Journal articles on the topic 'Fluorescence Recovery while photobleaching'
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1
Combs, Christian A., and Robert S. Balaban. "Enzyme-Dependant Fluorescence Recovery After Photobleaching (ED-FRAP): Application to Imaging Dehydrogenase Activity in Living Single Cells." Microscopy and Microanalysis 7, S2 (August 2001): 18–19. http://dx.doi.org/10.1017/s1431927600026167.
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Fluorescent recovery from photobleaching coupled with confocal microscopy was explored as a potential high-resolution method of imaging the distribution of enzyme activity in single living cardiac myocytes without relying on steady state measurements of fluorescence. On a fundamental level, much remains to be determined regarding how local conditions within a cell affect metabolism. Many studies have suggested that energy metabolism in muscle cells cannot be accurately described assuming a homogenous system of enzymatic reactions [1,2]. The autofluorescence of NADH has been used in many studies as a quantitative assay of mitochondrial energy metabolism [3,4], but studies of steady state fluorescence cannot distinguish between changes in energy production or utilization.In this study conditions were created where the fluorescent recovery of a probe would be solely dependent on cellular enzymatic activity (Enzyme Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)). Experiments examining the inherent fluorescence of NADH (351nm excitation, 450 nm emission) were conducted on small droplets (less than 325 μm diameter) containing NADH alone, in droplets containing an enzyme system capable of synthesis of NADH (Figure 1A) and on isolated rabbit cardiac myocytes (Figure 1D). Photobleaching of the entire cell or droplet eliminated diffusion or bulk transport of NADH from non-bleached regions. Droplets containing NADH alone did not recover, while droplets containing enzyme were shown to recover exponentially (Figure 1B) with a rate constant of fluorescent recovery (kf) that was proportional to enzyme concentration (Figure 1C).
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Wüstner, Daniel. "Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells." Sensors 22, no. 13 (June 23, 2022): 4731. http://dx.doi.org/10.3390/s22134731.
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The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.
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DeBiasio, R. L., L. L. Wang, G. W. Fisher, and D. L. Taylor. "The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts." Journal of Cell Biology 107, no. 6 (December 1, 1988): 2631–45. http://dx.doi.org/10.1083/jcb.107.6.2631.
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The formation of protrusions at the leading edge of the cell is an essential step in fibroblast locomotion. Using fluorescent analogue cytochemistry, ratio imaging, multiple parameter analysis, and fluorescence photobleaching recovery, the distribution of actin and myosin was examined in the same protrusions at the leading edge of live, locomoting cells during wound-healing in vitro. We have previously defined two temporal stages of the formation of protrusions: (a) initial protrusion and (b) established protrusion (Fisher et al., 1988). Actin was slightly concentrated in initial protrusions, while myosin was either totally absent or present at extremely low levels at the base of the initial protrusions. In contrast, established protrusions contained diffuse actin and actin microspikes, as well as myosin in both diffuse and structured forms. Actin and myosin were also localized along concave transverse fibers near the base of initial and established protrusions. The dynamics of myosin penetration into a relatively stable, established protrusion was demonstrated by recording sequential images over time. Myosin was shown to be absent from an initial protrusion, but diffuse and punctate myosin was detected in the same protrusion within 1-2 min. Fluorescence photobleaching recovery indicated that myosin was 100% immobile in the region behind the leading edge containing transverse fibers, in comparison to the 21% immobile fraction detected in the perinuclear region. Possible explanations of the delayed penetration of myosin into established protrusions and the implications on the mechanism of protrusion are discussed.
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Gorbsky, G. J., and G. G. Borisy. "Microtubules of the kinetochore fiber turn over in metaphase but not in anaphase." Journal of Cell Biology 109, no. 2 (August 1, 1989): 653–62. http://dx.doi.org/10.1083/jcb.109.2.653.
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In previous work we injected mitotic cells with fluorescent tubulin and photobleached them to mark domains on the spindle microtubules. We concluded that chromosomes move poleward along kinetochore fiber microtubules that remain stationary with respect to the pole while depolymerizing at the kinetochore. In those experiments, bleached zones in anaphase spindles showed some recovery of fluorescence with time. We wished to determine the nature of this recovery. Was it due to turnover of kinetochore fiber microtubules or of nonkinetochore microtubules or both? We also wished to investigate the question of turnover of kinetochore microtubules in metaphase. We microinjected cells with x-rhodamine tubulin (x-rh tubulin) and photobleached spindles in anaphase and metaphase. At various times after photobleaching, cells were detergent lysed in a cold buffer containing 80 microM calcium, conditions that led to the disassembly of almost all nonkinetochore microtubules. Quantitative analysis with a charge coupled device image sensor revealed that the bleached zones in anaphase cells showed no fluorescence recovery, suggesting that these kinetochore fiber microtubules do not turn over. Thus, the partial fluorescence recovery seen in our earlier anaphase experiments was likely due to turnover of nonkinetochore microtubules. In contrast fluorescence in metaphase cells recovered to approximately 70% the control level within 7 min suggesting that many, but perhaps not all, kinetochore fiber microtubules of metaphase cells do turn over. Analysis of the movements of metaphase bleached zones suggested that a slow poleward translocation of kinetochore microtubules occurred. However, within the variation of the data (0.12 +/- 0.24 micron/min), it could not be determined whether the apparent movement was real or artifactual.
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Kamioka, Hiroshi, Yoshihito Ishihara, Hans Ris, Sakhr A. Murshid, Yasuyo Sugawara, Teruko Takano-Yamamoto, and Soo-Siang Lim. "Primary Cultures of Chick Osteocytes Retain Functional Gap Junctions between Osteocytes and between Osteocytes and Osteoblasts." Microscopy and Microanalysis 13, no. 2 (February 15, 2007): 108–17. http://dx.doi.org/10.1017/s143192760707016x.
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The inaccessibility of osteocytes due to their embedment in the calcified bone matrix in vivo has precluded direct demonstration that osteocytes use gap junctions as a means of intercellular communication. In this article, we report successfully isolating primary cultures of osteocytes from chick calvaria, and, using anti-connexin 43 immunocytochemistry, demonstrate gap junction distribution to be comparable to that found in vivo. Next, we demonstrate the functionality of the gap junctions by (1) dye coupling studies that showed the spread of microinjected Lucifer Yellow from osteoblast to osteocyte and between adjacent osteocytes and (2) analysis of fluorescence replacement after photobleaching (FRAP), in which photobleaching of cells loaded with a membrane-permeable dye resulted in rapid recovery of fluorescence into the photobleached osteocyte, within 5 min postbleaching. This FRAP effect did not occur when cells were treated with a gap junction blocker (18α-glycyrrhetinic acid), but replacement of fluorescence into the photobleached cell resumed when it was removed. These studies demonstrate that gap junctions are responsible for intercellular communication between adjacent osteocytes and between osteoblasts and osteocytes. This role is consistent with the ability of osteocytes to respond to and transmit signals over long distances while embedded in a calcified matrix.
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Kindermann, Stefan, and Štěpán Papáček. "On Data Space Selection and Data Processing for Parameter Identification in a Reaction-Diffusion Model Based on FRAP Experiments." Abstract and Applied Analysis 2015 (2015): 1–17. http://dx.doi.org/10.1155/2015/859849.
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Fluorescence recovery after photobleaching (FRAP) is a widely used measurement technique to determine the mobility of fluorescent molecules within living cells. While the experimental setup and protocol for FRAP experiments are usually fixed, data (pre)processing represents an important issue. The aim of this paper is twofold. First, we formulate and solve the problem ofrelevantFRAP data selection. The theoretical findings are illustrated by the comparison of the results of parameter identification when the full data set was used and the case when theirrelevant data set(data with negligible impact on the confidence interval of the estimated parameters) was removed from the data space. Second, we analyze and compare two approaches of FRAP data processing. Our proposition, surprisingly for the FRAP community, claims that the data set represented by the FRAP recovery curves in form of a time series (integrated data approachcommonly used by the FRAP community) leads to a larger confidence interval compared to thefull(spatiotemporal)data approach.
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Waterman-Storer, C. M., and W. C. Salmon. "Fluorescent Speckle Microscopy in Studies of Cytoskeletal Dynamics During Cell Motility." Microscopy and Microanalysis 7, S2 (August 2001): 6–7. http://dx.doi.org/10.1017/s1431927600026106.
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We have discovered a new method, Fluorescent Speckle Microscopy (FSM), for analyzing the dynamic movement and turnover of macromolecular protein assemblies, such as the cytoskeleton, in living cells (Waterman-Storer et al., 1998). FSM compliments or replaces the techniques of fluorescence recovery after photobleaching or photoactivation of fluorescence for measuring protein dynamics in vivo. For FSM, cells are microinjected with a very low fraction of fluorescently labeled subunits that co-assemble with unlabeled subunits to give a structure with a fluorescent speckled appearance in diffraction-limited wide-field or confocal digital fluorescence images. At low fractions of fluorescent subunits relative to unlabeled subunits, fluorescent speckles vary randomly in intensity according to the number of fluorescent subunits within a diffraction-limited region. in time-lapse FSM image series, movement of the fluorescent speckle pattern indicates translocation of structures, while changes in speckle intensity indicate subunit turnover. We have used FSM to study microtubule and actin behavior in interphase and mitotic cells. We use kymograph analysis to quantitate the movement of speckled structures (Fig 1) and are currently developing analysis procedures to quantify subunit turnover in structures.We have applied these methods to the study of microtubule and actin cytoskeletal dynamics in migrating vertebrate cells in culture. Interactions between the microtubule and actin cytoskeletons underlie fundamental cellular processes such as cytokinesis and cell locomotion, but are poorly understood.
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Riquelme, Meritxell, Salomon Bartnicki-García, Juan Manuel González-Prieto, Eddy Sánchez-León, Jorge A. Verdín-Ramos, Alejandro Beltrán-Aguilar, and Michael Freitag. "Spitzenkörper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of Neurospora crassa." Eukaryotic Cell 6, no. 10 (July 20, 2007): 1853–64. http://dx.doi.org/10.1128/ec.00088-07.
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ABSTRACT The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.
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Salas, P. J., D. E. Vega-Salas, J. Hochman, E. Rodriguez-Boulan, and M. Edidin. "Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity." Journal of Cell Biology 107, no. 6 (December 1, 1988): 2363–76. http://dx.doi.org/10.1083/jcb.107.6.2363.
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We have studied the role of restrictions to lateral mobility in the segregation of proteins to apical and basolateral domains of MDCK epithelial cells. Radioimmunoassay and semiquantitative video analysis of immunofluorescence on frozen sections showed that one apical and three basolateral glycoproteins, defined by monoclonal antibodies and binding of beta-2-microglobulin, were incompletely extracted with 0.5% Triton X-100 in a buffer that preserves the cortical cytoskeleton (Fey, E. G., K. M. Wan, and S. Penman. 1984. J. Cell Biol. 98:1973-1984; Nelson, W. T. and P. J. Veshnock. 1986. J. Cell Biol. 103:1751-1766). The marker proteins were preferentially extracted from the "incorrect" domain (i.e., the apical domain for a basolateral marker), indicating that the cytoskeletal anchoring was most effective on the "correct" domain. The two basolateral markers were unpolarized and almost completely extractable in cells prevented from establishing cell-cell contacts by incubation in low Ca++ medium, while an apical marker was only extracted from the basal surface under the same conditions. Procedures were developed to apply fluorescent probes to either the apical or the basolateral surface of live cells grown on native collagen gels. Fluorescence recovery after photobleaching of predominantly basolateral antigens showed a large percent of cells (28-52%) with no recoverable fluorescence on the basal domain but normal fluorescence recovery on the apical surface of most cells (92-100%). Diffusion coefficients in cells with normal fluorescence recovery were in the order of 1.1 x 10(-9) cm2/s in the apical domain and 0.6-0.9 x 10(-9) cm2/s in the basal surface, but the difference was not significant. The data from both techniques indicate (a) the existence of mobile and immobile protein fractions in both plasma membrane domains, and (b) that linkage to a domain specific submembrane cytoskeleton plays an important role in the maintenance of epithelial cell surface polarity.
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Wu, Yin, Hisaya Kawate, Keizo Ohnaka, Hajime Nawata, and Ryoichi Takayanagi. "Nuclear Compartmentalization of N-CoR and Its Interactions with Steroid Receptors." Molecular and Cellular Biology 26, no. 17 (September 1, 2006): 6633–55. http://dx.doi.org/10.1128/mcb.01534-05.
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ABSTRACT The repression mechanisms by the nuclear receptor corepressor (N-CoR) of steroid hormone receptor (SHR)-mediated transactivation were examined. Yellow fluorescent protein (YFP)-N-CoR was distributed as intranuclear discrete dots, while coexpression of androgen receptor (AR), glucocorticoid receptor α, and estrogen receptor α ligand-dependently triggered redistribution of YFP-N-CoR. In fluorescence recovery after photobleaching analysis, mobility of the N-CoR was reduced by 5α-dihydrotestosterone (DHT)-bound AR. The middle region of N-CoR mostly contributed to the interaction with agonist-bound SHRs and the suppression of their transactivation function. N-CoR impaired the DHT-induced N-C interaction of AR, and the impaired interaction was dose-dependently recovered by coexpression of SRC-1 and CBP. N-CoR also impaired the intranuclear complete (distinct) focus formation of SHRs. Coexpression of SRC-1 or CBP released YFP-N-CoR or endogenous N-CoR from incomplete foci and simultaneously recovered complete foci of AR-green fluorescent protein. These results indicate that the relative ratio of coactivators and corepressors determines the conformational equilibrium between transcriptionally active and inactive SHRs in the presence of agonists. The intranuclear foci formed by agonist-bound SHRs were completely destroyed by actinomycin D and α-amanitin, indicating that the focus formation does not precede the transcriptional activation. The focus formation may reflect the accumulation of SHR/coactivator complexes released from the transcriptionally active sites and thus be a mirror of transcriptionally active complex formation.
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Wier, M. L., and M. Edidin. "Effects of cell density and extracellular matrix on the lateral diffusion of major histocompatibility antigens in cultured fibroblasts." Journal of Cell Biology 103, no. 1 (July 1, 1986): 215–22. http://dx.doi.org/10.1083/jcb.103.1.215.
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We have studied the effect of cell density on the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membranes of fibroblasts using fluorescence recovery after photobleaching. The percent recovery of fluorescence was decreased in fibroblasts grown in confluent cultures. While recovery of fluorescence was measurable in greater than 90% of the cells from sparse cultures, measurable recovery was detected in only 60-80% of the cells from dense cultures; no mobile antigens were detectable in 20-40% of cells examined. The diffusion coefficient on human skin fibroblast cells that did show recovery was the same for cells grown in sparse or dense conditions. In WI-38, VA-2, and c1 1d cultures the diffusion coefficients of mobile antigens were smaller in cells from dense cultures. Changes in lateral diffusion occurred with increased cell-cell contact and with age of cell culture but were not observed in growth-arrested cells or in sparse cells cultured in medium conditioned by confluent cells. Decreased mobile fractions of MHC antigens were observed when cells were plated on extracellular matrix materials derived from confluent cultures. Treatment of the extracellular matrix materials with a combination of proteolytic enzymes or by enzymes that degrade proteoglycans abolished this effect. Matrices produced by cells from other cell lines were less effective in inducing changes in mobile fractions and purified matrix components alone did not induce changes in lateral diffusion. Finally, there were no differences in the proportion of MHC antigens that were resistant to Triton X-100 extraction in sparse and dense cells. These results suggest that cell-cell interactions mediated through extracellular matrix materials can influence the lateral diffusion of at least part of the population of MHC antigens.
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Jones, Laura A., and Peter E. Sudbery. "Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties." Eukaryotic Cell 9, no. 10 (August 6, 2010): 1455–65. http://dx.doi.org/10.1128/ec.00109-10.
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ABSTRACT During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.
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Anobile, Jonathan M., Vaithilingaraja Arumugaswami, Danielle Downs, Kirk Czymmek, Mark Parcells, and Carl J. Schmidt. "Nuclear Localization and Dynamic Properties of the Marek's Disease Virus Oncogene Products Meq and Meq/vIL8." Journal of Virology 80, no. 3 (February 1, 2006): 1160–66. http://dx.doi.org/10.1128/jvi.80.3.1160-1166.2006.
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ABSTRACT Marek's disease virus (MDV) is an avian herpesvirus that causes T-cell lymphomas and immune suppression in susceptible chickens. At least one gene product, MDV Eco Q-encoded protein (Meq), is essential for the oncogenicity of MDV. Alternative splicing permits the meq gene to give rise to two major transcripts encoding proteins designated Meq and Meq/vIL8. Meq is a basic leucine zipper protein capable of modulating transcription. The Meq/vIL8 protein retains a modified leucine zipper, along with the mature receptor-binding portion of vIL8, but lacks the domain of Meq responsible for transcriptional modulation. In this report, we describe studies using fusions between either Meq or Meq/vIL8 and fluorescent proteins to characterize the distribution and properties of these products in chicken embryo fibroblasts (CEFs). Meq and Meq/vIL8 both localized to the nucleoplasm, nucleoli, and Cajal bodies of transfected cells. Similar distributions were found for fluorescent fusion proteins and native Meq or Meq/vIL8. Fluorescence recovery after photobleaching and photoactivatable green fluorescent protein revealed that Meq exhibited mobility properties similar to those of other transcription factors, while Meq/vIL8 was far less mobile. In addition, fluorescence resonance energy transfer studies indicated the formation of Meq/vIL8 homodimers in CEFs. Time lapse studies revealed the coordinated elimination of a portion of Meq and Meq/vIL8 from the nucleus. Our data provide new insight regarding the dynamic cellular properties of two forms of a herpesvirus-encoded oncoprotein and suggest that these forms may have fundamentally different functions in MDV-infected cells.
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Boudjemaa, Rym, Romain Briandet, Matthieu Revest, Cédric Jacqueline, Jocelyne Caillon, Marie-Pierre Fontaine-Aupart, and Karine Steenkeste. "New Insight into Daptomycin Bioavailability and Localization in Staphylococcus aureus Biofilms by Dynamic Fluorescence Imaging." Antimicrobial Agents and Chemotherapy 60, no. 8 (June 13, 2016): 4983–90. http://dx.doi.org/10.1128/aac.00735-16.
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ABSTRACTStaphylococcus aureusis one of the most frequent pathogens responsible for biofilm-associated infections (BAI), and the choice of antibiotics to treat these infections remains a challenge for the medical community. In particular, daptomycin has been reported to fail against implant-associatedS. aureusinfections in clinical practice, while its association with rifampin remains a good candidate for BAI treatment. To improve our understanding of such resistance/tolerance toward daptomycin, we took advantage of the dynamic fluorescence imaging tools (time-lapse imaging and fluorescence recovery after photobleaching [FRAP]) to locally and accurately assess the antibiotic diffusion reaction in methicillin-susceptible and methicillin-resistantS. aureusbiofilms. To provide a realistic representation of daptomycin action, we optimized anin vitromodel built on the basis of our recently publishedin vivomouse model of prosthetic vascular graft infections. We demonstrated that at therapeutic concentrations, daptomycin was inefficient in eradicating biofilms, while the matrix was not a shield to antibiotic diffusion and to its interaction with its bacterial target. In the presence of rifampin, daptomycin was still present in the vicinity of the bacterial cells, allowing prevention of the emergence of rifampin-resistant mutants. Conclusions derived from this study strongly suggest thatS. aureusbiofilm resistance/tolerance toward daptomycin may be more likely to be related to a physiological change involving structural modifications of the membrane, which is a strain-dependent process.
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Vohnoutka, Rishel B., Anushree C. Gulvady, Gregory Goreczny, Kyle Alpha, Samuel K. Handelman, Jonathan Z. Sexton, and Christopher E. Turner. "The focal adhesion scaffold protein Hic-5 regulates vimentin organization in fibroblasts." Molecular Biology of the Cell 30, no. 25 (December 1, 2019): 3037–56. http://dx.doi.org/10.1091/mbc.e19-08-0442.
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Focal adhesion (FA)-stimulated reorganization of the F-actin cytoskeleton regulates cellular size, shape, and mechanical properties. However, FA cross-talk with the intermediate filament cytoskeleton is poorly understood. Genetic ablation of the FA-associated scaffold protein Hic-5 in mouse cancer-associated fibroblasts (CAFs) promoted a dramatic collapse of the vimentin network, which was rescued following EGFP-Hic-5 expression. Vimentin collapse correlated with a loss of detergent-soluble vimentin filament precursors and decreased vimentin S72/S82 phosphorylation. Additionally, fluorescence recovery after photobleaching analysis indicated impaired vimentin dynamics. Microtubule (MT)-associated EB1 tracking and Western blotting of MT posttranslational modifications indicated no change in MT dynamics that could explain the vimentin collapse. However, pharmacological inhibition of the RhoGTPase Cdc42 in Hic-5 knockout CAFs rescued the vimentin collapse, while pan-formin inhibition with SMIFH2 promoted vimentin collapse in Hic-5 heterozygous CAFs. Our results reveal novel regulation of vimentin organization/dynamics by the FA scaffold protein Hic-5 via modulation of RhoGTPases and downstream formin activity.
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Stenoien, David L., Anne C. Nye, Maureen G. Mancini, Kavita Patel, Martin Dutertre, Bert W. O'Malley, Carolyn L. Smith, Andrew S. Belmont, and Michael A. Mancini. "Ligand-Mediated Assembly and Real-Time Cellular Dynamics of Estrogen Receptor α-Coactivator Complexes in Living Cells." Molecular and Cellular Biology 21, no. 13 (July 1, 2001): 4404–12. http://dx.doi.org/10.1128/mcb.21.13.4404-4412.2001.
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ABSTRACT Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.
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Centonze, V. E., and G. G. Borisy. "Pole-to-chromosome movements induced at metaphase: sites of microtubule disassembly." Journal of Cell Science 100, no. 1 (September 1, 1991): 205–11. http://dx.doi.org/10.1242/jcs.100.1.205.
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Metaphase spindles can be induced to shrink by treating cells with microtubule-depolymerizing agents. During treatment, the paired sister chromatids remain at the metaphase plate and the poles move toward them. The question we asked is whether this pole-to-chromosome movement was accompanied by a loss of subunits from the kinetochore ends of the microtubules, the polar ends, or both ends. LLC-PK cells were injected at late prometaphase with Xrhodamine tubulin and at metaphase the fluorescent spindles were marked by photobleaching a bar between one pole and the chromosomes. Nocodazole at low concentrations was briefly applied to the cells to induce the shortening of the spindle and movement of the poles inward toward the chromosomes. In the induced shortening, the distance between the photobleached bar and the chromosomes decreased substantially while the distance between the bar and the pole showed a smaller change. Upon reversal from nocodazole, new polymer was added to the spindle as determined by recovery of fluorescence, and the cells progressed through mitosis and cytokinesis. We conclude that the movement of the poles to the chromosomes induced by nocodazole treatment during metaphase is similar to the chromosome-to-pole movement occurring during anaphase in that under both conditions the primary site for kinetochore microtubule disassembly is at the kinetochore.
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Ghosh, Anindya S., and Kevin D. Young. "Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane of Escherichia coli." Journal of Bacteriology 187, no. 6 (March 15, 2005): 1913–22. http://dx.doi.org/10.1128/jb.187.6.1913-1922.2005.
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ABSTRACT In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen of E. coli 2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane of E. coli has a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod.
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Caffaro, Carolina E., and John C. Boothroyd. "Evidence for Host Cells as the Major Contributor of Lipids in the Intravacuolar Network of Toxoplasma-Infected Cells." Eukaryotic Cell 10, no. 8 (June 17, 2011): 1095–99. http://dx.doi.org/10.1128/ec.00002-11.
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ABSTRACT The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.
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Bettayeb, Karima, Jerry C. Chang, Wenjie Luo, Suvekshya Aryal, Dante Varotsis, Lisa Randolph, William J. Netzer, Paul Greengard, and Marc Flajolet. "δ-COP modulates Aβ peptide formation via retrograde trafficking of APP." Proceedings of the National Academy of Sciences 113, no. 19 (April 25, 2016): 5412–17. http://dx.doi.org/10.1073/pnas.1604156113.
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The components involved in cellular trafficking and protein recycling machinery that have been associated with increased Alzheimer’s disease (AD) risk belong to the late secretory compartments for the most part. Here, we hypothesize that these late unavoidable events might be the consequence of earlier complications occurring while amyloid precursor protein (APP) is trafficking through the early secretory pathway. We investigated the relevance to AD of coat protein complex I (COPI)-dependent trafficking, an early step in Golgi-to-endoplasmic reticulum (ER) retrograde transport and one of the very first trafficking steps. Using a complex set of imaging technologies, including inverse fluorescence recovery after photobleaching (iFRAP) and photoactivatable probes, coupled to biochemical experiments, we show that COPI subunit δ (δ-COP) affects the biology of APP, including its subcellular localization and cell surface expression, its trafficking, and its metabolism. These findings demonstrate the crucial role of δ-COP in APP metabolism and, consequently, the generation of amyloid-β (Aβ) peptide, providing previously nondescribed mechanistic explanations of the underlying events.
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Dequidt, Caroline, Lydia Danglot, Philipp Alberts, Thierry Galli, Daniel Choquet, and Olivier Thoumine. "Fast Turnover of L1 Adhesions in Neuronal Growth Cones Involving Both Surface Diffusion and Exo/Endocytosis of L1 Molecules." Molecular Biology of the Cell 18, no. 8 (August 2007): 3131–43. http://dx.doi.org/10.1091/mbc.e06-12-1101.
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We investigated the interplay between surface trafficking and binding dynamics of the immunoglobulin cell adhesion molecule L1 at neuronal growth cones. Primary neurons were transfected with L1 constructs bearing thrombin-cleavable green fluorescent protein (GFP), allowing visualization of newly exocytosed L1 or labeling of membrane L1 molecules by Quantum dots. Intracellular L1–GFP vesicles showed preferential centrifugal motion, whereas surface L1–GFP diffused randomly, revealing two pathways to address L1 to adhesive sites. We triggered L1 adhesions using microspheres coated with L1–Fc protein or anti-L1 antibodies, manipulated by optical tweezers. Microspheres coupled to the actin retrograde flow at the growth cone periphery while recruiting L1–GFP molecules, of which 50% relied on exocytosis. Fluorescence recovery after photobleaching experiments revealed a rapid recycling of L1–GFP molecules at L1–Fc (but not anti-L1) bead contacts, attributed to a high lability of L1–L1 bonds at equilibrium. L1–GFP molecules truncated in the intracellular tail as well as neuronal cell adhesion molecules (NrCAMs) missing the clathrin adaptor binding sequence showed both little internalization and reduced turnover rates, indicating a role of endocytosis in the recycling of mature L1 contacts at the base of the growth cone. Thus, unlike for other molecules such as NrCAM or N-cadherin, diffusion/trapping and exo/endocytosis events cooperate to allow the fast renewal of L1 adhesions.
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Kretzschmar, Anja, Jan-Philip Schülke, Mercè Masana, Katharina Dürre, Marianne B. Müller, Andreas R. Bausch, and Theo Rein. "The Stress-Inducible Protein DRR1 Exerts Distinct Effects on Actin Dynamics." International Journal of Molecular Sciences 19, no. 12 (December 11, 2018): 3993. http://dx.doi.org/10.3390/ijms19123993.
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Cytoskeletal dynamics are pivotal to memory, learning, and stress physiology, and thus psychiatric diseases. Downregulated in renal cell carcinoma 1 (DRR1) protein was characterized as the link between stress, actin dynamics, neuronal function, and cognition. To elucidate the underlying molecular mechanisms, we undertook a domain analysis of DRR1 and probed the effects on actin binding, polymerization, and bundling, as well as on actin-dependent cellular processes. Methods: DRR1 domains were cloned and expressed as recombinant proteins to perform in vitro analysis of actin dynamics (binding, bundling, polymerization, and nucleation). Cellular actin-dependent processes were analyzed in transfected HeLa cells with fluorescence recovery after photobleaching (FRAP) and confocal microscopy. Results: DRR1 features an actin binding site at each terminus, separated by a coiled coil domain. DRR1 enhances actin bundling, the cellular F-actin content, and serum response factor (SRF)-dependent transcription, while it diminishes actin filament elongation, cell spreading, and actin treadmilling. We also provide evidence for a nucleation effect of DRR1. Blocking of pointed end elongation by addition of profilin indicates DRR1 as a novel barbed end capping factor. Conclusions: DRR1 impacts actin dynamics in several ways with implications for cytoskeletal dynamics in stress physiology and pathophysiology.
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Ochoa, Gian-Carlo, Vladimir I. Slepnev, Lynn Neff, Niels Ringstad, Kohji Takei, Laurie Daniell, Warren Kim, et al. "A Functional Link between Dynamin and the Actin Cytoskeleton at Podosomes." Journal of Cell Biology 150, no. 2 (July 24, 2000): 377–90. http://dx.doi.org/10.1083/jcb.150.2.377.
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Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2–binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aaG273D abolished podosomes while GFP-dynaminK44A was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.
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Bulinski, J. Chloë, David J. Odde, Bonnie J. Howell, Ted D. Salmon, and Clare M. Waterman-Storer. "Rapid dynamics of the microtubule binding of ensconsin in vivo." Journal of Cell Science 114, no. 21 (November 1, 2001): 3885–97. http://dx.doi.org/10.1242/jcs.114.21.3885.
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Microtubule-associated proteins (MAPs) are proteins that reversibly bind to and regulate microtubule dynamics and functions in vivo. We examined the dynamics of binding of a MAP called ensconsin (E-MAP-115) to microtubules in vivo. We used 5×GFP-EMTB, a construct in which the microtubule-binding domain of ensconsin (EMTB) is fused to five copies of green fluorescent protein (GFP), as a reporter molecule amenable to the use of fluorescent speckle microscopy. Fluorescent speckle microscopy (FSM) sequences and kymograph analyses showed rapid dynamics of speckles comprised of 5×GFP-EMTB in untreated cells. By contrast, in detergent-lysed cytoskeletons, speckles were not dynamic. Since detergent-lysed cytoskeletons differ from living cells in that they lack both ATP and dynamic microtubules, we used azide treatment to substantially reduce the level of ATP in living cells and we used Taxol to halt microtubule dynamics. Both treatments slowed the dynamics of 5×GFP-EMTB speckles observed by FSM. We also used fluorescence recovery after photobleaching (FRAP) to quantify the half-time of binding and dissociation of the 5×GFP-EMTB chimera and to compare this half-time to that of the full-length MAP molecule. In untreated cells, the tg of either 5×GFP-EMTB or full-length GFP-ensconsin was similarly rapid (∼4 seconds), while in ATP-reduced and Taxol-treated cells, tg was increased to 210 seconds and 40 seconds, respectively. In detergent-extracted cells no recovery was seen. Consistent with the rapid dynamics of 5×GFP-EMTB measured with fluorescent speckle microscopy and FRAP, we estimated that the affinity of the MAP for microtubules is ∼40 μM in untreated living cells, compared with ∼1 μM in vitro. However, KD,app was not significantly changed in the presence of azide and was increased to 110 μM in the presence of Taxol. To test whether changes in the phosphorylation state of cellular proteins might be responsible for altering the dynamics of ensconsin binding, we used FSM to monitor staurosporine-treated cells. Staurosporine treatment substantially halted dynamics of 5×GFP-EMTB speckles along MTs. Our results show that ensconsin is highly dynamic in its association with microtubules, and its microtubule association can be altered by in vivo phosphorylation events.
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Cheng, Vicki A., and Lynn M. Walker. "Transport of nanoparticulate material in self-assembled block copolymer micelle solutions and crystals." Faraday Discussions 186 (2016): 435–54. http://dx.doi.org/10.1039/c5fd00122f.
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Water soluble poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) [PEO–PPO–PEO] triblock copolymers self-assemble into thermoreversible micellar crystals comprised of periodically spaced micelles. The micelles have PPO cores surrounded by hydrated PEO coronas and the dimensions of the unit cell of the organized micelles is on the order of several to tens of nanometers. Fluorescence recovery after photobleaching (FRAP) is used to quantify nanoparticle transport in these nanostructured polymer micelle systems. Diffusivity of bovine serum albumin (BSA, Dh ∼ 7 nm) is quantified across a wide range of polymer, or micelle, concentrations covering both the disordered fluid as well as the structured micellar crystal to understand the effects of nanoscale structure on particle transport. Measured particle diffusivity in these micellar systems is reduced by as much as four orders of magnitude when compared to diffusivity in free solution. Diffusivity in the disordered micellar fluid is best understood in terms of diffusion through a polymeric solution, while transport in the structured micellar phase is possibly due to hopping between interstitial sites. These results not only show that the nanoscale structures of the micelles have a measureable impact on particle diffusivity, but also demonstrate the ability to tune nanoscale transport in self-assembled materials.
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Finnegan, Catherine M., Satinder S. Rawat, Edward H. Cho, Danielle L. Guiffre, Stephen Lockett, Alfred H. Merrill, and Robert Blumenthal. "Sphingomyelinase Restricts the Lateral Diffusion of CD4 and Inhibits Human Immunodeficiency Virus Fusion." Journal of Virology 81, no. 10 (March 7, 2007): 5294–304. http://dx.doi.org/10.1128/jvi.02553-06.
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ABSTRACT Previously, we reported that treatment of cells with sphingomyelinase inhibits human immunodeficiency virus type 1 (HIV-1) entry. Here, we determined by measuring fluorescence recovery after photobleaching that the lateral diffusion of CD4 decreased 4-fold following sphingomyelinase treatment, while the effective diffusion rate of CCR5 remained unchanged. Notably, sphingomyelinase treatment of cells did not influence gp120 binding, HIV-1 attachment, or fluid-phase and receptor-mediated endocytosis. Furthermore, sphingomyelinase treatment did not affect the membrane disposition of the HIV receptor proteins CD4, CXCR4, and CCR5, as determined by Triton X-100 extraction. Restriction of CD4 diffusion by antibody cross-linking also inhibited HIV infection. We therefore interpret the decrease in CD4 lateral mobility following sphingomyelinase treatment in terms of clustering of CD4 molecules. Examination of fusion intermediates indicated that sphingomyelinase treatment inhibited HIV at a step in the fusion process after CD4 engagement. Maximal inhibition of fusion was observed following short coculture times and with target cells that express low levels of CD4. As HIV entry into cells requires the sequential engagement of viral envelope protein with CD4 and coreceptor, we propose that sphingomyelinase inhibits HIV infection by inducing CD4 clustering that prevents coreceptor engagement and HIV fusion.
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Roettger, B. F., R. U. Rentsch, E. M. Hadac, E. H. Hellen, T. P. Burghardt, and L. J. Miller. "Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell." Journal of Cell Biology 130, no. 3 (August 1, 1995): 579–90. http://dx.doi.org/10.1083/jcb.130.3.579.
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Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell could be particularly effective to protect the cell from processes which might initiate pancreatitis, while providing for the rapid resensitization of this receptor to ensure appropriate pancreatic secretion to aid in nutrient assimilation for the organism.
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Pauls, Samantha, Arnab Ray, and Aaron Marshall. "Control of the molecular behavior of SHIP by FcγRIIB and Nck (P1122)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 64.8. http://dx.doi.org/10.4049/jimmunol.190.supp.64.8.
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Abstract Leukocyte activity is controlled by balancing activating and inhibitory signals in order to generate effective immune responses while avoiding tissue damage and autoimmunity. This project examines an important regulatory circuit in B cells that involves the lipid phosphatase SHIP. Localization, phosphorylation, and binding to interaction partners are important factors in the regulation of this protein, however knowledge of their inter-relationships is incomplete. Here, we examine the influence of two interaction partners on the molecular behaviour and function of SHIP. First, we examine in detail how the inhibitory receptor FcγRIIB controls SHIP localization dynamics. We have observed that co-engagement of FcγRIIB along with the B cell receptor in A20 cells does not influence the magnitude or kinetics of SHIP-EGFP recruitment to the membrane as assessed by confocal microscopy, however it does alter mobility at the cell periphery as measured by fluorescence recovery after photobleaching. Next, we probe the influence of a novel binding partner, the adaptor protein Nck. We have demonstrated an interaction between SHIP and Nck by both Biacore affinity analysis and pull-down assays. Functional relevance will be addressed through mutagenesis experiments. With these aims we hope to enhance our understanding of an interaction that is already recognized as relevant and investigate the function of a novel interaction that occurs at a previously uncharacterized regulatory site.
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Levitus, Marcia, and Suman Ranjit. "Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments." Quarterly Reviews of Biophysics 44, no. 1 (November 26, 2010): 123–51. http://dx.doi.org/10.1017/s0033583510000247.
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AbstractThe breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis–trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.
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Popescu, Narcis I., Cristina Lupu, and Florea Lupu. "Calcium Ionophore-Induced Tissue Factor (TF) Decryption Induces TF Immobilization Into Lipid Rafts and Negative Regulation of TF Procoagulant Activity." Blood 116, no. 21 (November 19, 2010): 1131. http://dx.doi.org/10.1182/blood.v116.21.1131.1131.
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Abstract Abstract 1131 Cell exposed tissue factor (TF), the physiologic initiator of blood coagulation, is normally expressed in a low procoagulant, or cryptic conformation, and requires activation, or decryption, to fully exhibit its procoagulant potential. TF decryption is not fully understood and multiple decrypting mechanisms have been proposed including phosphatidylserine (PS) exposure, TF monomerization, association with lipid rafts and redox modulation of TF. Calcium ionophores have been extensively used as TF decrypting agents, and both PS-dependent and independent mechanisms have been associated with ionophore-induced TF decryption. In the present study we analyzed the changes that occur in the lateral mobility of cell exposed TF during calcium ionophore-induced decryption, using a TF chimera with monomeric yellow fluorescent protein (YFP-TF). The YFP-TF expressed by endothelial cells (EC) retains TF procoagulant activity, is mainly exposed on the cell surface and can be decrypted similarly with endogenous TF by the calcium ionophore ionomycin. We analyzed the changes in TF membrane mobility during decryption using live cell imaging of YFP-TF expressed in EC. Fluorescence recovery after photobleaching (FRAP) analysis revealed a decreased mobility of TF in EC treated with the decrypting agent ionomycin. The YFP-TF fluorescence in the region of interest was more easily bleached in ionomycin–treated cells as compared with controls. The observed maximum recovery (Rmax) of YFP-TF fluorescence in the bleached region of interest was significantly higher in control cells (80.84% recovery) as compared with ionomycin treated EC (39.29% recovery). These correlated with a decrease in YFP-TF mobile fraction from 50% for the control cells to 18% for the ionomycin treated EC. The lateral diffusion of the YFP-TF mobile fraction was similar between the two conditions, with halftime of fluorescence recovery of 7.69 sec in ionophore-treated cells and 10.69 sec in controls. These results suggest an immobilization of YFP-TF during decryption, which can be achieved by either lipid raft translocation or cytoskeleton floating. Similar to previous observations where TF cytoplasmic domain did not influence TF decryption, deletion of the TF cytoplasmic domain did not affect the lateral mobility of YFP-TF in FRAP analysis. To analyze decryption-induced changes in TF association with lipid domains, membrane fractions were isolated on a discontinuous Opti-Prep density gradient. Ionomycin treatment induced YFP-TF translocation from higher density, non-raft membrane fractions toward higher-buoyancy, raft fractions. Furthermore, the observed TF translocation into lipid rafts occurs without the formation of the quaternary complex with coagulation factors FVIIa, FXa and tissue factor pathway inhibitor (TFPI), as previously described. To address the functional modulation of TF procoagulant potential in response to lipid raft translocation, cell membrane cholesterol was either depleted with methyl-β-cyclodextrine (MβCD) or supplemented from an aqueous mixture of cholesterol-MβCD. Membrane cholesterol depletion decrypted TF in EC, likely through PS exposure, while also enhancing the procoagulant potential of ionomycin-decrypted TF. In contrast, cholesterol supplementation decreases the procoagulant potential of ionomycin-decrypted TF. Taken together, these observations support the model of tonic inhibition of TF procoagulant activity by the lipid raft environment. In conclusion, by live cell imaging we show that TF membrane mobility changes during calcium-ionophore induced decryption resulting in an immobilization of TF in lipid rafts. The immobilization is not influenced by the cytoplasmic domain of TF and does not require the formation of the TF-FVIIa-FXa-TFPI quaternary complex. Translocation into lipid rafts provides tonic inhibition of TF procoagulant potential and, as a consequence, we show for the first time that decrypting agents can also initiate negative regulation of TF procoagulant function. This negative feedback loop may help convert the decrypted TF back to its cryptic, low coagulant form. Disclosures: No relevant conflicts of interest to declare.
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Lin, Hongkun, Xiaoping Guo, Jingjing Liu, Peiyi Liu, Guibin Mei, Hongxia Li, Dan Li, et al. "Improving Lipophagy by Restoring Rab7 Cycle: Protective Effects of Quercetin on Ethanol-Induced Liver Steatosis." Nutrients 14, no. 3 (February 4, 2022): 658. http://dx.doi.org/10.3390/nu14030658.
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Chronic alcohol consumption retards lipophagy, which contributes to the pathogenesis of liver steatosis. Lipophagy-related Rab7 has been presumed as a crucial regulator in the progression of alcohol liver disease despite elusive mechanisms. More importantly, whether or not hepatoprotective quercetin targets Rab7-associated lipophagy disorder is unknown. Herein, alcoholic fatty liver induced by chronic-plus-single-binge ethanol feeding to male C57BL/6J mice was manifested by hampering autophagosomes formation with lipid droplets and fusion with lysosomes compared with the normal control, which was normalized partially by quercetin. The GST-RILP pulldown assay of Rab7 indicated an improved GTP-Rab7 as the quercetin treatment for ethanol-feeding mice. HepG2 cells transfected with CYP2E1 showed similar lipophagy dysfunction when exposed to ethanol, which was blocked when cells were transfected with siRNA-Rab7 in advance. Ethanol-induced steatosis and autophagic flux disruption were aggravated by the Rab7-specific inhibitor CID1067700 while alleviated by transfecting with the Rab7Wt plasmid, which was visualized by immunofluorescence co-localization analysis and mCherry-GFP-LC3 transfection. Furthermore, TBC1D5, a Rab GTPase-activating protein for the subsequent normal circulation of Rab7, was downregulated after alcohol administration but regained by quercetin. Rab7 circulation retarded by ethanol and corrected by quercetin was further revealed by fluorescence recovery after photobleaching (FRAP). Altogether, quercetin attenuates hepatic steatosis by normalizing ethanol-imposed Rab7 turnover disorders and subsequent lipophagy disturbances, highlighting a novel mechanism and the promising prospect of quercetin-like phytochemicals against the crucial first hit from alcohol.
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Tole, Soumitra, Anne M. Durkan, Yi-Wei Huang, Guang Ying Liu, Alexander Leung, Laura L. Jones, Jasmine A. Taylor, and Lisa A. Robinson. "Thromboxane prostanoid receptor stimulation induces shedding of the transmembrane chemokine CX3CL1 yet enhances CX3CL1-dependent leukocyte adhesion." American Journal of Physiology-Cell Physiology 298, no. 6 (June 2010): C1469—C1480. http://dx.doi.org/10.1152/ajpcell.00380.2009.
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In atherosclerosis, chemokines recruit circulating mononuclear leukocytes to the vascular wall. A key factor is CX3CL1, a chemokine with soluble and transmembrane species that acts as both a chemoattractant and an adhesion molecule. Thromboxane A2 and its receptor, TP, are also critical to atherogenesis by promoting vascular inflammation and consequent leukocyte recruitment. We examined the effects of TP stimulation on processing and function of CX3CL1, using CX3CL1-expressing human ECV-304 cells and primary human vascular endothelial cells. TP agonists promoted rapid shedding of cell surface CX3CL1, which was inhibited by pharmacological inhibitors or specific small interfering RNA targeting tumor necrosis factor-α-converting enzyme (TACE). Because it reduced cell surface CX3CL1, we predicted that TP stimulation would inhibit adhesion of leukocytes expressing the CX3CL1 cognate receptor but, paradoxically, saw enhanced adhesion. We questioned whether the enhanced ability of the remaining membrane-associated CX3CL1 to bind targets was caused by changes in its lateral mobility. Using fluorescence recovery after photobleaching, we found that plasmalemmal CX3CL1 was initially tethered but ultimately mobilized by TP agonists. TP stimulation provoked clustering of transmembrane CX3CL1 at sites of contact with adherent leukocytes. These data demonstrate that TP stimulation induces two distinct effects: a rapid cleavage of surface CX3CL1, thereby releasing the soluble chemoattractant, plus mobilization of the remaining transmembrane CX3CL1 to enhance the avidity of interactions with adherent leukocytes. The dual effect of TP allows CX3CL1 to recruit leukocytes to sites of vascular inflammation while enhancing their adhesion once recruited.
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García-Martínez, S., R. Latorre, M. A. Sánchez-Hurtado, F. M. Sánchez-Margallo, N. Bernabò, R. Romar, O. López-Albors, and P. Coy. "Mimicking the temperature gradient between the sow’s oviduct and uterus improves in vitro embryo culture output." Molecular Human Reproduction 26, no. 10 (July 9, 2020): 748–59. http://dx.doi.org/10.1093/molehr/gaaa053.
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Abstract This work was designed to determine temperature conditions within the reproductive tract of the female pig and study their impact on ARTs. Temperatures were recorded using a laparo-endoscopic single-site surgery assisted approach and a miniaturized probe. Sows and gilts were used to address natural cycle and ovarian stimulation treatments, respectively. According to in vivo values, IVF was performed at three temperature conditions (37.0°C, 38.5°C and 39.5°C) and presumptive zygotes were cultured in these conditions for 20 h, while further embryo culture (EC) (21–168 h post-insemination) was maintained at 38.5°C. After 20 h, different fertility parameters were assessed. During EC, cleavage and blastocyst stages were evaluated. Sperm membrane fluidity at the experimental temperatures was studied by using differential scanning calorimetry and fluorescence recovery after photobleaching techniques. An increasing temperature gradient of 1.5°C was found between the oviduct and uterus of sows (P < 0.05) and when this gradient was transferred to pig in vitro culture, the number of poly-nuclear zygotes after IVF was reduced and the percentage of blastocysts was increased. Moreover, the temperature transition phase for the boar sperm membrane (37.0°C) coincided with the temperature registered in the sow oviduct, and sperm membranes were more fluid at 37.0°C compared with those of sperm incubated at higher temperatures (38.5°C and 39.5°C). These data suggest that there may be an impact of physiological temperature gradients on human embryo development.
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Kong, Cherrie H. T., Eva A. Rog-Zielinska, Peter Kohl, Clive H. Orchard, and Mark B. Cannell. "Solute movement in the t-tubule system of rabbit and mouse cardiomyocytes." Proceedings of the National Academy of Sciences 115, no. 30 (July 10, 2018): E7073—E7080. http://dx.doi.org/10.1073/pnas.1805979115.
Full textAbstract:
Cardiac transverse (t-) tubules carry both electrical excitation and solutes toward the cell center but their ability to transport small molecules is unclear. While fluorescence recovery after photobleaching (FRAP) can provide an approach to measure local solute movement, extraction of diffusion coefficients is confounded by cell and illumination beam geometries. In this study, we use measured cellular geometry and detailed computer modeling to derive the apparent diffusion coefficient of a 1-kDa solute inside the t-tubular system of rabbit and mouse ventricular cardiomyocytes. This approach shows that diffusion within individual t-tubules is more rapid than previously reported. T-tubule tortuosity, varicosities, and the presence of longitudinal elements combine to substantially reduce the apparent rate of solute movement. In steady state, large (>4 kDa) solutes did not freely fill the t-tubule lumen of both species and <50% of the t-tubule volume was available to solutes >70 kDa. Detailed model fitting of FRAP data suggests that solute diffusion is additionally restricted at the t-tubular entrance and this effect was larger in mouse than in rabbit. The possible structural basis of this effect was investigated using electron microscopy and tomography. Near the cell surface, mouse t-tubules are more tortuous and filled with an electron-dense ground substance, previously identified as glycocalyx and a polyanionic mesh. Solute movement in the t-tubule network of rabbit and mouse appears to be explained by their different geometric properties, which impacts the use of these species for understanding t-tubule function and the consequences of changes associated with t-tubule disease.
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Wist, Martin, Laura Meier, Orit Gutman, Jennifer Haas, Sascha Endres, Yuan Zhou, Reinhild Rösler, et al. "Noncatalytic Bruton's tyrosine kinase activates PLCγ2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells." Journal of Biological Chemistry 295, no. 17 (March 17, 2020): 5717–36. http://dx.doi.org/10.1074/jbc.ra119.011946.
Full textAbstract:
Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.
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Foeglein, Ágnes, Eva M. Loucaides, Manuela Mura, Helen M. Wise, Wendy S. Barclay, and Paul Digard. "Influence of PB2 host-range determinants on the intranuclear mobility of the influenza A virus polymerase." Journal of General Virology 92, no. 7 (July 1, 2011): 1650–61. http://dx.doi.org/10.1099/vir.0.031492-0.
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Avian influenza A viruses often do not propagate efficiently in mammalian cells. The viral polymerase protein PB2 is important for this host restriction, with amino-acid polymorphisms at residue 627 and other positions acting as ‘signatures’ of avian- or human-adapted viruses. Restriction is hypothesized to result from differential interactions (either positive or inhibitory) with unidentified cellular factors. We applied fluorescence recovery after photobleaching (FRAP) to investigate the mobility of the viral polymerase in the cell nucleus using A/PR/8/34 and A/Turkey/England/50-92/91 as model strains. As expected, transcriptional activity of a polymerase with the avian PB2 protein was strongly dependent on the identity of residue 627 in human but not avian cells, and this correlated with significantly slower diffusion of the inactive polymerase in human but not avian nuclei. In contrast, the activity and mobility of the PR8 polymerase was affected much less by residue 627. Sequence comparison followed by mutagenic analyses identified residues at known host-range-specific positions 271, 588 and 701 as well as a novel determinant at position 636 as contributors to host-specific activity of both PR8 and Turkey PB2 proteins. Furthermore, the correlation between poor transcriptional activity and slow diffusional mobility was maintained. However, activity did not obligatorily correlate with predicted surface charge of the 627 domain. Overall, our data support the hypothesis of a host nuclear factor that interacts with the viral polymerase and modulates its activity. While we cannot distinguish between positive and inhibitory effects, the data have implications for how such factors might operate.
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Dale, R. E. "Depolarized fluorescence photobleaching recovery." European Biophysics Journal 14, no. 3 (January 1987): 179–93. http://dx.doi.org/10.1007/bf00253843.
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Burrows, Sarah, Janet Patterson-Kane, David Becker, and Roland Fleck. "Fluorescence Recovery After Photobleaching." Imaging & Microscopy 8, no. 4 (November 2006): 62–64. http://dx.doi.org/10.1002/imic.200790074.
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Wang, Lili, Alice Sandmeyer, Wolfgang Hübner, Hongru Li, Thomas Huser, and Benjamin K. Chen. "A Replication-Competent HIV Clone Carrying GFP-Env Reveals Rapid Env Recycling at the HIV-1 T Cell Virological Synapse." Viruses 14, no. 1 (December 25, 2021): 38. http://dx.doi.org/10.3390/v14010038.
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HIV-1 infection is enhanced by cell–cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Combined use of surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, enabled the visualization of the targeted accumulation and sustained recycling of Env between endocytic compartments (EC) and the VS. We observed dynamic exchange of Env at the VS, while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse support that the trafficking and/or retention of a majority of Env towards the VS is not maintained by entrapment by a Gag lattice or immobilization by binding to CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is not required for accumulation at the VS, but is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus, but that this recycling is largely dispensable for VS formation and viral transfer across the VS.
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McSpadden, Luke C., Robert D. Kirkton, and Nenad Bursac. "Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level." American Journal of Physiology-Cell Physiology 297, no. 2 (August 2009): C339—C351. http://dx.doi.org/10.1152/ajpcell.00024.2009.
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Understanding how electrotonic loading of cardiomyocytes by unexcitable cells alters cardiac impulse conduction may be highly relevant to fibrotic heart disease. In this study, we optically mapped electrical propagation in confluent, aligned neonatal rat cardiac monolayers electrotonically loaded with cardiac fibroblasts, control human embryonic kidney (HEK-293) cells, or HEK-293 cells genetically engineered to overexpress the gap junction proteins connexin-43 or connexin-45. Gap junction expression and function were assessed by immunostaining, immunoblotting, and fluorescence recovery after photobleaching and were correlated with the optically mapped propagation of action potentials. We found that neonatal rat ventricular fibroblasts negative for the myofibroblast marker smooth muscle α-actin expressed connexin-45 rather than connexin-43 or connexin-40, weakly coupled to cardiomyocytes, and, without significant depolarization of cardiac resting potential, slowed cardiac conduction to 75% of control only at high (>60%) coverage densities, similar to loading effects found from HEK-293 cells expressing similar levels of connexin-45. In contrast, HEK-293 cells with connexin-43 expression similar to that of cardiomyocytes significantly decreased cardiac conduction velocity and maximum capture rate to as low as 22% and 25% of control values, respectively, while increasing cardiac action potential duration to 212% of control and cardiac resting potential from −71.6 ± 4.9 mV in controls to −65.0 ± 3.8 mV. For all unexcitable cell types and coverage densities, velocity anisotropy ratio remained unchanged. Despite the induced conduction slowing, none of the loading cell types increased the proportion of spontaneously active monolayers. These results signify connexin isoform and expression level as important contributors to potential electrical interactions between unexcitable cells and myocytes in cardiac tissue.
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Tweardy, David John, and Ying Huang. "Stat3 Isoforms, α and β, Demonstrate Distinct Intracellular Dynamics: Prolonged Nuclear Retention of Stat3β Maps to Its Unique C-Terminal End." Blood 108, no. 11 (November 16, 2006): 4348. http://dx.doi.org/10.1182/blood.v108.11.4348.4348.
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Abstract Two isoforms of signal transducer and activator of transcription (STAT) 3 are expressed in all cells—alpha] (p92) and β (p83)—both derived from a single gene by alternative mRNA splicing. Structurally, Stat3α contains a 55-residue C-terminal transactivation domain (TAD), which is deleted in Stat3β and replaced by 7 unique C-terminal residues (CT7) whose function remains uncertain. Functionally, Stat3α is transcriptionally active while Stat3β is not despite binding DNA more tightly than Stat3α. Stat3α is an oncogene activated in many cancers including multiple myeloma, acute myelogenous leukemia and lymphoma, while Stat3β appears to antagonize the oncogenic functions of Stat3α. To gain additional insight into the distinct biological and oncogenic functions of Stat3α and β at the single-cell level and, in particular, to determine the function of the unique CT7 domain of Stat3β, we subcloned the open reading frames of Stat3α, Stat3β and Stat3β missing the CT7 domain (ΔStat3β) into the C-terminus of GFP. Immunoblot analysis and DNA binding studies demonstrated that each GFP-tagged construct encoded proteins of the expected size that bound DNA. Similar to their non-tagged counterparts, GFP-Stat3α, but neither GFP-Stat3β nor GFP-ΔStat3β, activated a reporter construct containing acute phase response elements. Each GFP-Stat3 construct was stably expressed in murine embryonic fibroblasts (MEF) in which Stat3 was deleted previously using Cre/lox technology. Following selection, clones were isolated and sorted to ensure levels of expression of each GFP-Stat3 construct similar to levels found in wild type MEF cells. Fluorescence microscopic analysis of unstimulated MEF/α, MEF/β and MEF/Δβ cells revealed predominantly diffuse cytoplasmic localization of each GFP-tagged protein. Stimulation of cells with IL-6 (200 ng/ml) and soluble IL-6 receptor (sIL-6R, 250 ng/ml) revealed similar kinetics of cytoplasmic-to-nuclear translocation for each GFP-Stat3 construct with translocation starting at 10 min and becoming maximal at 30 min. High throughput microscopy analysis of 1,000 or more cells following removal of IL-6/sIL-6R revealed a nuclear-to-cytoplasmic translocation half life of 15 min for GFP-Stat3α and >3hr for GFP-Stat3β. In contrast to GFP-Stat3β, GFP-ΔStat3β had a nuclear-to-cytoplasmic translocation half life of <30 min similar to GFP-Stat3α indicating that the CT7 domain was responsible for prolonged nuclear retention of GFP-Stat3β. The intranuclear mobility of the GFP-Stat3 constructs was determined by fluorescence recovery after photobleaching (FRAP). The mobility of GFP-Stat3α assessed by fluorescence recovery half-time (t1/2) increased 33% with IL-6/sIL-6R stimulation (t1/2=1.07 ± 0.58 s before stimulation; t1/2=0.72 ± 0.45 s after stimulation; p<0.05). The mobility of GFP-Stat3β in unstimulated cells (1.89 ± 0.51 s) was 77% slower than GFP-Stat3α without stimulation (p<0.05) and was slowed 52% further following IL-6/sIL-6R simulation (2.88 ± 0.60 s; p<0.05). While deletion of the unique CT7 domain from Stat3β eliminated prolonged nuclear retention, it did not substantially reduce its intranuclear mobility (1.48 ± 0.47 s without stimulation; 2.63 ± 0.65 s following stimulation). Thus, Stat3α and Stat3β have distinct intracellular dynamics with Stat3β exhibiting prolonged nuclear retention and reduced intranuclear mobility both without and with stimulation; prolonged nuclear retention, but not reduced intranuclear mobility, map to the CT7 domain of Stat3β.
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Elson, E. L. "Fluorescence Correlation Spectroscopy and Photobleaching Recovery." Annual Review of Physical Chemistry 36, no. 1 (October 1985): 379–406. http://dx.doi.org/10.1146/annurev.pc.36.100185.002115.
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Fire, E., D. E. Zwart, M. G. Roth, and Y. I. Henis. "Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits." Journal of Cell Biology 115, no. 6 (December 15, 1991): 1585–94. http://dx.doi.org/10.1083/jcb.115.6.1585.
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Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.
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Ljungquist-Höddelius, Pia, Margareta Lirvall, Åke Wasteson, and Karl-Eric Magnusson. "Lateral diffusion of PDGF β-receptors in human fibroblasts." Bioscience Reports 11, no. 1 (February 1, 1991): 43–52. http://dx.doi.org/10.1007/bf01118604.
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When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum or PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding “normal” and “starved” cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0×10−10 cm2s−1 and >90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor, and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.
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Segal, G., W. Lee, P. D. Arora, M. McKee, G. Downey, and C. A. McCulloch. "Involvement of actin filaments and integrins in the binding step in collagen phagocytosis by human fibroblasts." Journal of Cell Science 114, no. 1 (January 1, 2001): 119–29. http://dx.doi.org/10.1242/jcs.114.1.119.
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In physiological conditions, collagen degradation by fibroblasts occurs primarily via phagocytosis, an intracellular pathway that is thought to require collagen receptors and actin assembly for fibril internalization and degradation. Currently it is unclear which specific steps of collagen phagocytosis in fibroblasts involve actin filament assembly. As studies of phagocytosis in fibroblasts are complicated by the relatively slow rate of particle internalization compared to professional phagocytes, we have examined the role of collagen receptors and actin only in the initial collagen binding step. Prior to the binding of collagen-coated fluorescent beads by human gingival fibroblasts, a cell type that is avidly phagocytic in vitro, cells were treated with cytochalasin D (actin filament barbed-end capping) or swinholide A (actin dimer sequestering and severing) or latrunculin B (actin monomer sequestering). Bead binding and immunostaining of (alpha)(2)(beta)(1) and (alpha)(3)(beta)(1) integrin collagen receptors were measured by flow cytometry. After 1–3 hours of coincubation with beads, cytochalasin D or swinholide A eliminated actin filaments stained by rhodamine-phalloidin and inhibited collagen bead binding (reductions of 25% and 50%, respectively), possibly because of cell rounding and restricted interactions with beads. In contrast, latrunculin enhanced binding dose-dependently over controls (twofold at 1 microM) and induced the formation of brightly staining aggregates of actin and the retention of long cytoplasmic extensions. Latrunculin also reduced surface (beta)(1), (alpha)(2) and (alpha)(3) integrin staining up to 40% in bead-free and bead-loaded cells, indicating that latrunculin enhanced collagen receptor internalization. As determined by fluorescence recovery after photobleaching, latrunculin increased the mobility of surface-bound (beta)(1) integrin. The stimulatory effect of latrunculin on collagen bead binding was reduced to control levels by treatment with a (beta)(1) integrin inactivating antibody while a (beta)(1) integrin blocking antibody abrogated both bead binding and the latrunculin-induced stimulation. Immunoblotting of bead-associated proteins showed that latrunculin completely eliminated binding of (beta)-actin to collagen beads but did not affect (beta)(1) integrin binding. These data indicate that latrunculin-induced sequestration of actin monomers facilitates the disengagement of actin from (beta)(1) integrin receptors, increases collagen bead binding and enhances collagen receptor mobility. We suggest that these alterations increase the probability of adhesive bead-to-cell interactions.
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Liu, Zhao, Keiko Ueda, Hye Jin Kim, and Janet R. Sparrow. "Photobleaching and Fluorescence Recovery of RPE Bisretinoids." PLOS ONE 10, no. 9 (September 14, 2015): e0138081. http://dx.doi.org/10.1371/journal.pone.0138081.
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Cadar, Adrian G., Tromondae K. Feaster, Kevin R. Bersell, Lili Wang, TingTing Hong, Joseph A. Balsamo, Zhentao Zhang, et al. "Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes." American Journal of Physiology-Cell Physiology 318, no. 1 (January 1, 2020): C163—C173. http://dx.doi.org/10.1152/ajpcell.00107.2019.
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Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.
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Srikantha, Nishanthan, Yurema Teijeiro-Gonzalez, Andrew Simpson, Naba Elsaid, Satyanarayana Somavarapu, Klaus Suhling, and Timothy L. Jackson. "Determining vitreous viscosity using fluorescence recovery after photobleaching." PLOS ONE 17, no. 2 (February 10, 2022): e0261925. http://dx.doi.org/10.1371/journal.pone.0261925.
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Purpose Vitreous humor is a complex biofluid whose composition determines its structure and function. Vitreous viscosity will affect the delivery, distribution, and half-life of intraocular drugs, and key physiological molecules. The central pig vitreous is thought to closely match human vitreous viscosity. Diffusion is inversely related to viscosity, and diffusion is of fundamental importance for all biochemical reactions. Fluorescence Recovery After Photobleaching (FRAP) may provide a novel means of measuring intravitreal diffusion that could be applied to drugs and physiological macromolecules. It would also provide information about vitreous viscosity, which is relevant to drug elimination, and delivery. Methods Vitreous viscosity and intravitreal macromolecular diffusion of fluorescently labelled macromolecules were investigated in porcine eyes using fluorescence recovery after photobleaching (FRAP). Fluorescein isothiocyanate conjugated (FITC) dextrans and ficolls of varying molecular weights (MWs), and FITC-bovine serum albumin (BSA) were employed using FRAP bleach areas of different diameters. Results The mean (±standard deviation) viscosity of porcine vitreous using dextran, ficoll and BSA were 3.54 ± 1.40, 2.86 ± 1.13 and 4.54 ± 0.13 cP respectively, with an average of 3.65 ± 0.60 cP. Conclusions FRAP is a feasible and practical optical method to quantify the diffusion of macromolecules through vitreous.
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Sullivan, Kelley D., Ania K. Majewska, and Edward B. Brown. "Single- and Two-Photon Fluorescence Recovery after Photobleaching." Cold Spring Harbor Protocols 2015, no. 1 (January 2015): pdb.top083519. http://dx.doi.org/10.1101/pdb.top083519.
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Kovaleski, John M., and Mary J. Wirth. "Peer Reviewed: Applications of Fluorescence Recovery after Photobleaching." Analytical Chemistry 69, no. 19 (October 1997): 600A—605A. http://dx.doi.org/10.1021/ac971792r.
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