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Live Cell Imaging (live + cell_imaging)
Selected AbstractsA FAK/Src chimera with gain-of-function properties promotes formation of large peripheral adhesions associated with dynamic actin assemblyCYTOSKELETON, Issue 1 2008Priscila M. F. Siesser Abstract Formation of a complex between the tyrosine kinases FAK and Src is a key integrin-mediated signaling event implicated in cell motility, survival, and proliferation. Past studies indicate that FAK functions in the complex primarily as a "scaffold," acting to recruit and activate Src within cell/matrix adhesions. To study the cellular impact of FAK-associated Src signaling we developed a novel gain-of-function approach that involves expressing a chimeric protein with the FAK kinase domain replaced by the Src kinase domain. This FAK/Src chimera is subject to adhesion-dependent activation and promotes tyrosine phosphorylation of p130Cas and paxillin to higher steady-state levels than is achieved by wild-type FAK. When expressed in FAK ,/, mouse embryo fibroblasts, the FAK/Src chimera resulted in a striking cellular phenotype characterized by unusual large peripheral adhesions, enhanced adhesive strength, and greatly reduced motility. Live cell imaging of the chimera-expressing FAK ,/, cells provided evidence that the large peripheral adhesions are associated with a dynamic actin assembly process that is sensitive to a Src-selective inhibitor. These findings suggest that FAK-associated Src kinase activity has the capacity to promote adhesion integrity and actin assembly. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Candida albicans ABG1 gene is involved in endocytosisFEMS YEAST RESEARCH, Issue 2 2009Verónica Veses Abstract The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis. [source] Symposium on ,Live cell imaging of neuronal and glial signalling'JOURNAL OF ANATOMY, Issue 6 2007Arthur M. Butt No abstract is available for this article. [source] Imaging ion flux and ion homeostasis in blood stage malaria parasitesBIOTECHNOLOGY JOURNAL, Issue 6 2009Petra Rohrbach Dr.Article first published online: 8 JUN 200 Abstract The steady-state regulation of intracellular levels of essential ions and ionic gradients is critical for almost all functions within a cell. Thus, it is not surprising to find that ions have been shown to play an important role in numerous parasitic processes, such as invasion, development and possibly drug resistance mechanisms. Live cell imaging has become a widespread technique to visualize and quantify several of these processes, including pH and Ca2+ homeostasis, in an effort to better understand the biology and physiology of cells. This is now also the case for many human pathogens. The aim of this review is to emphasize the importance of this technique and provide an overview of what we have learned so far, using the malaria parasite Plasmodium falciparum as a paradigm. [source] Effects of hydrogen peroxide and apolipoprotein E isoforms on apolipoprotein E trafficking in HepG2 cellsCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 12 2009Tharani Sabaretnam Summary 1.,The major source of apolipoprotein E (apoE) is the liver. In the present study, the effects of oxidative stress and apoE isoforms on apoE distribution and trafficking were established using the HepG2 liver tumour cell line. 2.,Hydrogen peroxide (0, 25, 250 and 1000 ,mol/L) was associated with rapid and concentration-dependent redistribution of apoE into the early endosomal compartment. This redistribution was achieved with a much lower concentration (25 ,mol/L) than that needed to induce changes in intracellular apoE mRNA expression, apoE protein levels and markers of oxidative stress (250,1000 ,mol/L). 3.,Live cell imaging of apoE3,green fluorescent protein revealed a significant decrease in traffic velocity in response to oxidative stress. 4.,The E4 isoform was associated with reduced trafficking velocity compared with the E3 isoform under basal conditions. 5.,The results indicate that oxidative stress and apoE isoforms influence apoE trafficking and distribution within HepG2 cells. Altered apoE hepatocyte trafficking may provide a mechanistic link between oxidative stress, ageing and some diseases in older people. [source] Centrioles are freed from cilia by severing prior to mitosis,CYTOSKELETON, Issue 7 2010Jeremy D.K. Parker Abstract Cilia are necessary for normal tissue development and homeostasis and are generally present during interphase, but not in mitosis. The precise mechanism of premitotic ciliary loss has been controversial, with data supporting either sequential disassembly through the transition zone or, alternatively, a severing event at the base of the cilia. Here we show by live cell imaging and immunofluoresence microscopy that resorbing flagella of Chlamydomonas leave remnants associated with the mother cell wall. We postulated that the remnants are the product of severing of doublet microtubules between the basal bodies and the flagellar transition zone, thereby freeing the centrioles to participate in spindle organization. We show via TEM that flagellar remnants are indeed flagellar transition zones encased in vesicles derived from the flagellar membrane. This transition zone vesicle can be lodged within the cell wall or it can be expelled into the environment. This process is observable in Chlamydomonas, first because the released flagellar remnants can remain associated with the cell by virtue of attachments to the cell wall, and second because the Chlamydomonas transition zone is particularly rich with electron-dense structure. However, release of basal bodies for spindle-associated function is likely to be conserved among the eukaryotes. © 2010 Wiley-Liss, Inc. [source] Microtubule-dependent motility and orientation of the cortical endoplasmic reticulum in elongating characean internodal cellsCYTOSKELETON, Issue 3 2009Ilse Foissner Abstract Motility of the endoplasmic reticulum (ER) is predominantly microtubule- dependent in animal cells but thought to be entirely actomyosin-dependent in plant cells. Using live cell imaging and transmission electron microscopy to examine ER motility and structural organization in giant internodal cells of characean algae, we discovered that at the onset of cell elongation, the cortical ER situated near the plasma membrane formed a tight meshwork of predominantly transverse ER tubules that frequently coaligned with microtubules. Microtubule depolymerization increased mesh size and decreased the dynamics of the cortical ER. In contrast, perturbing the cortical actin array with cytochalasins did not affect the transverse orientation but decreased mesh size and increased ER dynamics. Our data suggest that myosin-dependent ER motility is confined to the ER strands in the streaming endoplasm, while the more sedate cortical ER uses microtubule-based mechanisms for organization and motility during early stages of cell elongation. We show further that the ER has an inherent, NEM-sensitive dynamics which can be altered via interaction with the cytoskeleton and that tubule formation and fusion events are cytoskeleton-independent. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Heterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(3,4)-bisphosphateCYTOSKELETON, Issue 9 2008Jennifer Giorgione Abstract Latex beads are the preferred phagocytic substrate in biochemical studies of phagosome composition and maturation. Using living Dictyostelium cells and fluorescent probes, we compared the properties of phagosomes formed to ingest latex beads or digestible prey. Significant differences were found during the initial steps of phagocytosis. During uptake of bacteria or yeast, PHcrac-GFP, a probe that binds to membranes enriched in PI(3,4,5)P3 and PI(3,4)P2, always labeled the nascent phagosome and faded shortly after it sealed. However, labeling of bead-containing phagosomes was highly variable. Beads were engulfed by phagosomes either lacking or displaying the PHcrac-GFP label, and that label, if present, often persisted for many minutes, revealing that early trafficking steps for bead-containing phagosomes are quite heterogeneous. Later stages of the endocytic pathway appeared more similar for phagosomes containing prey and latex beads. Both types of phagosomes fused with acidic endosomes while undergoing transport along microtubules, both acquired the V-ATPase and lost it prior to exocytosis, and both bound the late endosome marker vacuolin B, which was transferred to the plasma membrane upon exocytosis. We conclude that caution is needed in extrapolating results from latex bead phagosomes to phagosomes containing physiological substances, especially in early stages of the endocytic pathway. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Correlative 3D Microscopy: CLSM and FIB/SEM TomographyIMAGING & MICROSCOPY (ELECTRONIC), Issue 3 2008A Study of Cellular Entry of Vaccinia Virus Abstract Subcellular structural investigation on single cells or tissue samples requires the coupling of optimal structural preservation with detailed imaging at the light and electron microscopic level. To apply light microscopy (FLM, CLSM) and electron microscopy (SEM, FIB/SEM, TEM) imaging modes to the identical sample area has become available with the establishment of chemical preparation, or freeze-substitution protocols after high pressure freezing, adapted to retain fluorophores. One and the same structure can now be investigated at mm to nm range in 2D and 3D in a multimodal set-up [1, 2]. In combination with live cell imaging prior to immobilisation, this approach becomes a powerful tool in life science, e.g. in the development of new anti-viral strategies, as this requires detailed information on the replication cycle of viruses and their interaction with their host cells. [source] Fluorescent proteins for live cell imaging: Opportunities, limitations, and challengesIUBMB LIFE, Issue 11 2009Jörg Wiedenmann Abstract The green fluorescent protein (GFP) from the jellyfish Aequorea victoria can be used as a genetically encoded fluorescence marker due to its autocatalytic formation of the chromophore. In recent years, numerous GFP-like proteins with emission colors ranging from cyan to red were discovered in marine organisms. Their diverse molecular properties enabled novel approaches in live cell imaging but also impose certain limitations on their applicability as markers. In this review, we give an overview of key structural and functional properties of fluorescent proteins that should be considered when selecting a marker protein for a particular application and also discuss challenges that lie ahead in the further optimization of the glowing probes. © 2009 IUBMB IUBMB Life, 61(11): 1029,1042, 2009 [source] Towards correlative imaging of plant cortical microtubule arrays: combining ultrastructure with real-time microtubule dynamicsJOURNAL OF MICROSCOPY, Issue 3 2009D.A. BARTON Summary There are a variety of microscope technologies available to image plant cortical microtubule arrays. These can be applied specifically to investigate direct questions relating to array function, ultrastructure or dynamics. Immunocytochemistry combined with confocal laser scanning microscopy provides low resolution "snapshots" of cortical microtubule arrays at the time of fixation whereas live cell imaging of fluorescent fusion proteins highlights the dynamic characteristics of the arrays. High-resolution scanning electron microscopy provides surface detail about the individual microtubules that form cortical microtubule arrays and can also resolve cellulose microfibrils that form the innermost layer of the cell wall. Transmission electron microscopy of the arrays in cross section can be used to examine links between microtubules and the plasma membrane and, combined with electron tomography, has the potential to provide a complete picture of how individual microtubules are spatially organized within the cortical cytoplasm. Combining these high-resolution imaging techniques with the expression of fluorescent cytoskeletal fusion proteins in live cells using correlative microscopy procedures will usher in an radical change in our understanding of the molecular dynamics that underpin the organization and function of the cytoskeleton. [source] Mechanisms of neurodegenerative diseases: Insights from live cell imagingJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2008Carina Weissmann Abstract Pathologic alterations in protein dynamics such as changes in protein degradation, accumulation of misfolded proteins, and deficits in cellular transport mechanisms are a common feature of most if not all neurodegenerative diseases. Live cell imaging studies promise to contribute to a better understanding of the molecular mechanisms underlying these diseases by visualizing the turnover, accumulation, and transport of proteins in a living cellular context in real time. In this review, we discuss recent work in which different live cell imaging approaches are applied in cellular models of amyotrophic lateral sclerosis, polyQ diseases, and tauopathies as paradigmatic examples of diseases with different types of alterations in protein dynamics. It becomes evident that live cell imaging studies provide new insights into different aspects of protein dynamics, such as the understanding that aggregates are not as static as concluded from previous studies but exhibit a remarkable molecular exchange and that the dynamicity state of the neuronal cytoskeleton might have a critical role in neuronal degeneration. It can be anticipated that live cell imaging studies will lead to a more dynamic view of protein turnover and aggregation, which may aid in identifying drugs that specifically interfere with disease-related changes. © 2007 Wiley-Liss, Inc. [source] Fluorescent proteins as markers in the plant secretory pathwayMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2006Sally L. Hanton Abstract The use of fluorescent proteins and live cell imaging has greatly increased our knowledge of cell biology in recent years. Not only can these technologies be used to study protein trafficking under different conditions, but they have also been of use in elucidating the relationships between different organelles in a noninvasive manner. The use of multiple different fluorochromes allows the observation of interactions between organelles and between proteins, making this one of the fastest-developing and exciting fields at this time. In this review, we discuss the multitude of fluorescent markers that have been generated to study the plant secretory pathway. Although these markers have been used to solve many mysteries in this field, some areas that require further discussion remain. Microsc. Res. Tech. 69:152,159, 2006. © 2006 Wiley-Liss, Inc. [source] Proteome analysis of apoptosis signaling by S -trityl- L -cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2008Frank Kozielski Abstract Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S -trityl- L -cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-,-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68,kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes. [source] Come in and take your coat off , how host cells provide endocytosis for virus entryCELLULAR MICROBIOLOGY, Issue 10 2010Mario Schelhaas Summary Viruses are intracellular parasites that rely upon the host cell machinery for their life cycle. Newly generated virus particles have to transmit their genomic information to uninfected cells/organisms. Viral entry is the process to gain access to viral replication sites within uninfected cells, a multistep course of events that starts with binding to target cells. Since viruses are simple in structure and composition and lack any locomotive capacity, viruses depend on hundreds of host cell proteins during entry. Most animal viruses take advantage of endocytosis to enter cells. Cell biological, morphological and biochemical studies, live cell imaging and systematic approaches have identified various new endocytic mechanisms besides clathrin-mediated endocytosis, macropinocytosis and caveolar/lipid raft-mediated endocytosis. Hence, studying virus entry has become ever more complex. This review provides a cell biological overview of the existing endocytic mechanisms and strategies used or potentially used by viruses to enter cells. [source] Local dynamic changes in confined extracellular environments within organsCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 10 2009Natasha Behrendorff Summary 1Herein we review past work that has studied the composition of luminal fluid in organs, with a focus on measures of calcium and pH in the exocrine glands. This luminal environment is ,external' to the mammalian body and is not subject to the usual mechanisms of homeostatic control. Instead, it is controlled by the behaviour of the cells that line the lumen. 2We discuss the likely possibility that rapid and local changes in calcium and pH occur within microdomains in the lumen. Further, we present preliminary evidence, using live cell imaging of intact pancreatic fragments, that supports the idea that pH changes do occur. Our evidence indicates that exocytosis of secretory granules in pancreatic acinar cells leads to a loss of protons from the granule and a subsequent local acidification of the lumen. 3These changes in luminal composition are placed in the context of diseases of the pancreas, such as cystic fibrosis and pancreatitis, both of which are known to result in perturbations of luminal fluid composition. [source] | |||||||||||||||||||||||||