scholarly journals Localization, Dynamics, and Function of Survivin Revealed by Expression of Functional SurvivinDsRed Fusion Proteins in the Living Cell

2003 ◽  
Vol 14 (1) ◽  
pp. 78-92 ◽  
Author(s):  
Achim Temme ◽  
Michael Rieger ◽  
Friedemann Reber ◽  
Dirk Lindemann ◽  
Bernd Weigle ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Laser Scanning ◽  
Tumor Therapy ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mutant Proteins ◽  
Apoptosis Protein ◽  
Chromosomal Passenger ◽  
And Function

Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.

scholarly journals Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

1997 ◽  
Vol 136 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Erik A.C. Wiemer ◽  
Thibaut Wenzel ◽  
Thomas J. Deerinck ◽  
Mark H. Ellisman ◽  
Suresh Subramani
Keyword(s):  
Mammalian Cells ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Small Subset ◽  
Subcellular Compartment ◽  
Directional Movement ◽  
Green Fluorescent

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

Laser Scanning Microscopy Applied to Studies of the Cell Cycle.

1997 ◽  
Vol 3 (S2) ◽  
pp. 235-236
Author(s):  
Ed Luther ◽  
Louis A. Kamentsky
Keyword(s):  
Cell Cycle ◽  
Laser Beam ◽  
Laser Scanning ◽  
Block Diagram ◽  
Data File ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
List Mode ◽  
Microscope Slide

Non-confocal Laser Scanning Microscopy (LSCM) was developed to allow applying the tenents of flow cytometery to specimens attached to a microscope slide (1). Areas of the slide are scanned, fluorescently labled cells are automatically segmented, and a list of features for each cell is calculated and stored in a list mode data file. Figure 1 shows a block diagram of the LSCM. A collimated laser beam scans in the y direction, and values from photomultiplier and photodiode detectors are digitized to .25 micron resolution. The automated stage advances in .5 micron steps in the x direction. When cascading memory banks are filled, the “image” is segmented. Figure 2 shows contours drawn as part of the segmentation process. The innermost contour is drawn around pixels that exceed a predetermined threshold, and is used to define events. Other contours are used to define integration areas, background calculation and correction, and integrating nuclear vs. peripheral fluorescence areas.

scholarly journals Cocktail Strategy Based on NK Cell-Derived Exosomes and Their Biomimetic Nanoparticles for Dual Tumor Therapy

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1560 ◽  
Author(s):  
Wang ◽  
Hu ◽  
Chen ◽  
Shou ◽  
Ye ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Nk Cell ◽  
Imaging System ◽  
Tumor Therapy ◽  
Killer Cell ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Shell Nanoparticles ◽  
Therapy Strategy

Successful cancer therapy requires drugs being precisely delivered to tumors. Nanosized drugs have attracted considerable recent attention, but their toxicity and high immunogenicity are important obstacles hampering their clinical translation. Here we report a novel “cocktail therapy” strategy based on excess natural killer cell-derived exosomes (NKEXOs) in combination with their biomimetic core–shell nanoparticles (NNs) for tumor-targeted therapy. The NNs were self- assembled with a dendrimer core loading therapeutic miRNA and a hydrophilic NKEXOs shell. Their successful fabrication was confirmed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). The resulting NN/NKEXO cocktail showed highly efficient targeting and therapeutic miRNA delivery to neuroblastoma cells in vivo, as demonstrated by two-photon excited scanning fluorescence imaging (TPEFI) and with an IVIS Spectrum in vivo imaging system (IVIS), leading to dual inhibition of tumor growth. With unique biocompatibility, we propose this NN/NKEXO cocktail as a new avenue for tumor therapy, with potential prospects for clinical applications.

Toxicity Evaluation of Two Dental Composites: Three-Dimensional Confocal Laser Scanning Microscopy Time-Lapse Imaging of Cell Behavior

2013 ◽  
Vol 19 (3) ◽  
pp. 596-607 ◽  
Author(s):  
Ghania Nina Attik ◽  
Nelly Pradelle-Plasse ◽  
Doris Campos ◽  
Pierre Colon ◽  
Brigitte Grosgogeat
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Cell Metabolism ◽  
Three Dimensional ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Dental Composites ◽  
Confocal Laser ◽  
Time Lapse Imaging

AbstractThe purpose of this study was to investigate thein vitrobiocompatibility of two dental composites (namely A and B) with similar chemical composition used for direct restoration using three-dimensional confocal laser scanning microscopy (CLSM) time-lapse imaging. Time-lapse imaging was performed on cultured human HGF-1 fibroblast-like cells after staining using Live/Dead®. Image analysis showed a higher mortality rate in the presence of composite A than composite B. The viability rate decreased in a time-dependent manner during the 5 h of exposure. Morphological alterations were associated with toxic effects; cells were enlarged and more rounded in the presence of composite A as shown by F-actin and cell nuclei staining. Resazurin assay was used to confirm the active potential of composites in cell metabolism; results showed severe cytotoxic effects in the presence of both no light-curing composites after 24 h of direct contact. However, extracts of polymerized composites induced a moderate decrease in cell metabolism after the same incubation period. Composite B was significantly better tolerated than composite A at all investigated end points and all time points. The finding confirmed that the used CLSM method was sufficiently sensitive to differentiate the biocompatibility behavior of two composites based on similar methacrylate monomers.

Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis

1999 ◽  
Vol 112 (22) ◽  
pp. 4031-4039 ◽  
Author(s):  
M. Koike ◽  
T. Awaji ◽  
M. Kataoka ◽  
G. Tsujimoto ◽  
T. Kartasova ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Laser Scanning ◽  
Growth Regulation ◽  
Strand Break ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Metaphase Chromosomes ◽  
Interphase Cells ◽  
Ku Protein

The Ku protein is a complex of two subunits, Ku70 and Ku80. Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells. In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G(2)/M phase, which does not require DNA-PKcs.

scholarly journals Hydrosol of Thymbra capitata Is a Highly Efficient Biocide against Salmonella enterica Serovar Typhimurium Biofilms

2016 ◽  
Vol 82 (17) ◽  
pp. 5309-5319 ◽  
Author(s):  
Foteini Karampoula ◽  
Efstathios Giaouris ◽  
Julien Deschamps ◽  
Agapi I. Doulgeraki ◽  
George-John E. Nychas ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Laser Scanning ◽  
High Efficiency ◽  
3D Structure ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Planktonic Cells ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTSalmonellais recognized as one of the most significant enteric foodborne bacterial pathogens. In recent years, the resistance of pathogens to biocides and other environmental stresses, especially when they are embedded in biofilm structures, has led to the search for and development of novel antimicrobial strategies capable of displaying both high efficiency and safety. In this direction, the aims of the present work were to evaluate the antimicrobial activity of hydrosol of the Mediterranean spiceThymbracapitataagainst both planktonic and biofilm cells ofSalmonella entericaserovar Typhimurium and to compare its action with that of benzalkonium chloride (BC), a commonly used industrial biocide. In order to achieve this, the disinfectant activity following 6-min treatments was comparatively evaluated for both disinfectants by calculating the concentrations needed to achieve the same log reductions against both types of cells. Their bactericidal effect against biofilm cells was also comparatively determined byin situand real-time visualization of cell inactivation through the use of time-lapse confocal laser scanning microscopy (CLSM). Interestingly, results revealed that hydrosol was almost equally effective against biofilms and planktonic cells, whereas a 200-times-higher concentration of BC was needed to achieve the same effect against biofilm compared to planktonic cells. Similarly, time-lapse CLSM revealed the significant advantage of the hydrosol to easily penetrate within the biofilm structure and quickly kill the cells, despite the three-dimensional (3D) structure ofSalmonellabiofilm.IMPORTANCEThe results of this paper highlight the significant antimicrobial action of a natural compound, hydrosol ofThymbra capitata, against both planktonic and biofilm cells of a common foodborne pathogen. Hydrosol has numerous advantages as a disinfectant of food-contact surfaces. It is an aqueous solution which can easily be rinsed out from surfaces, it does not have the strong smell of the essential oil (EO) and it is a byproduct of the EO distillation procedure without any industrial application until now. Consequently, hydrosol obviously could be of great value to combat biofilms and thus to improve product safety not only for the food industries but probably also for many other industries which experience biofilm-related problems.

scholarly journals Expression and immunolocalization of the calpain–calpastatin system during parthenogenetic activation and fertilization in the rat egg

Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 35-43 ◽  
Author(s):  
K Haim ◽  
I Ben-Aharon ◽  
R Shalgi
Keyword(s):  
Laser Scanning ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Meiotic Spindle ◽  
Tissue Type ◽  
Egg Activation ◽  
Confocal Laser ◽  
Membrane Area ◽  
Rat Eggs ◽  
And Function

Calpastatin is an intrinsic intracellular inhibitor of calpain, a Ca2+-dependent thiol protease. The calpain–calpastatin system constitutes one functional proteolytic unit whose presence and function has already been investigated in various cell types, but not in the egg. We have previously shown that calpain is expressed in rat eggs and is activated upon egg activation. The present study was designed to investigate the calpain–calpastatin interplay throughout the process.Western blot analysis revealed two main calpastatin isoforms, the erythrocyte type (77 kDa) and the muscle tissue type (110 kDa). By immunohistochemistry and confocal laser scanning microscopy, we demonstrated that the 110 kDa calpastatin was localized at the membrane area and highly abundant at the meiotic spindle in eggs at the first and second meiotic divisions. The 77 kDa calpastatin isoform appeared to be localized as a cortical sphere of clusters. The 110kDa calpastatin and β-tubulin have both been localized to the spindle of metaphase II eggs, both being scattered all through the cytoplasm following spindle disruption by nocodazole treatment, implying a dynamic interaction between calpastatin and microtubule elements. Upon egg activation, membranous calpastatin translocated to the cortex whereas cortical millimolar (m)-calpain shifted towards the membrane. Spindle calpastatin and calpain remained static.We suggest that calpastatin serves as a regulator of m-calpain. The counter translocation of m-calpain and calpastatin could serve as a means of calpain escape from calpastatin inhibition and may reflect a step in the process of calpain activation, throughout egg activation, that is required for calpain to exert its proteolytic activity.

scholarly journals The Hyaluronan Pericellular Coat and Cold Atmospheric Plasma Treatment of Cells

2020 ◽  
Vol 10 (15) ◽  
pp. 5024
Author(s):  
Claudia Bergemann ◽  
Anna-Christin Waldner ◽  
Steffen Emmert ◽  
J. Barbara Nebe
Keyword(s):  
Plasma Treatment ◽  
Laser Scanning ◽  
Tumor Therapy ◽  
Impedance Measurement ◽  
Laser Scanning Microscopy ◽  
Atmospheric Plasma ◽  
Confocal Laser ◽  
Hacat Cells ◽  
Cold Atmospheric Plasma ◽  
Medicine Research

In different tumors, high amounts of hyaluronan (HA) are correlated with tumor progression. Therefore, new tumor therapy strategies are targeting HA production and degradation. In plasma medicine research, antiproliferative and apoptosis-inducing effects on tumor cells were observed using cold atmospheric plasma (CAP) or plasma-activated media (PAM). Until now, the influence of PAM on the HA pericellular coat has not been the focus of research. PAM was generated by argon-plasma treatment of Dulbecco’s modified Eagle’s Medium via the kINPen®09 plasma jet. The HA expression on PAM-treated HaCaT cells was determined by flow cytometry and confocal laser scanning microscopy. Changes in the adhesion behavior of vital cells in PAM were observed by impedance measurement using the xCELLigence system. We found that PAM treatment impaired the HA pericellular coat of HaCaT cells. The time-dependent adhesion was impressively diminished. However, a disturbed HA coat alone was not the reason for the inhibition of cell adhesion because cells enzymatically treated with HAdase did not lose their adhesion capacity completely. Here, we showed for the first time that the plasma-activated medium (PAM) was able to influence the HA pericellular coat.

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