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Synthesis of water-soluble dye-cored poly(amidoamine) dendrimers for long-term live cell imaging

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Science China Materials 2018年第11期61卷 1475-1483页

作者： Yang Cai Chendong Ji Shaobo Zhang Zhiqiang Su Meizhen Yin State Key Laboratory of Chemical Resource Engineering Beijing Laboratory of Biomedical MaterialsBeijing University of Chemical TechnologyBeijing 100029China

Hydrophilic dendrimers, especially poly（amidoamine）（PAMAM） dendrimers are widely applied in modifying fluorescent dyes to endow them with water solubility and biocompatibility for biologic fluorescence *** preparation strategies of fluorescent dendrimers including encapsulating dyes or attaching dyes at periphery of dendrimers might cause uncertain constituent and lower biocompatibility. Here, we have developed a series of watersoluble fluorescent dendrimers with dye as core and fanshaped PAMAM as arms. Carboxylated perylene bisimides（PBI） dye and squarylium indocyanine（SICy） dye were conjugated with PAMAM dendrons by amidation to obtain a series of fluorescent dendrimers with enhanced water-solubility. Two PBI based dendrimers（PBI-G2.5 and PBI-G1.5）were chosen as model compounds for further optical, selfassembly and biological studies. In aqueous environment,PBI-G2.5 exhibited strong fluorescence, small size（~30 nm）and slightly positive surface charge（~2.46 mV）, which are ideal for biomedical applications. In vitro assays demonstrated that PBI-G2.5 nanoparticles accumulated in the cytoplasm of He La cells with rapid cellular uptake. The strong fluorescence in He La cells remained for over 48 h. To conclude, our study provides an effective strategy for preparing water-soluble fluorescent dendrimers towards long-term live cell imaging.

关键词： fluorescent dendrimers fan-shaped PAMAM perylene bisimides water solubility live cell imaging

An Activity-Based Probe for live cell imaging of MGMT

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An Activity-Based Probe for Live Cell Imaging of MGMT

live imaging of the effects of fucoidan on cell proliferation for laboratory instruction

2011年全国药物化学学术会议——药物的源头创新

作者： Xin Li,Shijing Qian,Bo Yang,Qiaojun He,and Yongzhou Hu~* College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310058

O-Methylguanine-DNA methyltransferase(MGMT) is a crucial biomarker for individual’s susceptibility to guanine O-targeting *** assays have been devised to examine its levels in tumor *** all the known methods suffer f... 详细信息

关键词： Fluorescent probe MGMT live cell imaging

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TMR Theory and Hypothesis 2018年第2期1卷 45-50页

作者： Hong Wu Laboratory of cell imaging Henan University of Chinese Medicine. Zhengzhou China

The aim of this study is to introduce live cell imaging and its applications for the evaluation of the effects of fucoidan, a fucose-enriched sulfated polysaccharide, on the proliferation of cultured cells in vitro. In this study, long-term time- lapse observation （87 h） of the effects of fucoidan was conducted using BioStation CT, an integrated cell culture observation system. In contrast, the effects of heparin, which has a similar structure to fucoidan, were observed to distinguish the differences between the two chemicals. At the same time, the viability of the floating cells detached by fucoidan in the medium was measured by culturing them again in the absence of fucoidan. Finally, total internal reflection fluorescence microscopy （TIRF） was used to confirm when the detachment of the cells by fucoidan occurred. The results indicate that the inhibitory effects of fucoidan on the proliferation of cells are dose-dependent （from 0.125 mg/ml to 1.0 mg/ml）. Fucoidan also causes cell detachment without killing all the cells within 24 hours. The cell detachment did not occur until after half an hour, as observed under the TIRF microscope. Combined with our previous study, the findings suggest that the inhibition of calcium responses by fucoidan may be one of the mechanisms underlying its inhibition of cell proliferation, which is responsible for the death of cancer cells. cell proliferation can be visualized in the real time and the images can provide important information regarding when and how the cells grow and proliferate.

关键词： live cell imaging Time-lapse cell proliferation Fucoidan

Expanding puromycin based analogues capable of live cell and cell type-selective imaging

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Expanding puromycin based analogues capable of live cell and...

The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features

第十届全国化学生物学学术会议

作者： Ge Jingyan Du Shubo Yao Shao Q. Key Laboratory of Bioorganic Synthesis of Zhejiang Province College of Biotechnology and Bioengineering Zhejiang University of Technology Department of Chemistry National University of Singapore

Global proteome analysis has provided important insights into diverse biological processes. Especially, newly synthesized proteins （NSP）, a subset of the proteome, are involved in every cellular process. However, existing chemical tools used to study them have shortcomings, including limited temporal resolution and incompatibility of live-cell imaging. Herein, we first reported a suite of cell-permeable puromycin analogues capable of being metabolically incorporated into newly synthesized proteins in both live cells and neurons. Furthermore, by taking advantage of"click-to-release" reaction, we developed a versatile puromycin-based strategy for imaging and profiling of newly synthesized proteomes in a cell-specific *** new methods making use of puromycin overcame major drawbacks of existing strategies, thus setting the stage to directly monitor NSP from specialized samples.

关键词： Newly synthesized protein Puromycin live cell imaging cell type-selective

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Protein & cell 2012年第7期3卷 545-558页

作者： Zhengpeng Wan Wanli Liu School of Life Sciences Tsinghua UniversityBeijing 100084China

B lymphocyte cell senses and acquires foreign anti-gens through clonal distributed B cell receptors(BCRs)expressed on the surface of plasma *** presentation formats of antigens are quite *** on their Brownian diffusion mobility,there are three forms:free mobile soluble antigens,lateral mobile membrane bound antigens,and fixed immobile ***,using high resolution high speed live cell imaging approaches,we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell’s encountering of antigens with each format of motion *** high speed live cell imaging,we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the *** propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.

关键词： BCR microcluster TIRFM live cell imaging fixed immobile antigen lateral mobile antigen free mobile antigen

Illuminating single genomic loci in live cells by reducing nuclear background fluorescence

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Science China(Life Sciences) 2021年第5期64卷 667-677页

作者： Song Lu Dianbing Wang Yu Hou Dongge Guo Yulin Deng Xian-En Zhang School of Life Sciences Beijing Institute of TechnologyBeijing 100081China National Laboratory of Biomacromolecules CAS Center for Excellence in BiomacromoleculesInstitute of BiophysicsChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China

The tagging of genomic loci in living cells provides visual evidence for the study of genomic spatial organization and gene ***/dCas9(clustered regularly interspaced short palindromic repeats/deactivated Cas9)labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic ***,the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent *** study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9-SunTag labeling system consisting of dCas9-SunTag-NLS(target module)and scFv-sfGFP-NLS(signal module).We removed the nuclear location sequence(NLS)of the signal module and inserted two copies of EGFP into the signal *** ratio of the fluorescent intensity of the nucleus to that of the cytoplasm(N/C ratio)was decreased by 71%,and the ratio of the signal to the background(S/B ratio)was increased by 1.6 *** system can stably label randomly selected genomic loci with as few as 9 repeat sequences.

关键词： CRISPR/dCas9 labeling background reduction low-repetitive loci live cell imaging

Quantitative imaging reveals the role of MpARF proteasomal degradation during gemma germination

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Plant Communications 2024年第11期5卷 116-127页

作者： Shubhajit Das Martijn de Roij Simon Bellows Melissa Dipp Alvarez Sumanth Mutte Wouter Kohlen Etienne Farcot Dolf Weijers Jan Willem Borst Laboratory of Biochemistry Wageningen University and ResearchStippeneng 46708WE Wageningenthe Netherlands School of Mathematical Sciences University of NottinghamUniversity ParkNG72RD NottinghamUK Laboratory of Molecular Biology Wageningen University and ResearchDroevendaalsesteeg 16708PB Wageningenthe Netherlands Present address:Institute of Science and Technology 3400 KlosterneuburgAustria

The auxin signaling molecule controls a variety of growth and developmental processes in land plants. Auxin regulates gene expression through a nuclear auxin signaling pathway (NAP) consisting of the ubiquitin ligase auxin receptor TIR1/AFB, its Aux/IAA degradation substrate, and DNA-binding ARF transcription factors. Although extensive qualitative understanding of the pathway and its interactions has been obtained, mostly by studying the flowering plant Arabidopsis thaliana, it remains unknown how these translate to quantitative system behavior in vivo, a problem that is confounded by the large NAP gene families in most species. Here, we used the minimal NAP of the liverwort Marchantia polymorpha to quantitatively map NAP protein accumulation and dynamics in vivo through the use of knockin fluorescent fusion proteins. Beyond revealing the dynamic native accumulation profile of the entire NAP protein network, we discovered that the two central ARFs, MpARF1 and MpARF2, are proteasomally degraded. This auxin-independent degradation tunes ARF protein stoichiometry to favor gene activation, thereby reprogramming auxin response during the developmental progression. Thus, quantitative analysis of the entire NAP has enabled us to identify ARF degradation and the stoichiometries of activator and repressor ARFs as a potential mechanism for controlling gemma germination.

关键词： auxin signaling ARF degradation Marchantia polymorpha live cell imaging

Recent advances in optical techniques for dynamically probing cellular mechanobiology

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Biomedical Engineering Communications 2024年第3期3卷 3-11页

作者： Fengqi Wang Qin Zhang Mo Yang Bohan Yin Siu Hong Dexter Wong School of Medicine and Pharmacy Ocean University of ChinaQingdao 266003China Laboratory for Marine Drugs and Bioproducts Qingdao Marine Science and Technology CenterQingdao 266237China Department of Biomedical Engineering The Hong Kong Polytechnic UniversityHong Kong 999077China.

cellular mechanotransduction characterized by the transformation of mechanical stimuli into biochemical signals,represents a pivotal and complex process underpinning a multitude of cellular *** process is integral to diverse biological phenomena,including embryonic development,cell migration,tissue regeneration,and disease pathology,particularly in the context of cancer metastasis and cardiovascular *** the profound biological and clinical significance of mechanotransduction,our understanding of this complex process remains *** recent development of advanced optical techniques enables in-situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside a *** this review,we delved into the current state-of-the-art techniques utilized to probe cellular mechanobiology,their principles,applications,and *** mainly examined optical methodologies to quantitatively measure the mechanical properties of cells during intracellular transport,cell adhesion,and *** provided an introductory overview of various conventional and optical-based techniques for probing cellular *** techniques have provided into the dynamics of mechanobiology,their potential to unravel mechanistic intricacies and implications for therapeutic intervention.

关键词： mechanobiology cell adhesion optical techniques live cell imaging cell fates

Epigenetic regulation and mechanobiology

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Biophysics Reports 2020年第Z1期6卷 33-48页

作者： Shitian Li Dingyi Yang Li Gao Yingxiao Wang Qin Peng Department of Bioengineering and Whitaker Institute of Biomedical Engineering University of California Key Laboratory for Biorheological Science and Technology of Ministry of Education (Chongqing University) Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital School of Chemical Engineering and Technology North University of China

Mechanical force regulates a variety of cellular functions through inducing modulations in nuclear chromatin structures and epigenetic landscapes(outside in). The epigenetic modifications, in turn,regulate gene expressions and affect phenotypic outcomes, including cytoskeletal organization and cell–cell/cell–ECM interactions(inside out). While there have been significant advances in the understanding of mechanotransduction in the nucleus, there is still a lack of knowledge on the potential mechanisms through which mechanical cues affect epigenetic and chromatin regulations to determine genetic outcomes. This review firstly focuses on the current understanding of epigenetic regulations and then summarizes how mechanotransduction and epigenetic modification couple together to regulate molecular and cellular functions, eventually causing functional phenotype changes e.g., ***, we introduce related technologies for mechanistic studies, particularly fluorescence resonance energy transfer(FRET) biosensors for the visualization of dynamic epigenetic regulations in single living cells, as well as the applications of FRET biosensors to visualize mechanotransduction events occurring in the nucleus. These studies could provide new insights into epigenetics in regulating the physiological and pathological processes in living cells under different mechanical environments.

关键词： Epigenetic modification Mechanobiology live cell imaging Fluorescence resonance energy transfer (FRET)