Wuhan, China
| | - Heng Kang
- Institute of Applied Mycology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
- Hubei Hongshan Laboratory, Wuhan, China
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Ionic liquids as protein stabilizers for biological and biomedical applications: A review. Biotechnol Adv 2022; 61:108055. [DOI: 10.1016/j.biotechadv.2022.108055] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 10/13/2022] [Accepted: 10/23/2022] [Indexed: 11/22/2022]
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Yamanashi Y, Ito T. A Minority Population of Non-dye-decolorizing Bacillus subtilis enhances the Azo Dye-decolorizing Activity of Enterococcus faecalis. Microbes Environ 2022; 37. [PMID: 35650111 PMCID: PMC9530732 DOI: 10.1264/jsme2.me21080] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
Microbes live in communities in biological wastewater treatment plants and in the intestines. However, limited information is currently available on the mechanisms by which minority bacterial populations assist other bacteria besides syntrophic relationships as well as on the microbial food web. Therefore, the present study investigated the effects of non-dye-decolorizing Bacillus subtilis strain S4ga at population levels ranging between 0.04 and 4% on the activity of dye-decolorizing Enterococcus faecalis strain T6a1 using a dye decolorization assay. The results obtained revealed that the minority population of B. subtilis S4ga enhanced the dye-decolorizing activity of E. faecalis T6a1, resulting in a shorter lag time and longer active time of dye decolorization. These effects were related to redox potential values rather than O2 concentrations. Comparisons of the extracellular metabolites in individual incubations of E. faecalis T6a1 and B. subtilis S4ga and a co-incubation suggested a mutual relationship through the cross-feeding of specific amino acids (tyrosine, methionine, tryptophan, phenylalanine, valine, and leucine from B. subtilis S4ga to E. faecalis T6a1; glutamine, histidine, aspartic acid, and proline from E. faecalis T6a1 to B. subtilis S4ga). An analysis of intracellular primary metabolites indicated that the arginine deiminase (ADI) pathway, an ATP-producing energy-generating process, was more strongly activated in co-incubated E. faecalis T6a1 than in E. faecalis T6a1 incubated alone. These results suggest that a co-incubation with B. subtilis S4ga promoted ATP production by E. faecalis T6a1 cells and enhanced its dye-decolorizing activity.
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Affiliation(s)
- Yu Yamanashi
- Department of Environmental Engineering Science, Gunma University
| | - Tsukasa Ito
- Department of Environmental Engineering Science, Gunma University
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Zhai L, Nakashima R, Shinoda H, Ike Y, Matsuda T, Nagai T. Structure-based analysis and evolution of a monomerized red-colored chromoprotein from the Olindias formosa jellyfish. Protein Sci 2022; 31:e4285. [PMID: 35481635 PMCID: PMC8994484 DOI: 10.1002/pro.4285] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Revised: 02/08/2022] [Accepted: 02/13/2022] [Indexed: 11/13/2022]
Abstract
GFP-like chromoproteins (CPs) with non-fluorescence ability have been used as bioimaging probes. Existing CPs have voids in the optical absorption window which limits their extensibility. The development of new CP color is therefore ongoing. Here, we cloned CPs from the jellyfish, Olindias formosa, and developed a completely non-fluorescent monomeric red CP, R-Velour, with an absorption peak at 528 nm. To analyze the photophysical properties from a structural aspect, we determined the crystal structure of R-Velour at a 2.1 Å resolution. R-Velour has a trans-chromophore similar to the green fluorescence protein, Gamillus, derived from the same jellyfish. However, in contrast to the two coplanar chromophoric rings in Gamillus, R-Velour has a large torsion inducing non-fluorescence property. Through site-directed mutagenesis, we surveyed residues surrounding the chromophore and found a key residue, Ser155, which contributes to the generation of four-color variants with the bathochromic and hypsochromic shift of the absorption peak, ranging from 506 to 554 nm. The recently proposed spectrum shift theory, based on the Marcus-Hush model, supports the spectrum shift of these mutants. These findings may support further development of R-Velour variants with useful absorption characteristics for bioimaging, including fluorescence lifetime imaging and photoacoustic imaging.
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Affiliation(s)
- Le Zhai
- Graduate School of Frontier BioscienceOsaka UniversitySuitaJapan
- SANKEN (The Institute of Scientific and Industrial Research)Osaka UniversityIbarakiJapan
| | - Ryosuke Nakashima
- SANKEN (The Institute of Scientific and Industrial Research)Osaka UniversityIbarakiJapan
| | - Hajime Shinoda
- SANKEN (The Institute of Scientific and Industrial Research)Osaka UniversityIbarakiJapan
| | - Yoshimasa Ike
- SANKEN (The Institute of Scientific and Industrial Research)Osaka UniversityIbarakiJapan
- Department of BiotechnologyGraduate School of Engineering, Osaka UniversitySuitaJapan
| | - Tomoki Matsuda
- SANKEN (The Institute of Scientific and Industrial Research)Osaka UniversityIbarakiJapan
- Department of BiotechnologyGraduate School of Engineering, Osaka UniversitySuitaJapan
| | - Takeharu Nagai
- Graduate School of Frontier BioscienceOsaka UniversitySuitaJapan
- SANKEN (The Institute of Scientific and Industrial Research)Osaka UniversityIbarakiJapan
- Department of BiotechnologyGraduate School of Engineering, Osaka UniversitySuitaJapan
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Seiler DK, Hay JC. Genetically encoded fluorescent tools: Shining a little light on ER-to-Golgi transport. Free Radic Biol Med 2022; 183:14-24. [PMID: 35272000 PMCID: PMC9097910 DOI: 10.1016/j.freeradbiomed.2022.03.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/14/2021] [Revised: 03/03/2022] [Accepted: 03/05/2022] [Indexed: 12/11/2022]
Abstract
Since the first fluorescent proteins (FPs) were identified and isolated over fifty years ago, FPs have become commonplace yet indispensable tools for studying the constitutive secretory pathway in live cells. At the same time, genetically encoded chemical tags have provided a new use for much older fluorescent dyes. Innovation has also produced several specialized methods to allow synchronous release of cargo proteins from the endoplasmic reticulum (ER), enabling precise characterization of sequential trafficking steps in the secretory pathway. Without the constant innovation of the researchers who design these tools to control, image, and quantitate protein secretion, major discoveries about ER-to-Golgi transport and later stages of the constitutive secretory pathway would not have been possible. We review many of the tools and tricks, some 25 years old and others brand new, that have been successfully implemented to study ER-to-Golgi transport in intact and living cells.
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Affiliation(s)
- Danette Kowal Seiler
- Division of Biological Sciences, Center for Structural & Functional Neuroscience, University of Montana, Missoula, MT, 59812, USA
| | - Jesse C Hay
- Division of Biological Sciences, Center for Structural & Functional Neuroscience, University of Montana, Missoula, MT, 59812, USA.
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29
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Zarowny L, Clavel D, Johannson R, Duarte K, Depernet H, Dupuy J, Baker H, Brown A, Royant A, Campbell RE. Cyan fluorescent proteins derived from mNeonGreen. Protein Eng Des Sel 2022; 35:gzac004. [PMID: 35417013 PMCID: PMC9083105 DOI: 10.1093/protein/gzac004] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Revised: 03/17/2022] [Accepted: 04/05/2022] [Indexed: 11/13/2022] Open
Abstract
mNeonGreen, an engineered green fluorescent protein (GFP) derived from lancelet, is one of the most brightly fluorescent homologs of Aequorea victoria jellyfish GFP (avGFP) yet reported. In this work, we investigated whether this bright fluorescence might be retained in homologs of mNeonGreen with modified chromophore structures and altered fluorescent hues. We found mNeonGreen to be generally less tolerant than avGFP to chromophore modification by substitution of the key chromophore-forming tyrosine residue with other aromatic amino acids. However, we were ultimately successful in creating a variant, designated as NeonCyan1, with a tryptophan-derived cyan fluorescent protein (CFP)-type chromophore, and two additional mutants with distinct spectral hues. Structural, computational, and photophysical characterization of NeonCyan1 and its variants provided insight into the factors that control the fluorescence emission color. Though not recommended as replacements for contemporary CFP variants, we demonstrate that NeonCyan1 variants are potentially suitable for live cell imaging applications.
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Affiliation(s)
- Landon Zarowny
- Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Canada
| | - Damien Clavel
- Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS), 38000 Grenoble, France
| | - Ryan Johannson
- Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Canada
| | - Kévin Duarte
- Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS), 38000 Grenoble, France
| | - Hadrien Depernet
- Structural Biology Group, European Synchrotron Radiation Facility, 38043 Grenoble, France
| | - Jérôme Dupuy
- Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS), 38000 Grenoble, France
| | - Heather Baker
- Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Canada
| | - Alex Brown
- Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Canada
| | - Antoine Royant
- Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS), 38000 Grenoble, France
- Structural Biology Group, European Synchrotron Radiation Facility, 38043 Grenoble, France
| | - Robert E Campbell
- Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Canada
- Department of Chemistry, University of Tokyo, Tokyo 113-0033, Japan
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30
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Lodovichi C, Ratto GM, Trevelyan AJ, Arosio D. Genetically encoded sensors for Chloride concentration. J Neurosci Methods 2022; 368:109455. [PMID: 34952088 DOI: 10.1016/j.jneumeth.2021.109455] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Revised: 12/11/2021] [Accepted: 12/18/2021] [Indexed: 12/12/2022]
Abstract
Insights into chloride regulation in neurons have come slowly, but they are likely to be critical for our understanding of how the brain works. The reason is that the intracellular Cl- level ([Cl-]i) is the key determinant of synaptic inhibitory function, and this in turn dictates all manner of neuronal network function. The true impact on the network will only be apparent, however, if Cl- is measured at many locations at once (multiple neurons, and also across the subcellular compartments of single neurons), which realistically, can only be achieved using imaging. The development of genetically-encoded anion biosensors (GABs) brings the additional benefit that Cl- imaging may be done in identified cell-classes and hopefully in subcellular compartments. Here, we describe the historical background and motivation behind the development of these sensors and how they have been used so far. There are, however, still major limitations for their use, the most important being the fact that all GABs are sensitive to both pH and Cl-. Disambiguating the two signals has proved a major challenge, but there are potential solutions; notable among these is ClopHensor, which has now been developed for in vivo measurements of both ion species. We also speculate on how these biosensors may yet be improved, and how this could advance our understanding of Cl- regulation and its impact on brain function.
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Affiliation(s)
- Claudia Lodovichi
- Neuroscience Institute-CNR, Depart. Biomedical Sciences, Unipd, Padova, Veneto Institute of Molecular Medicine, Padova Neuroscience Center, Padova, Italy.
| | - Gian Michele Ratto
- National Enterprise for nanoScience and nanoTechnology (NEST), Istituto Nanoscienze, Consiglio Nazionale delle Ricerche (CNR) and Scuola Normale Superiore Pisa, 56127 Pisa, Italy
| | - Andrew J Trevelyan
- Newcastle University Biosciences Institute, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK
| | - Daniele Arosio
- Consiglio Nazionale delle Ricerche (CNR), Istituto di Biofisica, 38123 Trento, Italy.
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31
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Post translational modifications of Trifolitoxin: a blue fluorescent peptide antibiotic. J Antibiot (Tokyo) 2022; 75:125-135. [PMID: 35022574 PMCID: PMC8816728 DOI: 10.1038/s41429-021-00497-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2021] [Revised: 12/13/2021] [Accepted: 12/13/2021] [Indexed: 11/09/2022]
Abstract
Trifolitoxin (TFX, C41H63N15O15S) is a selective, ribosomally-synthesized, post-translationally modified, peptide antibiotic, produced by Rhizobium leguminosarum bv. trifolii T24. TFX specifically inhibits α-proteobacteria, including the plant symbiont Rhizobium spp., the plant pathogen Agrobacterium spp. and the animal pathogen Brucella abortus. TFX-producing strains prevent legume root nodulation by TFX-sensitive rhizobia. TFX has been isolated as a pair of geometric isomers, TFX1 and TFX2, which are derived from the biologically inactive primary amino acid sequence: Asp-Ile-Gly-Gly-Ser-Arg-Gln-Gly-Cys-Val-Ala. Gly-Cys is present as a thiazoline ring and the Arg-Gln-Gly sequence is extensively modified to a UV absorbing, blue fluorescent chromophore. The chromophore consists of a conjugated, 5-membered heterocyclic ring and side chain of modified glutamine.
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32
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Adhikari DP, Biener G, Stoneman MR, Badu DN, Paprocki JD, Eis A, Park PSH, Popa I, Raicu V. Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions. SPECTROCHIMICA ACTA. PART A, MOLECULAR AND BIOMOLECULAR SPECTROSCOPY 2021; 262:120133. [PMID: 34243141 DOI: 10.1016/j.saa.2021.120133] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/22/2021] [Revised: 06/17/2021] [Accepted: 06/28/2021] [Indexed: 06/13/2023]
Abstract
Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photo-switching, is critical to the development of criteria for their selection as tags for fluorescent-based biological applications. While two-photon excitation imaging techniques have steadily gained popularity - due to comparatively deeper penetration depth, reduced out-of-focus photobleaching, and wide separation between emission spectra and two-photon excitation spectra -, most studies reporting on the photophysical properties of FPs tend to remain focused on single-photon excitation. Here, we report our investigation of the photophysical properties of several commonly used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. Our measurements indicate that not only the excitation (and sometimes emission) spectra of FPs may be markedly different between single-photon and two-photon excitation, but also their relative brightness and their photo-stability. A good understanding of the photophysical properties of FPs under two-photon excitation is essential for choosing the right tag(s) for a desired experiment.
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Affiliation(s)
- Dhruba P Adhikari
- Department of Physics, University of Wisconsin-Milwaukee, WI 53211, USA
| | - Gabriel Biener
- Department of Physics, University of Wisconsin-Milwaukee, WI 53211, USA
| | | | - Dammar N Badu
- Department of Physics, University of Wisconsin-Milwaukee, WI 53211, USA
| | - Joel D Paprocki
- Department of Physics, University of Wisconsin-Milwaukee, WI 53211, USA
| | - Annie Eis
- Department of Physics, University of Wisconsin-Milwaukee, WI 53211, USA
| | - Paul S-H Park
- Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH 44106, USA
| | - Ionel Popa
- Department of Physics, University of Wisconsin-Milwaukee, WI 53211, USA
| | - Valerică Raicu
- Department of Physics, University of Wisconsin-Milwaukee, WI 53211, USA; Department of Biological Sciences, University of Wisconsin-Milwaukee, WI 53211, USA.
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Agisha VN, Ashwin NMR, Vinodhini RT, Nalayeni K, Ramesh Sundar A, Malathi P, Viswanathan R. Protoplast-mediated transformation in Sporisorium scitamineum facilitates visualization of in planta developmental stages in sugarcane. Mol Biol Rep 2021; 48:7921-7932. [PMID: 34655406 DOI: 10.1007/s11033-021-06823-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2021] [Accepted: 10/08/2021] [Indexed: 02/05/2023]
Abstract
BACKGROUND Sporisorium scitamineum is the causative agent of smut disease in sugarcane. The tricky life cycle of S. scitamineum consists of three distinct growth stages: diploid teliospores, haploid sporidia and dikaryotic mycelia. Compatible haploid sporidia representing opposite mating types (MAT-1 and MAT-2) of the fungus fuse to form infective dikaryotic mycelia in the host tissues, leading to the development of a characteristic whip shaped sorus. In this study, the transition of distinct stages of in vitro life cycle and in planta developmental stages of S. scitamineum are presented by generating stable GFP transformants of S. scitamineum. METHODS AND RESULTS Haploid sporidia were isolated from the teliospores of Ss97009, and the opposite mating types (MAT-1 and MAT-2) were identified by random mating assay and mating type-specific PCR. Both haploid sporidia were individually transformed with pNIIST plasmid, harboring an enhanced green fluorescent protein (eGFP) gene and hygromycin gene by a modified protoplast-based PEG-mediated transformation method. Thereafter, the distinct in vitro developmental stages including fusion of haploid sporidia and formation of dikaryotic mycelia expressing GFP were demonstrated. To visualize in planta colonization, transformed haploids (MAT-1gfp and MAT-2gfp) were fused and inoculated onto the smut susceptible sugarcane cultivar, Co 97009 and examined microscopically at different stages of colonization. GFP fluorescence-based analysis presented an extensive fungal colonization of the bud surface as well as inter- and intracellular colonization of the transformed S. scitamineum in sugarcane tissues during initial stages of disease development. Noticeably, the GFP-tagged S. scitamineum led to the emergence of smut whips, which established their pathogenicity, and demonstrated initial colonization, active sporogenesis and teliospore maturation stages. CONCLUSION Overall, for the first time, an efficient protoplast-based transformation method was employed to depict clear-cut developmental stages in vitro and in planta using GFP-tagged strains for better understanding of S. scitamineum life cycle development.
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Affiliation(s)
- V N Agisha
- Plant Pathology Section, Division of Crop Protection, ICAR-Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, 641007, India
| | - N M R Ashwin
- Plant Pathology Section, Division of Crop Protection, ICAR-Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, 641007, India
| | - R T Vinodhini
- Plant Pathology Section, Division of Crop Protection, ICAR-Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, 641007, India
| | - Kumaravel Nalayeni
- Plant Pathology Section, Division of Crop Protection, ICAR-Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, 641007, India
| | - Amalraj Ramesh Sundar
- Plant Pathology Section, Division of Crop Protection, ICAR-Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, 641007, India.
| | - Palaniyandi Malathi
- Plant Pathology Section, Division of Crop Protection, ICAR-Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, 641007, India
| | - Rasappa Viswanathan
- Plant Pathology Section, Division of Crop Protection, ICAR-Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, 641007, India
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Pe KBA, Yatsuzuka K, Hakariya H, Kida T, Katsuda Y, Fukuda M, Sato SI. RNA-based cooperative protein labeling that permits direct monitoring of the intracellular concentration change of an endogenous protein. Nucleic Acids Res 2021; 49:e132. [PMID: 34581825 PMCID: PMC8682759 DOI: 10.1093/nar/gkab839] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Revised: 08/24/2021] [Accepted: 09/09/2021] [Indexed: 11/13/2022] Open
Abstract
Imaging the dynamics of proteins in living cells is a powerful means for understanding cellular functions at a deeper level. Here, we report a versatile method for spatiotemporal imaging of specific endogenous proteins in living mammalian cells. The method employs a bifunctional aptamer capable of selective protein recognition and fluorescent probe-binding, which is induced only when the aptamer specifically binds to its target protein. An aptamer for β-actin protein preferentially recognizes its monomer forms over filamentous forms, resulting in selective G-actin staining in both fixed and living cells. Through actin-drug treatment, the method permitted direct monitoring of the intracellular concentration change of endogenous G-actin. This protein-labeling method, which is highly selective and non-covalent, provides rich insights into the study of spatiotemporal protein dynamics in living cells.
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Affiliation(s)
| | - Kenji Yatsuzuka
- Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
| | - Hayase Hakariya
- Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
| | - Tomoki Kida
- Division of Materials Science and Chemistry, Faculty of Advanced Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan
| | - Yousuke Katsuda
- Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8501, Japan.,Division of Materials Science and Chemistry, Faculty of Advanced Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan
| | - Masatora Fukuda
- Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
| | - Shin-Ichi Sato
- Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
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35
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Coughlan NJA, Stockett MH, Kjær C, Ashworth EK, Bulman Page PC, Meech SR, Brøndsted Nielsen S, Blancafort L, Hopkins WS, Bull JN. Action spectroscopy of the isolated red Kaede fluorescent protein chromophore. J Chem Phys 2021; 155:124304. [PMID: 34598549 DOI: 10.1063/5.0063258] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Incorporation of fluorescent proteins into biochemical systems has revolutionized the field of bioimaging. In a bottom-up approach, understanding the photophysics of fluorescent proteins requires detailed investigations of the light-absorbing chromophore, which can be achieved by studying the chromophore in isolation. This paper reports a photodissociation action spectroscopy study on the deprotonated anion of the red Kaede fluorescent protein chromophore, demonstrating that at least three isomers-assigned to deprotomers-are generated in the gas phase. Deprotomer-selected action spectra are recorded over the S1 ← S0 band using an instrument with differential mobility spectrometry coupled with photodissociation spectroscopy. The spectrum for the principal phenoxide deprotomer spans the 480-660 nm range with a maximum response at ≈610 nm. The imidazolate deprotomer has a blue-shifted action spectrum with a maximum response at ≈545 nm. The action spectra are consistent with excited state coupled-cluster calculations of excitation wavelengths for the deprotomers. A third gas-phase species with a distinct action spectrum is tentatively assigned to an imidazole tautomer of the principal phenoxide deprotomer. This study highlights the need for isomer-selective methods when studying the photophysics of biochromophores possessing several deprotonation sites.
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Affiliation(s)
- Neville J A Coughlan
- Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
| | - Mark H Stockett
- Department of Physics, Stockholm University, SE-10691 Stockholm, Sweden
| | - Christina Kjær
- Department of Physics and Astronomy, Aarhus University, Aarhus 8000, Denmark
| | - Eleanor K Ashworth
- School of Chemistry, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, United Kingdom
| | - Philip C Bulman Page
- School of Chemistry, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, United Kingdom
| | - Stephen R Meech
- School of Chemistry, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, United Kingdom
| | | | - Lluís Blancafort
- Institut de Química Computacional i Catálisi and Departament de Química, Universitat de Girona, C/M.A. Capmany 69, 17003 Girona, Spain
| | - W Scott Hopkins
- Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
| | - James N Bull
- School of Chemistry, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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Evoglow-Pp1 and mCherry proteins: a dual fluorescent labeling system for lactic acid bacteria. Appl Microbiol Biotechnol 2021; 105:7367-7378. [PMID: 34536099 DOI: 10.1007/s00253-021-11537-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2021] [Revised: 07/30/2021] [Accepted: 08/19/2021] [Indexed: 10/20/2022]
Abstract
Fluorescent proteins are widely used for cell and protein tracking. Most of these proteins show a high signal and need the presence of oxygen to emit fluorescence. Among them, the fluorescent protein mCherry stands up because of its bright signal and fast maturation. Furthermore, the anaerobic cyan-green fluorescent protein Evoglow-Pp1 allows fluorescent detection under anaerobic conditions. In this work, we modified the pNZ:TuR.aFP plasmid, which harbors the gene encoding Evoglow-Pp1 and the promoter of elongation factor Tu from Limosilactobacillus reuteri CECT925, to obtain a plasmid containing the mrfp gene encoding the monomeric mCherry (pNZ:TuR.mCherry). Moreover, both genes were cloned together (pNZ:TuR.aFP.mCherry) developing a chimeric protein; and with a stop codon between them (pNZ:TuR.aFP.STOP.mCherry) resulting in the expression of both Evoglow-Pp1 and mCherry proteins separately under the influence of the same promoter. Lactococcus lactis, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and L. reuteri strains were transformed with the previously mentioned plasmids, showing an excellent red (pNZ:TuR.mCherry), green (pNZ:TuR.aFP), and red combined with green (pNZ:TuR.aFP.mCherry and pNZ:TuR.aFP.STOP.mCherry) fluorescence signal. Both fluorescence emissions were stable in strains transformed with pNZ:TuR.aFP.STOP.mCherry, while differences in the red or green fluorescence emission were observed in some of the strains harboring pNZ:TuR.aFP.mCherry. Moreover, these plasmids allowed strains differentiation in a complex environment, such as fecal microbiota. Hence, we present the plasmid pNZ:TuR.aFP.STOP.mCherry as a useful tool for the labeling of lactobacilli strains, which would be functional under anoxic conditions, thanks to Evoglow-Pp1, while having the high brightness and good photostability of mCherry. KEY POINTS: • LAB transformed with pNZ:TuR.mCherry expressed the red fluorescent protein mCherry. • LAB transformed with pNZ:TuR.aFP.mCherry developed a fusion of both proteins Evoglow-Pp1 and mCherry. • LAB with pNZ:TuR.aFP.STOP.mCherry expressed both fluorescent proteins separately.
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Split Green Fluorescent Protein-Based Contact Site Sensor (SPLICS ) for Heterotypic Organelle Juxtaposition as Applied to ER -Mitochondria Proximities. Methods Mol Biol 2021. [PMID: 34118050 DOI: 10.1007/978-1-0716-1262-0_23] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/16/2024]
Abstract
In the last decades, membrane contact sites (MCSs) have been the object of intense investigation in different fields of cell physiology and pathology and their importance for the correct functioning of the cell is now widely recognized. MCS between any known intercellular organelles, including endoplasmic reticulum (ER), mitochondria, Golgi, endosomes, peroxisomes, lysosomes, lipid droplets, and the plasma membrane (PM), have been largely documented and in some cases the molecules responsible for the tethering also identified. They represent specific membrane hubs where a tightly coordinated exchange of ions, lipids, nutrients, and factors required to maintain proper cellular homeostasis takes place. Their delicate, dynamic, and sometimes elusive nature prevented and/or delayed the development of tools to easily image interorganelle proximity under physiological conditions and in living organisms. Nowadays, this aspect received great momentum due to the finding that MCSs' dysregulation is involved in several pathological conditions. We have recently developed modular, split-GFP-based contact site sensors (SPLICS) engineered to fluoresce when homo- and heterotypic juxtapositions between ER and mitochondria occur over a range of specific distances. Here we describe in detail, by highlighting strengths and weaknesses, the use and the application of these novel genetically encoded SPLICS sensors and how to properly quantify short- and long-range ER-mitochondria interactions.
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Morikawa TJ, Nishiyama M, Yoshizawa K, Fujita H, Watanabe TM. Glycine insertion modulates the fluorescence properties of Aequorea victoria green fluorescent protein and its variants in their ambient environment. Biophys Physicobiol 2021; 18:145-158. [PMID: 34178565 PMCID: PMC8214926 DOI: 10.2142/biophysico.bppb-v18.016] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2021] [Accepted: 04/27/2021] [Indexed: 12/04/2022] Open
Abstract
The green fluorescent protein (GFP) derived from Pacific Ocean jellyfish is an essential tool in biology. GFP-solvent interactions can modulate the fluorescent property of GFP. We previously reported that glycine insertion is an effective mutation in the yellow variant of GFP, yellow fluorescent protein (YFP). Glycine insertion into one of the β-strands comprising the barrel structure distorts its structure, allowing water molecules to invade near the chromophore, enhancing hydrostatic pressure or solution hydrophobicity sensitivity. However, the underlying mechanism of how glycine insertion imparts environmental sensitivity to YFP has not been elucidated yet. To unveil the relationship between fluorescence and β-strand distortion, we investigated the effects of glycine insertion on the dependence of the optical properties of GFP variants named enhanced-GFP (eGFP) and its yellow (eYFP) and cyan (eCFP) variants with respect to pH, temperature, pressure, and hydrophobicity. Our results showed that the quantum yield decreased depending on the number of inserted glycines in all variants, and the dependence on pH, temperature, pressure, and hydrophobicity was altered, indicating the invasion of water molecules into the β-barrel. Peak shifts in the emission spectrum were observed in glycine-inserted eGFP, suggesting a change of the electric state in the excited chromophore. A comparative investigation of the spectral shift among variants under different conditions demonstrated that glycine insertion rearranged the hydrogen bond network between His148 and the chromophore. The present results provide important insights for further understanding the fluorescence mechanism in GFPs and suggest that glycine insertion could be a potent approach for investigating the relationship between water molecules and the intra-protein chromophore.
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Affiliation(s)
- Takamitsu J Morikawa
- Laboratory for Comprehensive Bioimaging, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo 650-0047, Japan.,Graduate School of Frontier Bioscience, Osaka University, Suita, Osaka 565-0871, Japan
| | - Masayoshi Nishiyama
- Department of Physics, Kindai University, Higashiosaka, Osaka 577-8502, Japan
| | - Keiko Yoshizawa
- Laboratory for Comprehensive Bioimaging, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo 650-0047, Japan
| | - Hideaki Fujita
- Laboratory for Comprehensive Bioimaging, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo 650-0047, Japan.,Department of Stem Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan
| | - Tomonobu M Watanabe
- Laboratory for Comprehensive Bioimaging, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo 650-0047, Japan.,Department of Stem Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan
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Pletneva NV, Maksimov EG, Protasova EA, Mamontova AV, Simonyan TR, Ziganshin RH, Lukyanov KA, Muslinkina L, Pletnev S, Bogdanov AM, Pletnev VZ. Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design. Comput Struct Biotechnol J 2021; 19:2950-2959. [PMID: 34136094 PMCID: PMC8163865 DOI: 10.1016/j.csbj.2021.05.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2021] [Revised: 05/06/2021] [Accepted: 05/09/2021] [Indexed: 01/07/2023] Open
Abstract
For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G - a variant of the enhanced yellow fluorescent protein - obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (~470 and ~530 nm), and the major peak at ~330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein. EYFP-F165G crystal structure revealed two general independent routes of post-translational chemistry, resulting in two main states of the polypeptide chain with the intact chromophore forming triad (~85%) and mature chromophore (~15%). Our experiments thus highlighted important stereochemical role of the 165th position strongly affecting spectral characteristics of the protein. On the basis of the determined EYFP-F165G three-dimensional structure, new variants with ~ 2-fold improved brightness were engineered.
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Key Words
- Ala (A), alanine
- Arg (R), arginine
- Asn (R), asparagine
- Chromophore maturation
- DTT, dithiothreitol
- EC, extinction coefficient
- EET, excitation energy transfer
- EGFP, enhanced green fluorescent protein
- ESET, excited-state electron transfer
- EYFP
- EYFP, enhanced yellow fluorescent protein
- Excitation energy transfer
- FLIM, fluorescence lifetime imaging microscopy
- FP, fluorescent protein
- FQY, fluorescence quantum yield
- FRET, Förster resonance energy transfer
- FTIR, Fourier-transform infrared (spectroscopy
- Femtosecond spectroscopy
- Fluorescent proteins
- GFP, green fluorescent protein
- GYG, glycine-tyrosine-glycine
- Gln (Q), glutamine
- Glu (E), glutamic acid
- Gly (G), glycine
- His (H), histidine
- IVA-cloning, in vivo assembly cloning
- Leu (L), leucine
- PBS, phosphate buffered saline
- PCR, polymerase chain reaction
- Phe (F), phenylalanine
- REACh, resonance energy-accepting chromoprotein
- Ser (S), serine
- Structure-guided mutagenesis
- Trp (W), tryptophan
- Tryptophan fluorescence
- Tyr (Y), tyrosine
- Val (V), valine
- X-ray structure
- avGFP, Aequorea victoria green fluorescent protein
- sfGFP, superfolder GFP
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Affiliation(s)
- Nadya V. Pletneva
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia
| | - Eugene G. Maksimov
- Faculty of Biology, Lomonosov Moscow State University, 119992 Moscow, Russia
| | - Elena A. Protasova
- Faculty of Biology, Lomonosov Moscow State University, 119992 Moscow, Russia
| | - Anastasia V. Mamontova
- Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 121205, Russia
| | - Tatiana R. Simonyan
- Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 121205, Russia
| | - Rustam H. Ziganshin
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia
| | - Konstantin A. Lukyanov
- Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 121205, Russia
| | - Liya Muslinkina
- Structural Biology Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Sergei Pletnev
- Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Alexey M. Bogdanov
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia,Corresponding authors at: Depatment of biophotonics (both), Laboratory of genetically encoded molecular tools ( A.M.B.), Laboratory of of X-ray study ( V.Z.P.).
| | - Vladimir Z. Pletnev
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia,Corresponding authors at: Depatment of biophotonics (both), Laboratory of genetically encoded molecular tools ( A.M.B.), Laboratory of of X-ray study ( V.Z.P.).
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40
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Sultana A, Tiash S. Improved DNA delivery using invasive E. coli DH10B in human cells by modified bactofection method. J Control Release 2021; 332:233-244. [PMID: 33561481 DOI: 10.1016/j.jconrel.2021.02.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2020] [Revised: 01/16/2021] [Accepted: 02/02/2021] [Indexed: 11/15/2022]
Abstract
E. coli mediated gene delivery faces a major drawback of low efficiency despite of being a safer alternative to viral vectors. This study showed a novel, simple and effective strategy to enhance invasive E. coli DH10B vector's efficiency in human epithelial cells. The bactofection efficiency of invasive E .coli vector was analyzed in nine cell lines. It demonstrated highest (16%) reporter gene (GFP) expression in cervical cells. Methods were employed to further enhance its efficiency by adding transfection reagents (trans-bactofection method) to promote entry into host cells, lysosomotropic reagents for escape from lysosomal degradation or antibiotics to lyse internalized bacteria. Increased bacterial entry, as elucidated from nil to 3% expression in liver cells, was obtained upon complexing bacteria with PULSin. Chloroquine mediated endosomal escape resulted in 7.2 folds increase whereas tetracycline addition to lyse internalized bacteria caused ≈90% of GFP in HeLa. Eventually, the combined effect of these three methods exhibited close to 100% GFP in cervical and remarkable increase of 138 folds in breast cells. This is the first study showing comparative study of vector's gene delivery ability in various epithelial cells of the human body with improving its delivery efficiency. These data demonstrated the potential of developed bactofection method to boost up the efficiency of other bacterial vectors also, which could further be used for effectual therapeutic gene delivery in human cells.
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Affiliation(s)
- Alviya Sultana
- School of Science, Monash University, Bandar Sunway, Malaysia.
| | - Snigdha Tiash
- Griffith Institute for Drug Discovery, Griffith University, Queensland, Australia.
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41
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Dhamija S, De AK. Elucidating Contributions from Multiple Species during Photoconversion of Enhanced Green Fluorescent Protein (EGFP) under Ultraviolet Illumination. Photochem Photobiol 2021; 97:980-990. [PMID: 33624317 DOI: 10.1111/php.13409] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2021] [Accepted: 02/21/2021] [Indexed: 11/30/2022]
Abstract
Photocycle in wild-type green fluorescent protein (wt-GFP) involves generation of a bright fluorescent deprotonated chromophore from feebly fluorescent protonated form via excited-state proton transfer. In addition to this usual photocycle, wt-GFP is also known to exhibit irreversible photoconversion upon illumination with ultraviolet and visible radiation. However, a detailed understanding of photoconversion in enhanced GFP (EGFP: S65T/F64L mutant of wt-GFP), which predominantly exists in deprotonated form, is yet to be explored. Using 254 nm irradiation, we study how photoconversion proceeds in EGFP. The key findings are observation of spreading out of an isosbestic point and existence of an initial lag phase in spectral kinetics of absorbance, indicative of sequential photoconversion through an intermediate. Fluorescence kinetics of EGFP and its photoproduct are estimated by assigning two unique fluorescence lifetimes which is further complicated by the fact that their fluorescence are spectrally inseparable, as evident from global analysis of fluorescence lifetime. Time-resolved fluorescence anisotropy studies further suggest minimal structural changes in protein scaffold upon photoconversion. Based on these findings, an analytic model is developed to account for the overall decay in fluorescence (as photoconversion proceeds) that inherently incorporates the initial lag phase and a summary of energetics and processes involved is provided.
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Affiliation(s)
- Shaina Dhamija
- Department of Chemical Sciences, Indian Institute of Science Education and Research Mohali, SAS Nagar, Punjab, India
| | - Arijit K De
- Department of Chemical Sciences, Indian Institute of Science Education and Research Mohali, SAS Nagar, Punjab, India
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42
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Soares BP, Santos JH, Martins M, Almeida MR, Santos NV, Freire MG, Santos-Ebinuma VC, Coutinho JA, Pereira JF, Ventura SP. Purification of green fluorescent protein using fast centrifugal partition chromatography. Sep Purif Technol 2021. [DOI: 10.1016/j.seppur.2020.117648] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
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43
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Functional Imaging Using Bioluminescent Reporter Genes in Living Subjects. Mol Imaging 2021. [DOI: 10.1016/b978-0-12-816386-3.00004-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
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44
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DeRose BT, Kelley RS, Ravi R, Kokona B, Beld J, Spiliotis ET, Padrick SB. Production and analysis of a mammalian septin hetero-octamer complex. Cytoskeleton (Hoboken) 2020; 77:485-499. [PMID: 33185030 DOI: 10.1002/cm.21643] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Revised: 10/26/2020] [Accepted: 11/08/2020] [Indexed: 01/19/2023]
Abstract
The septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.
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Affiliation(s)
- Barry T DeRose
- Department of Biochemistry and Molecular Biology, Drexel University, Philadelphia, Pennsylvania, USA
| | - Robert S Kelley
- Department of Biochemistry and Molecular Biology, Drexel University, Philadelphia, Pennsylvania, USA.,VCU Health System, Richmond, Virginia, USA
| | - Roshni Ravi
- Department of Biochemistry and Molecular Biology, Drexel University, Philadelphia, Pennsylvania, USA.,WuXi Advanced Therapies, Philadelphia, Pennsylvania, USA
| | - Bashkim Kokona
- Department of Chemistry, Haverford College, Haverford, Pennsylvania, USA
| | - Joris Beld
- Department of Microbiology and Immunology, Drexel University, Philadelphia, Pennsylvania, USA
| | - Elias T Spiliotis
- Department of Biology, Drexel University, Philadelphia, Pennsylvania, USA
| | - Shae B Padrick
- Department of Biochemistry and Molecular Biology, Drexel University, Philadelphia, Pennsylvania, USA
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45
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Cambré A, Aertsen A. Bacterial Vivisection: How Fluorescence-Based Imaging Techniques Shed a Light on the Inner Workings of Bacteria. Microbiol Mol Biol Rev 2020; 84:e00008-20. [PMID: 33115939 PMCID: PMC7599038 DOI: 10.1128/mmbr.00008-20] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The rise in fluorescence-based imaging techniques over the past 3 decades has improved the ability of researchers to scrutinize live cell biology at increased spatial and temporal resolution. In microbiology, these real-time vivisections structurally changed the view on the bacterial cell away from the "watery bag of enzymes" paradigm toward the perspective that these organisms are as complex as their eukaryotic counterparts. Capitalizing on the enormous potential of (time-lapse) fluorescence microscopy and the ever-extending pallet of corresponding probes, initial breakthroughs were made in unraveling the localization of proteins and monitoring real-time gene expression. However, later it became clear that the potential of this technique extends much further, paving the way for a focus-shift from observing single events within bacterial cells or populations to obtaining a more global picture at the intra- and intercellular level. In this review, we outline the current state of the art in fluorescence-based vivisection of bacteria and provide an overview of important case studies to exemplify how to use or combine different strategies to gain detailed information on the cell's physiology. The manuscript therefore consists of two separate (but interconnected) parts that can be read and consulted individually. The first part focuses on the fluorescent probe pallet and provides a perspective on modern methodologies for microscopy using these tools. The second section of the review takes the reader on a tour through the bacterial cell from cytoplasm to outer shell, describing strategies and methods to highlight architectural features and overall dynamics within cells.
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Affiliation(s)
- Alexander Cambré
- KU Leuven, Department of Microbial and Molecular Systems, Faculty of Bioscience Engineering, Leuven, Belgium
| | - Abram Aertsen
- KU Leuven, Department of Microbial and Molecular Systems, Faculty of Bioscience Engineering, Leuven, Belgium
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46
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Vasilopoulos V, Pitou M, Fekas I, Papi R, Ouranidis A, Pavlidou E, Patsalas P, Choli-Papadopoulou Τ. Graphene-Wrapped Copper Nanoparticles: An Antimicrobial and Biocompatible Nanomaterial with Valuable Properties for Medical Uses. ACS OMEGA 2020; 5:26329-26334. [PMID: 33110960 PMCID: PMC7581071 DOI: 10.1021/acsomega.0c00834] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/25/2020] [Accepted: 06/04/2020] [Indexed: 06/11/2023]
Abstract
The great demand for antibacterial, biocompatible, and easily manufactured nanostructures has led to the design and development of graphene-wrapped copper nanoparticles (CuNPs) supported on Si wafers. In this study, we investigated the antibacterial properties of graphene/CuNPs nanostructures against Gram-positive and Gram-negative bacteria. Additional experiments regarding graphene/CuNPs nanostructures behavior against mouse fibroblast cell line L929 indicated their biocompatibility and consequently render them as model biomaterials for medical uses. Biofunctionalization of graphene/CuNPs nanostructures with a high-molecular-weight protein (green fluorescent protein), which retains its functionality after a "tight binding" on the nanostructure's surface, opens the way for attaching and other proteins, or biomolecules of great biological interest, to prepare novel biomaterials.
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Affiliation(s)
- Vasileios Vasilopoulos
- Department
of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Maria Pitou
- Department
of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Ilias Fekas
- Department
of Physics, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Rigini Papi
- Department
of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Andreas Ouranidis
- Department
of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Eleni Pavlidou
- Department
of Physics, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
| | - Panos Patsalas
- Department
of Physics, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
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47
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Lam CKC, Truong K. Engineering a Synthesis-Friendly Constitutive Promoter for Mammalian Cell Expression. ACS Synth Biol 2020; 9:2625-2631. [PMID: 32927951 DOI: 10.1021/acssynbio.0c00310] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Since cell-based therapies require the constitutive and stable expression of therapeutic transgenes, lentiviral infection is commonly used to integrate gene material regulated by standard constitutive promoters. Unfortunately, none of the standard or synthetic constitutive promoters can be easily synthesized at low cost due to the presence of repeated subsequences. Thus, in this paper, we designed a synthetic constitutive promoter (named SFCp) that can drive the expression of fluorescent proteins that subsequently trafficked to intended subcellular localizations and the expression of synthetic proteins that rewired the cellular response of Ca2+ to cell morphology changes. Furthermore, SFCp can be used to avoid sequence homology that can theoretically result in loss of genetic material by homologous recombination in tandem constructs. As gene synthesis becomes an indispensable tool in the arsenal of synthetic biology, it is essential to develop a toolbox of gene synthesis friendly components for cell engineering such as constitutive promoters.
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Affiliation(s)
- Chee Ka Candice Lam
- Institute of Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada
| | - Kevin Truong
- Institute of Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada
- Edward S. Rogers, Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King’s College Circle, Toronto, Ontario M5S 3G4, Canada
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48
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Masso M. Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants. Protein Eng Des Sel 2020; 33:5905305. [DOI: 10.1093/protein/gzaa022] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2020] [Revised: 07/16/2020] [Accepted: 08/04/2020] [Indexed: 11/13/2022] Open
Abstract
Abstract
A computational mutagenesis technique was used to characterize the structural effects associated with over 46 000 single and multiple amino acid variants of Aequorea victoria green fluorescent protein (GFP), whose functional effects (fluorescence levels) were recently measured by experimental researchers. For each GFP mutant, the approach generated a single score reflecting the overall change in sequence-structure compatibility relative to native GFP, as well as a vector of environmental perturbation (EP) scores characterizing the impact at all GFP residue positions. A significant GFP structure–function relationship (P < 0.0001) was elucidated by comparing the sequence-structure compatibility scores with the functional data. Next, the computed vectors for GFP mutants were used to train predictive models of fluorescence by implementing random forest (RF) classification and tree regression machine learning algorithms. Classification performance reached 0.93 for sensitivity, 0.91 for precision and 0.90 for balanced accuracy, and regression models led to Pearson’s correlation as high as r = 0.83 between experimental and predicted GFP mutant fluorescence. An RF model trained on a subset of over 1000 experimental single residue GFP mutants with measured fluorescence was used for predicting the 3300 remaining unstudied single residue mutants, with results complementing known GFP biochemical and biophysical properties. In addition, models trained on the subset of experimental GFP mutants harboring multiple residue replacements successfully predicted fluorescence of the single residue GFP mutants. The models developed for this study were accurate and efficient, and their predictions outperformed those of several related state-of-the-art methods.
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Affiliation(s)
- Majid Masso
- Laboratory for Structural Bioinformatics, School of Systems Biology, George Mason University, 10900 University Boulevard MS 5B3, Manassas, VA 20110, USA
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49
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Koveal D, Díaz-García CM, Yellen G. Fluorescent Biosensors for Neuronal Metabolism and the Challenges of Quantitation. Curr Opin Neurobiol 2020; 63:111-121. [PMID: 32559637 PMCID: PMC7646541 DOI: 10.1016/j.conb.2020.02.011] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2020] [Revised: 02/18/2020] [Accepted: 02/25/2020] [Indexed: 01/08/2023]
Abstract
Over the past decade, genetically encoded fluorescent biosensors that report metabolic changes have become valuable tools for understanding brain metabolism. These sensors have been targeted to specific brain regions and cell types in different organisms to track multiple metabolic processes at single cell (and subcellular) resolution. Here, we review genetically encoded biosensors used to study metabolism in the brain. We particularly focus on the principles needed to use these sensors quantitatively while avoiding false inferences from variations in sensor fluorescence that arise from differences in expression level or environmental influences such as pH or temperature.
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Affiliation(s)
- Dorothy Koveal
- Department of Neurobiology, Harvard Medical School, Boston, MA, USA
| | | | - Gary Yellen
- Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
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50
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Sheshukova EV, Ershova NM, Kamarova KA, Dorokhov YL, Komarova TV. The Tobamoviral Movement Protein: A "Conditioner" to Create a Favorable Environment for Intercellular Spread of Infection. FRONTIERS IN PLANT SCIENCE 2020; 11:959. [PMID: 32670343 PMCID: PMC7328123 DOI: 10.3389/fpls.2020.00959] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/18/2020] [Accepted: 06/10/2020] [Indexed: 06/11/2023]
Abstract
During their evolution, viruses acquired genes encoding movement protein(s) (MPs) that mediate the intracellular transport of viral genetic material to plasmodesmata (Pd) and initiate the mechanisms leading to the increase in plasmodesmal permeability. Although the current view on the role of the viral MPs was primarily formed through studies on tobacco mosaic virus (TMV), the function of its MP has not been fully elucidated. Given the intercellular movement of MPs independent of genomic viral RNA (vRNA), this characteristic may induce favorable conditions ahead of the infection front for the accelerated movement of the vRNA (i.e. the MP plays a role as a "conditioner" of viral intercellular spread). This idea is supported by (a) the synthesis of MP from genomic vRNA early in infection, (b) the Pd opening and the MP transfer to neighboring cells without formation of the viral replication complex (VRC), and (c) the MP-mediated movement of VRCs beyond the primary infected cell. Here, we will consider findings that favor the TMV MP as a "conditioner" of enhanced intercellular virus movement. In addition, we will discuss the mechanism by which TMV MP opens Pd for extraordinary transport of macromolecules. Although there is no evidence showing direct effects of TMV MP on Pd leading to their dilatation, recent findings indicate that MPs exert their influence indirectly by modulating Pd external and structural macromolecules such as callose and Pd-associated proteins. In explaining this phenomenon, we will propose a mechanism for TMV MP functioning as a conditioner for virus movement.
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Affiliation(s)
| | - Natalia M. Ershova
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
| | - Kamila A. Kamarova
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
- Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia
| | - Yuri L. Dorokhov
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
| | - Tatiana V. Komarova
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
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