共查询到20条相似文献,搜索用时 15 毫秒
1.
Carlo A. Beretta Nicolas Dross Ulrike Engel Matthias Carl 《Journal of visualized experiments : JoVE》2016,(108)
The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species. 相似文献
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Peter J. Bosch Ivan R. Corrêa Jr. Michael H. Sonntag Jenny Ibach Luc Brunsveld Johannes S. Kanger Vinod Subramaniam 《Biophysical journal》2014
Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines. 相似文献
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Fatma M. Youniss Gobalakrishnan Sundaresan Laura J. Graham Li Wang Collin R. Berry Gajanan K. Dewkar Purnima Jose Harry D. Bear Jamal Zweit 《PloS one》2014,9(10)
The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge can be imaged in vivo. 相似文献
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Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility. 相似文献
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Various proteins are modified post-translationally to localize them at the cell membrane. Among them, hedgehog-family proteins are modified by cholesterol at the C-terminal. In this study, green fluorescent protein (GFP) modified with cholesterol (GFP-Chol) at the C-terminal was prepared semisynthetically and investigated. This semi-synthesis was performed using the following native chemical ligation: GFP-Cα-thioester was prepared using the intein-mediated thioester exchange reaction and was ligated to Cys-NH-diethylene glycol-NHCO-cholesterol in the presence of a detergent. After removal of the detergent, the GFP-Chol was applied to mouse live cells. Confocal laser fluorescent microscopy confirmed localization of GFP-Chol at the cell membrane. The findings suggest that modifying proteins with cholesterol at the C-terminal is useful for targeting the proteins to the cell membrane of live cells. 相似文献
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Andreas Anderluh Enrico Klotzsch Alexander W. A. F. Reismann Mario Brameshuber Oliver Kudlacek Amy Hauck Newman Harald H. Sitte Gerhard J. Schütz 《The Journal of biological chemistry》2014,289(7):4387-4394
The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane. 相似文献
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Paclitaxel (PTX), a chemotherapeutic drug, affects microtubule dynamics and influences endocytic trafficking. However, the mechanism and the dynamics of altered endocytic trafficking by paclitaxel treatment in single living cells still remain elusive. By labeling quantum dots (QDs) to the epidermal growth factor (EGF), we continuously tracked the endocytosis and post-endocytic trafficking of EGF receptors (EGFRs) in A549 cells for a long time interval. A single-cell analysis method was introduced to quantitatively study the dynamics of endocytic trafficking. Compared with the control cells, the velocity of directed motion was reduced by 30% due to the suppression of high speed movements of EGF-QDs along the microtubules in PTX-treated cells. The endocytic trafficking in PTX-treated cells was mainly via super-diffusive mode of motion, whereas in control cells, it was mostly via sub-diffusive mode of motion. Moreover, PTX shortened endosomal trafficking and prevented EGF-QDs from moving to the perinuclear area via the rapid delivery of EGF-QDs into the peripheral lysosomes. The present study may shed light on the mechanism of the effect of PTX on the treatment of lung cancer. 相似文献
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《Journal of biological education》2012,46(4):319-323
Some aspects of the selection and teaching of options of the Joint Matriculation Board's Advanced Level Biology syllabus were investigated by means of a questionnaire. The teachers' opinions of the options and their aims in teaching them are considered. Options seem to be regarded favourably by most of the teachers. 相似文献
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We report the characterization of three-dimensional membrane waves for migrating single and collective cells and describe their propagation using wide-field optical profiling technique with nanometer resolution. We reveal the existence of small and large membrane waves the amplitudes of which are in the range of ∼3–7 nm to ∼16–25 nm respectively, through the cell. For migrating single-cells, the amplitude of these waves is about 30 nm near the cell edge. Two or more different directions of propagation of the membrane nanowaves inside the same cell can be observed. After increasing the migration velocity by BMP-2 treatment, only one wave direction of propagation exists with an increase in the average amplitude (more than 80 nm near the cell edge). Furthermore for collective-cell migration, these membrane nanowaves are attenuated on the leader cells and poor transmission of these nanowaves to follower cells was observed. After BMP-2 treatment, the membrane nanowaves are transmitted from the leader cell to several rows of follower cells. Surprisingly, the vast majority of the observed membrane nanowaves is shared between the adjacent cells. These results give a new view on how single and collective-cells modulate their motility. This work has significant implications for the therapeutic use of BMPs for the regeneration of skin tissue. 相似文献
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Yuce Merve Ozkirimli Elif Sariyar Akbulut Berna Ulgen Kutlu 《International journal of peptide research and therapeutics》2021,27(3):2003-2013
International Journal of Peptide Research and Therapeutics - Cell penetrating peptides (CPPs) are unique molecules with the ability to pass through biological membranes as they carry their cargoes... 相似文献
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《Cell cycle (Georgetown, Tex.)》2013,12(20):2563-2570
In this study we have explored the use of hyperspectral imaging (HSI) to determine the cell-cycle status of live cells in culture. Live cancer cell lines in culture were either synchronized by release from nocodazole or arrested in various cell-cycle phases with serum starvation (G1), aphidicolin (S), or nocodazole (G2/M). The live cells were then stained with the fluorescent DNA binding dyes Heochst 33342 or Dyecycle orange along with propidium iodide or Mitotracker green. Microscopic HSI data was then collected using the PARISS HSI system. Classified spectra were incorporated into spectral libraries; and all spectra acquired from each sample were correlated with library spectra to a user-determined confidence threshold, generating a unique spectral signature for each sample. Examination of these spectral signatures revealed that all cell cycle phases could be objectively differentiated. Ongoing studies employing other viable cell fluorescent dyes, and dyes in combination may provide more robust spectral signatures defining the status and condition of living cells. 相似文献
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Cell Membrane Disruption Stimulates NO/PKG Signaling and Potentiates Cell Membrane Repair in Neighboring Cells 总被引:1,自引:0,他引:1
T Togo 《PloS one》2012,7(8):e42885
Resealing of a disrupted plasma membrane at the micron-diameter range requires Ca(2+)-regulated exocytosis. Repeated membrane disruptions reseal more quickly than the initial wound, and this potentiation of membrane resealing persists for at least 24 hours after the initial wound. Long-term potentiation of membrane resealing requires CREB-dependent gene expression, which is activated by the PKC- and p38 MAPK-dependent pathway in a wounded cell. The present study demonstrates that membrane resealing is potentiated in both wounded and neighboring cells in MDCK cells. Wounding of cells expressing CREB133, a mutant variant of CREB, does not show the potentiated response of cell membrane resealing in either wounded or neighboring cells. Furthermore, wounding of cells induces CREB phosphorylation, not only in wounded cells, but also in neighboring cells. Inhibition of the nitric oxide/PKG signaling pathway suppresses CREB phosphorylation in neighboring cells, but not in wounded cells. The potentiation of membrane resealing in neighboring cells is suppressed if the nitric oxide/PKG pathway is inhibited during the initial wound. Together, these results suggest that the nitric oxide/PKG pathway stimulates CREB phosphorylation in neighboring cells so that subsequent cell membrane disruptions of the neighboring cells reseal more quickly. 相似文献
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Hugo McGuire Mark R. P. Aurousseau Derek Bowie Rikard Blunck 《The Journal of biological chemistry》2012,287(43):35912-35921
Elucidating subunit stoichiometry of neurotransmitter receptors is preferably carried out in a mammalian expression system where the rules of native protein assembly are strictly obeyed. Although successful in Xenopus oocytes, single subunit counting, manually counting photobleaching steps of GFP-tagged subunits, has been hindered in mammalian cells by high background fluorescence, poor control of expression, and low GFP maturation efficiency. Here, we present a fully automated single-molecule fluorescence counting method that separates tagged proteins on the plasma membrane from background fluorescence and contaminant proteins in the cytosol or the endoplasmic reticulum and determines the protein stoichiometry. Lower GFP maturation rates observed in cells cultured at 37 °C were partly offset using a monomeric version of superfolder GFP. We were able to correctly identify the stoichiometry of GluK2 and α1 glycine receptors. Our approach permits the elucidation of stoichiometry for a wide variety of plasma membrane proteins in mammalian cells with any commercially available TIRF microscope. 相似文献
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We have developed an imaging system that combines the soft compliance of an optical trap with the sensitivity of single particle tracking to image forces on/in live cells using a single molecule probe. The probe used is a single (or few) molecule of interest that is conjugated with a single 40 nm colloidalgold probe. The colloidal gold/membrane protein complex, freely diffusing on a live cell, is held in a laser trap while the cell is scanned underneath. Computer control allows for synchronization of the cell scan and capture of the probe position. Resistance to the dragging of the probe images a fine structure of barriers in the membrane of live cells. 相似文献
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Alexander B. Owczarczak Stephen O. Shuford Scott T. Wood Sandra Deitch Delphine Dean 《Journal of visualized experiments : JoVE》2012,(61)
Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15 相似文献
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Membrane pearling in live cells is observed when the plasma membrane is depleted of its support, the cortical actin network. Upon efficient depolymerization of actin, pearls of variable size are formed, which are connected by nanotubes of ∼40 nm diameter. We show that formation of the membrane tubes and their transition into chains of pearls do not require external tension, and that they neither depend on microtubule-based molecular motors nor pressure generated by myosin-II. Pearling thus differs from blebbing. The pearling state is stable as long as actin is prevented from polymerizing. When polymerization is restored, the pearls are retracted into the cell, indicating continuity of the membrane. Our data suggest that the alternation of pearls and strings is an energetically favored state of the unsupported plasma membrane, and that one of the functions of the actin cortex is to prevent the membrane from spontaneously assuming this configuration. 相似文献
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Takashi Ushiki Shinae Kizaka-Kondoh Eishi Ashihara Shotaro Tanaka Masayoshi Masuko Hideyo Hirai Shinya Kimura Yoshifusa Aizawa Taira Maekawa Masahiro Hiraoka 《PloS one》2010,5(6)