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Fluorescence Intensity Decay Shape Analysis Microscopy （FIDSAM） for Quantitative and Sensitive Live-Cell Imaging： A Novel Technique for Fluorescence Microscopy of Endogenously Expressed Fusion-proteins

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Molecular Plant 2010年第3期3卷 555-562页

作者： Frank Schleifenbaum Kirstin Elgass Marcus Sackrow Katharina Caesar Kenneth Berendzen Alfred J. Meixner Klaus Hatter Center for Plant Molecular Biology Department of Plant Physiology University of Tubingen Auf der Morgenstelle 1 72076 Tubingen Germany Institute of Physical and Theoretical Chemistry University of Tubingen Auf der Morgenstelle 8 72076 Tubingen Germany

Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy （FIDSAM） to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay （fluorescence lifetime curve） and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b （LTI6b-eGFP） to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophoretagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.

关键词： Cell structure cell walls membrane proteins high-resolution fluorescence microscopy.

Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor

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Acta Pharmaceutica Sinica B 2022年第7期12卷 3113-3123页

作者： Yuhong Cao Jiahao Fang Yiwei Shi Hui Wang Xiaofei Chen Yue Liu Zhenyu Zhu Yan Cao Zhanying Hong Yifeng Chai School of Pharmacy Second Military Medical UniversityShanghai Key Laboratory for Pharmaceutical Metabolites ResearchShanghai 200433China Zhejiang Institute for Food and Drug Control Hangzhou 310057China

P-glycoprotein(P-gp)highly expressed in cancer cells can lead to multidrug resistance(MDR)and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer *** this study,we established a label-free and detergent-free system combining surface plasmon resonance(SPR)biosensor with styrene maleic acid(SMA)polymer membrane proteins(MPs)stabilization technology to screen potential P-gp ***,P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes(SMALPs).Following that,SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system,and the affinity between P-gp and small molecule ligand was *** methodological investigation proved that the screening system had good specificity and *** P-gp ligands were screened out from 50 natural products,and their affinity constants with P-gp were also *** in vitro cell verification experiments demonstrated that tetrandrine,fangchinoline,praeruptorin B,neobaicalein,and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin(Adr).Moreover,tetrandrine,praeruptorin B,and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of *** is the first time that SMALPs-based stabilization strategy was applied to SPR analysis *** polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of *** developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.

关键词： Styrene maleic acid P-Glycoprotein Surface plasmon resonance membrane proteins Inhibitor screening Affinity calculation Natural products Multidrug resistance

Design,synthesis,and biological evaluation of multiple targeting antimalarials

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Acta Pharmaceutica Sinica B 2021年第9期11卷 2900-2913页

作者： Yiqing Yang Tongke Tang Xiaolu Li Thomas Michel Liqin Ling Zhenghui Huang Maruthi Mulaka Yue Wu Hongying Gao Liguo Wang Jing Zhou Brigitte Meunier Hangjun Ke Lubin Jiang Yu Rao MOE Key Laboratory of Protein Sciences School of Pharmaceutical SciencesMOE Key Laboratory of Bioorganic Phosphorus Chemistry&Chemical BiologyTsinghua UniversityBeijing 100084China Unit of Human Parasite Molecular and Cell Biology Key Laboratory of Molecular Virology and ImmunologyInstitut Pasteur of ShanghaiUniversity of Chinese Academy of SciencesChinese Academy of SciencesShanghai 200031China School of Life Science and Technology ShanghaiTech UniversityShanghai 201210China Department of Biochemistry and Molecular Biology State Key Laboratory of Medical Molecular BiologyInstitute of Basic Medical SciencesChinese Academy of Medical SciencesPeking Union Medical CollegeBeijing 100005China Institute for Integrative Biology of the Cell(I2BC) CEACNRSUniversite´Paris-SaclayGif-sur-Yvette 91198France Center for Molecular Parasitology Department of Microbiology and ImmunologyDrexel University College of MedicinePhiladelphiaPA 19129USA Department of Laboratory Medicine West China HospitalSichuan UniversityChengdu 610041China The Nanjing Unicorn Academy of Innovation Institut Pasteur of ShanghaiChinese Academy of SciencesNanjing 211135China

Malaria still threatens global health seriously *** the current discoveries of antimalarials are almost totally focused on single mode-of-action inhibitors,multi-targeting inhibitors are highly desired to overcome the increasingly serious drug ***,we performed a structure-based drug design on mitochondrial respiratory chain of Plasmodium falciparum and identified an extremely potent molecule,RYL-581,which binds to multiple protein binding sites of *** simultaneously(allosteric site of type Ⅱ NADH dehydrogenase,Q_(o) and Q_(i) sites of cytochrome bc_(1)).Antimalarials with such multiple targeting mechanism of action have never been reported ***-581 kills various drug-resistant strains in vitro and shows good solubility as well as in vivo *** structurebased strategy for designing RYL-581 from starting compound may be helpful for other medicinal chemistry projects in the future,especially for drug discovery on membrane-associated targets.

关键词： Drug design Multiple targeting compounds Antimalarial inhibitors Mechanism of action membrane proteins

Cellulose synthesis in land plants

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Molecular Plant 2023年第7期16卷 206-231页

作者： Gustav B.Pedersen Leonard Blaschek Kristian E.H.Frandsen Lise C.Noack Staffan Persson Copenhagen Plant Science Center(CPSC) Department of Plant&Environmental SciencesUniversity of CopenhagenThorvaldsensvej 401871Frederiksberg CDenmark Joint International Research Laboratory of Metabolic&Developmental Sciences State Key Laboratory of Hybrid RiceSJTU-University of Adelaide Joint Centre for Agriculture and HealthSchool of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghaiChina

All plant cells are surrounded by a cell wall that provides cohesion,protection,and a means of directional growth to *** microfibrils contribute the main biomechanical scaffold for most of these *** biosynthesis of cellulose,which typically is the most prominent constituent of the cell wall and therefore Earth’s most abundant biopolymer,is finely attuned to developmental and environmental *** understanding of the machinery that catalyzes and regulates cellulose biosynthesis has substantially improved due to recent technological advances in,for example,structural biology and ***,we provide a comprehensive overview of the structure,function,and regulation of the cellulose synthesis machinery and its regulatory *** aim to highlight important knowledge gaps in the field,and outline emerging approaches that promise a means to close those gaps.

关键词： plant cell wall cellulose microfibrils cellulose synthases membrane proteins cytoskeleton protein interaction

Proteomic Analysis of the Proplastid Envelope membrane Provides Novel Insights into Small Molecule and Protein Transport across Proplastid membranes

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Molecular Plant 2009年第6期2卷 1247-1261页

作者： Andrea Brautigam Andreas RM. Weber Institut fur Biochemie der Pflanzen Heinrich Heine Universitat Universitatsstrasse 1 D-40225 Dusseldorf Germany Genetics Program Michigan State University East Lansing MI USA Department of Plant Biology Michigan State University East Lansing MI USA

Proplastids are undifferentiated plastids of meristematic tissues that synthesize amino acids for protein synthesis, fatty acids for membrane lipid production, and purines and pyrimidines for DNA and RNA synthesis. Unlike chloroplasts, proplastids depend on supply, with reducing power, energy, and precursor metabolites from the remainder of the cell. Comparing proplastid and chloroplast envelope proteomes and the corresponding transcriptomes of leaves and shoot apex revealed a clearly distinct composition of the proplastid envelope. It is geared towards import of metabolic precursors and export of product metabolites for the rapidly dividing cell. The analysis also suggested a new role for the triosephosphate translocator in meristematic tissues, identified the route of organic nitrogen import into proplastids, and detected an adenine nucleotide exporter. The protein import complex contains the import receptors Toc120 and Toc132 and lacks the redox sensing complex subunits of Tic32, Tic55, and Tic62, which mirrors the expression patterns of the corresponding genes in leaves and the shoot apex. We further show that the protein composition of the internal membrane system is similar to etioplasts, as it is dominated by the ATP synthase complex and thus remarkably differs from that of chloroplast thylakoids.

关键词： membrane biochemistry proteomics transporters chloroplast biology membrane proteins transcriptome analysis.

Nanoscale interactions between the nicotinic acetylcholine receptor and cholesterol

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BIOCELL 2021年第6期45卷 1479-1484页

作者： FRANCISCO J.BARRANTES Laboratory of Molecular Neurobiology Institute of Biomedical Research(BIOMED)UCA-CONICETFaculty of Medical SciencesBuenos AiresArgentina

Cholesterol is a major lipid in biological *** not only plays a structural role but also modulates a wide range of functional properties of neurotransmitter and hormone receptors and ion *** membraneembedded segments of the paradigm neurotransmitter receptor for acetylcholine(nAChR)contain linear sequences of amino acids with the capacity to recognize *** cholesterol consensus domains have been designated as“CARC”and its mirror sequence“CRAC”.CARC preferentially occurs in the exoplasmic-facing membrane leaflet,and CRAC,in the cytoplasmic-facing *** motifs are highly conserved among ion-channel and neurotransmitter receptor proteins in vertebrate nervous systems,where they recognize cholesterol,and in prokaryotic homologues in bacteria,where they recognize *** phylogenetically conserved trait is an indication that the hopanoids in some bacteria and cholesterol in eukaryotes subserve analogous functions,probably contributing to the stability of membrane-embedded protein *** studies from our laboratory using superresolution optical microscopy(“nanoscopy”)have disclosed other interrelated functional and structural properties exerted by cholesterol on the *** neutral lipid content at the cell surface influences both the macromolecular organization of the receptor and its translational mobility(diffusion)in the plane of the membrane.

关键词： Cholesterol Pentameric ligand-gated ion channel Nicotinic acetylcholine receptor membrane proteins Evolution Nanoscopy Cholesterol-recognition domains

Solid-state NMR of the retinal protonated Schiff base in microbial rhodopsins

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Magnetic Resonance Letters 2024年第3期4卷 11-18页

作者： Sari Kumagai Izuru Kawamura Graduate School of Engineering Science Yokohama National UniversityYokohama240-8501Japan

Rhodopsin is a seven-helical transmembrane protein with a retinal chromophore covalently bound to a conserved lysine in helix G via a retinal protonated Schiff base(RPSB).Microbial rhodopsins absorb light through chromophore and play a fundamental role in *** microbial rhodopsins have been discovered,contributing to diverse functions and ***-state NMR spectroscopy has been instrumental in elucidating the conformation of chromophores and the three-dimensional structure of microbial *** review focuses on the 15N chemical shift values of RPSB and summarizes recent progress in the *** displayed the correlation between the 15N isotropic chemical shift values of RPSB and the maximum absorption wavelength of rhodopsin using solid-state NMR spectroscopy.

关键词： membrane proteins Microbial rhodopsin Retinal Solid-state NMR Protonated Schiff base

FISSION1A, an Arabidopsis Tail-Anchored Protein, Is Localized to Three Subcellular Compartments

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Molecular Plant 2014年第8期7卷 1393-1396页

作者： Cristina Ruberti Alex Costa Emanuela Pedrazzini Fiorella Lo Schiavo Michela Zottini Department of Biology University of Padua Via U. Bassi 58/B I-35131 Padua Italy MSU-DOE Plant Research Laboratory and Department of Plant Biology Michigan State University 612 WilsonRoad East Lansing MI 48824 USA Department of Biosciences University of Milan Via G. Celoria 26 I-20133 Milan Italy Institute of Biophysics National Research Council (CNR) Via G. Celoria 26 I-20133 Milan Italy Institute of Agricultural Biology and Biotechnology National Research Council (CNR) via Bassini 15 I-20133 Milan Italy

Dear Editor, The multiple targeting proteins that have been described in yeast, mammals, and plants have generated intriguing areas of inquiry, including the evolutionary relevance of common proteins shared by different organelles, the mechanisms that determine targeting to each organelle, and, finally, the extent of this phenomenon. Several dual-targeted proteins have been identified in eukaryotic cells.

关键词： PLANT proteins ARABIDOPSIS thaliana membrane proteins CHLOROPLASTS FISSION (Biology)

How Sugars Might Coordinate Chloroplast and Nuclear Gene Expression during Acclimation to High Light Intensities

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Molecular Plant 2014年第7期7卷 1121-1137页

作者： Rainer E. Haiusler Luisa Heinrichs Jessica Schmitz Ulf-Ingo Flugge Department of Botany II Cologne Biocenter University of Cologne ZuIpicherstr. 47b 50674 Cologne Germany Present address: Plant Molecular Physiology and Biotechnology Heinrich-Heine-University Universitatsstrasse 1 40225 D~sseldorfGermany

The concept of retrograde control of nuclear gene expression assumes the generation of signals inside the chloroplasts, which are either released from or sensed inside of the organelle. In both cases, downstream signaling path- ways lead eventually to a differential regulation of nuclear gene expression and the production of proteins required in the chloroplast. This concept appears reasonable as the majority of the over 3000 predicted plastidial proteins are encoded by nuclear genes. Hence, the nucleus needs information on the status of the chloroplasts, such as during acclimation responses, which trigger massive changes in the protein composition of the thylakoid membrane and in the stroma. Here, we propose an additional control mechanism of nuclear- and plastome-encoded photosynthesis genes, taking advantage of pathways involved in sugar- or hormonal signaling. Sugars are major end products of photosynthesis and their con- tents respond very sensitively to changes in light intensities. Based on recent findings, we ask the question as to whether the carbohydrate status outside the chloroplast can be directly sensed within the chloroplast stroma. Sugars might syn- chronize the responsiveness of both genomes and thereby help to coordinate the expression of piastome- and nuclear- encoded photosynthesis genes in concert with other, more specific retrograde signals.

关键词： acclimation--physiological assimilate allocation/partitioning carbohydrate metabolism molecular physiology membrane proteins Arabidopsis.