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1
Volkov, I. A., N. V. Frigo, L. F. Znamenskaya, and O. R. Katunina. "Application of Confocal Laser Scanning Microscopy in Biology and Medicine." Vestnik dermatologii i venerologii 90, no. 1 (February 24, 2014): 17–24. http://dx.doi.org/10.25208/0042-4609-2014-90-1-17-24.
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Fluorescence confocal laser scanning microscopy and reflectance confocal laser scanning microscopy are up-to-date highend study methods. Confocal microscopy is used in cell biology and medicine. By using confocal microscopy, it is possible to study bioplasts and localization of protein molecules and other compounds relative to cell or tissue structures, and to monitor dynamic cell processes. Confocal microscopes enable layer-by-layer scanning of test items to create demonstrable 3D models. As compared to usual fluorescent microscopes, confocal microscopes are characterized by a higher contrast ratio and image definition.
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Eggeling, Christian. "Super-resolution optical microscopy of lipid plasma membrane dynamics." Essays in Biochemistry 57 (February 6, 2015): 69–80. http://dx.doi.org/10.1042/bse0570069.
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Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.
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WANG, XIAO-PING, HUAI-NA YU, and TONG-SHENG CHEN. "QUANTITATIVE FRET MEASUREMENT BASED ON CONFOCAL MICROSCOPY IMAGING AND PARTIAL ACCEPTOR PHOTOBLEACHING." Journal of Innovative Optical Health Sciences 05, no. 03 (July 2012): 1250015. http://dx.doi.org/10.1142/s1793545812500150.
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Fluorescence resonance energy transfer (FRET) technology had been widely used to study protein–protein interactions in living cells. In this study, we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest (ROI) function of confocal microscope and partial acceptor photobleaching. We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3, and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine (STS)-induced apoptosis. Our results for the first demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.
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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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Yang, Kun, Shu Bai, and Yan Sun. "Protein adsorption dynamics in cation-exchange chromatography quantitatively studied by confocal laser scanning microscopy." Chemical Engineering Science 63, no. 16 (August 2008): 4045–54. http://dx.doi.org/10.1016/j.ces.2008.05.013.
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Olmsted, J. B., K. R. Olson, M. L. Gonzalez-Garay, and F. Cabral. "Green fluorescent protein: Use of GFP-chimeras in the analysis of microtubule-associated protein 4 domains and microtubule dynamics." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 888–89. http://dx.doi.org/10.1017/s0424820100166907.
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Green Fluorescent Protein (GFP) is an endogenous 27 kDa fluorophore of the jellyfish, Aequorea victoria. Chalfie et al., first described the exogenous expression of this molecule in bacteria, and its utility as a reporter in higher eukaryotes. Potential applications of GFP have been expanded through the construction of variants with enhanced brightness and/or different spectral properties.We have explored using GFP for the analysis of the real-time behaviors of microtubules and their associated proteins. Constructs of microtubule-associated protein 4 (MAP 4) or β-tubulin were generated in pRC/CMV vectors and used in either transient or stable transfection assays in a variety of cultured cell lines (3T3, PtKl, BHK, CHO, Cos). The GFP-chimeras were visualized using conventional fluorescence microscopy and confocal laser scanning microscopy. Unusual features of the GFP reporter are that fluorescence intensity increased 2-10 fold upon illumination, and that phototoxicity was low.
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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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Chiodi, I., M. Biggiogera, M. Denegri, M. Corioni, F. Weighardt, F. Cobianchi, S. Riva, and G. Biamonti. "Structure and dynamics of hnRNP-labelled nuclear bodies induced by stress treatments." Journal of Cell Science 113, no. 22 (November 15, 2000): 4043–53. http://dx.doi.org/10.1242/jcs.113.22.4043.
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We have previously described HAP, a novel hnRNP protein that is identical both to SAF-B, a component of the nuclear scaffold, and to HET, a transcriptional regulator of the gene for heat shock protein 27. After heat shock, HAP is recruited to a few nuclear bodies. Here we report the characterisation of these bodies, which are distinct from other nuclear components such as coiled bodies and speckles. The formation of HAP bodies is part of a general cell response to stress agents, such as heat shock and cadmium sulfate, which also affect the distribution of hnRNP protein M. Electron microscopy demonstrates that in untreated cells, similar to other hnRNP proteins, HAP is associated to perichromatin fibrils. Instead, in heat shocked cells the protein is preferentially associated to clusters of perichromatin granules, which correspond to the HAP bodies observed in confocal microscopy. Inside such clusters, perichromatin granules eventually merge into a highly packaged ‘core’. HAP and hnRNP M mark different districts of these structures. HAP is associated to perichromatin granules surrounding the core, while hnRNP M is mostly detected within the core. BrU incorporation experiments demonstrate that no transcription occurs within the stress-induced clusters of perichromatin granules, which are depots for RNAs synthesised both before and after heat shock.
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Aymerich, María S., J. López-Azcárate, J. Bonaventura, G. Navarro, D. Fernández-Suárez, V. Casadó, F. Mayor, et al. "Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics." Scientific World JOURNAL 11 (2011): 1995–2010. http://dx.doi.org/10.1100/2011/690858.
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Understanding the trafficking of G-protein-coupled receptors (GPCRs) and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 μm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.
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Wilkins, Ngozi A., Brian Storrie, and Jeffrey A. Kamykowski. "Characterization of Platelet Alpha-Granule Dynamics." Blood 116, no. 21 (November 19, 2010): 327. http://dx.doi.org/10.1182/blood.v116.21.327.327.
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Abstract Abstract 327 Background: Platelets, anucleated cells that play a critical role in blood clotting, store proteins and small molecules in alpha-granules and dense granules, respectively, for secretion. Alpha-granules contain several proteins including von Willebrand factor and fibrinogen and dense granules contain serotonin. Rab4, a marker for the early endosomes has been implicated in regulating alpha granule secretions (Sirakawa et al, 2010). Previous fluorescence microscopy mapping of alpha-granule protein distributions suggested that there are either two different alpha-granule types or subdomains within a single granule population (Storrie and Seghal, 2007; Italiano et al, 2008). More recent work based on electron tomography (Kamykowski et al, manuscript in preparation) indicates that human platelets are comprised of one alpha granule population. We hypothesized that there was a single population of alpha-granules in which all fibrinogen is similarly compartmentalized. Hence, fibrinogen endocytocized by guinea pig megakaryocytes and platelets in vivo at 4 h (short label) and 24 h (long label) would map to the same location. Aims: We carried out several experiments to form a basis for future high-resolution (5 nm) electron tomography to establish packaging of HRP-conjugated fibrinogen or nanogold conjugated fibrinogen into platelet alpha-granules. (a) Using PD-10 columns, we prepared Cy3 conjugated fibrinogen. Using an in vivo guinea pig model to test the ability of guinea pig platelets to take up fluorescently labeled fibrinogen, we injected 10 mg/ml of Cy3 conjugated fibrinogen (short label, 4 h) and 10 mg/ml of commercially purchased AlexaFluor 488 conjugated fibrinogen (long label, 28 h) into guinea pigs. Platelets were then fixed, purified and confocal microscopy performed. (b) Using triple immunofluorescence, serotonin antibody was applied to fixed and purified resting state human and guinea pig platelets and immunofluorescence microscopy was performed to provide whole platelet information on the staining pattern of the dense granules in comparison to the alpha-granules and early endosomes. (c) Preliminary Electron Microscopy fixation conditions were also tested on guinea pig platelets. Results: For the uptake experiment, spinning-disk confocal microscopy was used to collect full platelet volume image stacks which were then deconvolved, pixel shift corrected for red and green channels and analyzed. Overlap of green and red fibrinogen conjugates was observed where the fluorescently tagged fibrinogens were taken up by structures presumed to be alpha-granules. For the triple labeling experiments, the distribution of serotonin, Rab4 and von Willebrand factor was observed in resting state platelets. Using spinning-disk confocal microscopy, full platelet volume image stacks were collected, deconvolved, pixel shift corrected for red, far red and green channels and analyzed. Serotonin antibody gave an abundant punctate staining pattern in both the triple-labeled human and guinea pig platelets. In both the human platelets and the guinea pig platelets, the serotonin positive punctate granules, presumed to be dense granules, had a more similar pattern to the von Willebrand factor positive punctate alpha granules, than to the Rab4 positive punctate granules, presumed to be the early endosomes. The triple label results were unexpected because previous electron microscopy studies have indicated that the dense granules in human platelets are fewer in number than the alpha-granules and fewer than the corresponding dense granules in guinea pig platelets. Results of the electron microscopy preparations are pending. Conclusions: Our results indicate that the guinea pig model, while its platelets are a much smaller size than human platelets, is a good system for loading alpha-granules with labeled proteins for electron tomography. The serotonin distribution results together with previous electron tomography also raise the question as to whether dense granules could be a specialized form of the alpha-granules. A summary of this research will be presented at the Promoting Minorities in Hematology event during the 2010 ASH meeting. Disclosures: No relevant conflicts of interest to declare.
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Laňková, Martina, Jana Humpolíčková, Stanislav Vosolsobě, Zdeněk Cit, Jozef Lacek, Martin Čovan, Milada Čovanová, Martin Hof, and Jan Petrášek. "Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy." Microscopy and Microanalysis 22, no. 2 (March 3, 2016): 290–99. http://dx.doi.org/10.1017/s1431927616000568.
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AbstractA number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.
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Shevchuk, Andrew I., Pavel Novak, Marcus Taylor, Ivan A. Diakonov, Azza Ziyadeh-Isleem, Marc Bitoun, Pascale Guicheney, et al. "An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy." Journal of Cell Biology 197, no. 4 (May 7, 2012): 499–508. http://dx.doi.org/10.1083/jcb.201109130.
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Current knowledge of the structural changes taking place during clathrin-mediated endocytosis is largely based on electron microscopy images of fixed preparations and x-ray crystallography data of purified proteins. In this paper, we describe a study of clathrin-coated pit dynamics in living cells using ion conductance microscopy to directly image the changes in pit shape, combined with simultaneous confocal microscopy to follow molecule-specific fluorescence. We find that 70% of pits closed with the formation of a protrusion that grew on one side of the pit, covered the entire pit, and then disappeared together with pit-associated clathrin–enhanced green fluorescent protein (EGFP) and actin-binding protein–EGFP (Abp1-EGFP) fluorescence. This was in contrast to conventionally closing pits that closed and cleaved from flat membrane sheets and lacked accompanying Abp1-EGFP fluorescence. Scission of both types of pits was found to be dynamin-2 dependent. This technique now enables direct spatial and temporal correlation between functional molecule-specific fluorescence and structural information to follow key biological processes at cell surfaces.
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Corry, Ben, Annette C. Hurst, Prithwish Pal, Takeshi Nomura, Paul Rigby, and Boris Martinac. "An improved open-channel structure of MscL determined from FRET confocal microscopy and simulation." Journal of General Physiology 136, no. 4 (September 27, 2010): 483–94. http://dx.doi.org/10.1085/jgp.200910376.
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Mechanosensitive channels act as molecular transducers of mechanical force exerted on the membrane of living cells by opening in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing, blood pressure regulation, and osmoregulation. Here, we determine the likely structure of the open state of the mechanosensitive channel of large conductance using a combination of patch clamp, fluorescence resonance energy transfer (FRET) spectroscopy, data from previous electron paramagnetic resonance experiments, and molecular and Brownian dynamics simulations. We show that structural rearrangements of the protein can be measured in similar conditions as patch clamp recordings while controlling the state of the pore in its natural lipid environment by modifying the lateral pressure distribution via the lipid bilayer. Transition to the open state is less dramatic than previously proposed, while the N terminus remains anchored at the surface of the membrane where it can either guide the tilt of or directly translate membrane tension to the conformation of the pore-lining helix. Combining FRET data obtained in physiological conditions with simulations is likely to be of great value for studying conformational changes in a range of multimeric membrane proteins.
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Zhao, Han, Liang Chen, Guisheng Zhong, Yina Huang, Xulai Zhang, Cenfeng Chu, Lin Chen, and Ming Wang. "Titanium Dioxide Nanoparticles Induce Mitochondrial Dynamic Imbalance and Damage in HT22 Cells." Journal of Nanomaterials 2019 (April 30, 2019): 1–16. http://dx.doi.org/10.1155/2019/4607531.
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Mitochondria, as dynamic organelles, are precisely regulated by fusion and fission. The dynamic balance of fusion and fission controls mitochondrial morphology and their subcellular location and function. Exposure to titanium dioxide nanoparticles (TiO2 NPs) may cause serious health problems. However, how TiO2 NPs affect the mitochondrial dynamics remains unclear. In the present study, we investigated the changes of mitochondrial dynamics in the TiO2NPs-treated HT22 cells by confocal and stimulated emission depletion (STED) microscopy. The confocal images demonstrated obvious changes in the average length and density of the mitochondria after TiO2 NPs treatment, while STED images further obtained the nanoscale submitochondrial structures of the mitochondria under TiO2 NPs insult. The fluorescence intensity distributions suggested that mitochondria fragmented in the TiO2 NPs-treated cells. TiO2 NPs treatment caused mitochondrial dynamic imbalance due to the imbalanced expression of dynamin-related protein 1 (Drp1) and optic atrophy 1 (Opa1). Furthermore, we examined the levels of oxidative stress and mitochondrial membrane potential (MMP) and the generation of adenosine triphosphate (ATP), which revealed the damage of mitochondria under TiO2 NPs exposure. Meanwhile, the significant changes of expressions of B-cell lymphoma 2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cytochrome c (Cyt C), and caspase 9 demonstrated that TiO2 NPs treatment activated the mitochondrial-related apoptosis pathway. These cellular events can be largely prevented via cell incubation with mitoTEMPO, a mitochondria-targeted superoxide scavenger. Our results confirm that TiO2 NPs targeted the mitochondria, inducing mitochondrial dynamic imbalance and damage in HT22 cells. Our study provides an insightful understanding of the mechanisms underlying TiO2 NPs cytotoxicity.
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Han, Ziying, Gordon Ruthel, Shantoshini Dash, Corbett T. Berry, Bruce D. Freedman, Ronald N. Harty, and Olena Shtanko. "Angiomotin regulates budding and spread of Ebola virus." Journal of Biological Chemistry 295, no. 25 (May 7, 2020): 8596–601. http://dx.doi.org/10.1074/jbc.ac120.013171.
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The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain–bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain–containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.
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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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Tolker-Nielsen, Tim, Ulla C. Brinch, Paula C. Ragas, Jens Bo Andersen, Carsten Suhr Jacobsen, and Søren Molin. "Development and Dynamics of Pseudomonassp. Biofilms." Journal of Bacteriology 182, no. 22 (November 15, 2000): 6482–89. http://dx.doi.org/10.1128/jb.182.22.6482-6489.2000.
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ABSTRACT Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.
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Li, Li, Bodan Su, Xueying Qi, Xi Zhang, Susheng Song, and Xiaoyi Shan. "JA-Induced Endocytosis of AtRGS1 Is Involved in G-Protein Mediated JA Responses." International Journal of Molecular Sciences 20, no. 15 (August 2, 2019): 3779. http://dx.doi.org/10.3390/ijms20153779.
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Arabidopsis heterotrimeric G proteins regulate diverse plant growth and defense processes by coupling to 7TM AtRGS1 proteins. Although G protein mutants display alterations in response to multiple plant hormones, the underlying mechanism by which G proteins participate in the regulation of hormone responses remains elusive. Here, we show that genetic disruption of Gα and Gβ subunits results in reduced sensitivity to JA treatment. Furthermore, using confocal microscopy, VA-TIRFM, and FRET-FLIM, we provide evidence that stimulation by JA induces phosphorylation- and C-terminus-dependent endocytosis of AtRGS1, which then promotes dissociation of AtRGS1 from AtGPA1. In addition, SPT analysis reveals that JA treatment affects the diffusion dynamics of AtRGS1 and AtRGS1-ΔCt. Taken together, these findings suggest that the JA signal activates heterotrimeric G proteins through the endocytosis of AtRGS1 and dissociation of AtRGS1 from AtGPA1, thus providing valuable insight into the mechanisms of how the G protein system perceives and transduces phytohormone signals.
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Chytilova, Eva, Jiri Macas, Elwira Sliwinska, Susanne M. Rafelski, Georgina M. Lambert, and David W. Galbraith. "Nuclear Dynamics in Arabidopsis thaliana." Molecular Biology of the Cell 11, no. 8 (August 2000): 2733–41. http://dx.doi.org/10.1091/mbc.11.8.2733.
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The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to β-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm. This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.
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McKenna, J. F., D. J. Rolfe, S. E. D. Webb, A. F. Tolmie, S. W. Botchway, M. L. Martin-Fernandez, C. Hawes, and J. Runions. "The cell wall regulates dynamics and size of plasma-membrane nanodomains inArabidopsis." Proceedings of the National Academy of Sciences 116, no. 26 (June 10, 2019): 12857–62. http://dx.doi.org/10.1073/pnas.1819077116.
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Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.
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Michele, Daniel E., Faris P. Albayya, and Joseph M. Metzger. "Thin Filament Protein Dynamics in Fully Differentiated Adult Cardiac Myocytes: Toward A Model of Sarcomere Maintenance." Journal of Cell Biology 145, no. 7 (June 28, 1999): 1483–95. http://dx.doi.org/10.1083/jcb.145.7.1483.
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Sarcomere maintenance, the continual process of replacement of contractile proteins of the myofilament lattice with newly synthesized proteins, in fully differentiated contractile cells is not well understood. Adenoviral-mediated gene transfer of epitope-tagged tropomyosin (Tm) and troponin I (TnI) into adult cardiac myocytes in vitro along with confocal microscopy was used to examine the incorporation of these newly synthesized proteins into myofilaments of a fully differentiated contractile cell. The expression of epitope-tagged TnI resulted in greater replacement of the endogenous TnI than the replacement of the endogenous Tm with the expressed epitope-tagged Tm suggesting that the rates of myofilament replacement are limited by the turnover of the myofilament bound protein. Interestingly, while TnI was first detected in cardiac sarcomeres along the entire length of the thin filament, the epitope-tagged Tm preferentially replaced Tm at the pointed end of the thin filament. These results support a model for sarcomeric maintenance in fully differentiated cardiac myocytes where (a) as myofilament proteins turnover within the cell they are rapidly exchanged with newly synthesized proteins, and (b) the nature of replacement of myofilament proteins (ordered or stochastic) is protein specific, primarily affected by the structural properties of the myofilament proteins, and may have important functional consequences.
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Carpenter, David A., Sohaib A. Khan, and Wallace Ip. "Discrimination of the assembly states of cytoskeletal proteins in cultured cells using confocal microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 51 (August 1, 1993): 270–71. http://dx.doi.org/10.1017/s0424820100147193.
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The cytoskeleton is a three-dimensional network of cytoplasmic filaments that mediates many processes involving motility, and the specification and maintenance of cell form. In recent years, it has become evident that all three major components of the cytoskeleton- microfilaments, microtubules, and intermediate filaments (IF)-are dynamic structures that undergo reversible assembly-disassembly as required by the physiologic needs of the cell. While the assembled form of the cytoskeleton-the filamentous network-is readily visible by conventional immunofluorescence microscopy, it is often difficult to visualize the nonfilamentous form of a cytoskeletal protein because the subunits or oligomeric assemblies are small and because the images tend to be diffused due to interference from fluorescently labelled subunits above and below the plane of focus. Confocal microscopy offers a convenient solution to this problem at the light microscope level, because optical sections of fluorescently immunolabelled cytoskeletal networks do not suffer from such out-of-focus interference.
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Santoso, Yusdi, Ling Chin Hwang, Ludovic Le Reste, and Achillefs N. Kapanidis. "Red light, green light: probing single molecules using alternating-laser excitation." Biochemical Society Transactions 36, no. 4 (July 22, 2008): 738–44. http://dx.doi.org/10.1042/bst0360738.
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Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein–nucleic acid interactions.
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McGhee, Eric, Juan Uruena, Alex McGhee, Duane Mitchell, Catherine Flores, and W. Gregory Sawyer. "TMIC-50. 3D QUANTIFICATION OF DYNAMIC CYTOKINE GRADIENTS PRODUCED BY PRIMARY TUMOR MODELS." Neuro-Oncology 21, Supplement_6 (November 2019): vi258—vi259. http://dx.doi.org/10.1093/neuonc/noz175.1084.
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Abstract BACKGROUND & SIGNIFICANCE The immune response is a coordinated effort directed by cytokine gradients and concentrations. High sensitivity and spatial resolution are necessary to resolve cytokine gradients in 3-dimensions. This system uses in situ confocal fluorescent microscopy with printed bead-based immunoassays. The combination of 3D printing of the beads and biofabrication of patient derived tumors allows for direct imaging, quantification, and movies of tumor cytokine secretion in response to challenges from immune cells, tumor associated fibroblasts, and chemotherapeutic agents. HYPOTHESIS: Cytokine dynamics can be measured in real-time by balancing concentrations of detection antibodies in solution with stationary immunoassay beads with measured capture and release rates. Favorable balancing of assay kinetics can facilitate measurements of concentrations of cytokines between 50 pg/mL to over 2,000 pg/mL. METHODS In situ confocal fluorescent microscopy identifies position, fluorescence, and type of bead relative to the in vitro tumor. Cytokines including IL2, IL6, IL8, IL11, and IFNg and growth factors such as VEGF and TGFb have been measured for a wide range of solid tumors (brain, sarcoma, and epithelial) under different stresses. RESULTS Dynamic gradients of IL8 were found across multiple tumor models showing local concentrations in excess of 2,000 pg/mL. Production rates were estimated to be over 1 protein per second for each cell, and inclusion of cancer associated support cells showed 10x increases in some cytokines. CD4+ cell production of IL2 was confirmed and quantified and showed strong sensitivity to tumor activity and antigen presentation. Multiplexed beads of CCL2, CCL5, CXCL8, CXCL9, and CXCL10 allow for simultaneous measurement of multiple chemokines. Uncertainties associated with fluorescence measurement and quantification of concentrations will be discussed. CONCLUSIONS This integrated system of 3D assay printing, biofabrication of tumors and immune cell constructs, and in situ confocal microscopy provides the first direct measurements of 3D cytokine gradients in response to tumor stress.
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Bleve, Gianluca, Giuseppe Zacheo, Maria Stella Cappello, Franco Dellaglio, and Francesco Grieco. "Subcellular localization and functional expression of the glycerol uptake protein 1 (GUP1) of Saccharomyces cerevisiae tagged with green fluorescent protein." Biochemical Journal 390, no. 1 (August 9, 2005): 145–55. http://dx.doi.org/10.1042/bj20042045.
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GFP (green fluorescent protein) from Aequorea victoria was used as an in vivo reporter protein when fused to the N- and C-termini of the glycerol uptake protein 1 (Gup1p) of Saccharomyces cerevisiae. The subcellular localization and functional expression of biologically active Gup1–GFP chimaeras was monitored by confocal laser scanning and electron microscopy, thus supplying the first study of GUP1 dynamics in live yeast cells. The Gup1p tagged with GFP is a functional glycerol transporter localized at the plasma membrane and endoplasmic reticulum levels of induced cells. The factors involved in proper localization and turnover of Gup1p were revealed by expression of the Gup1p–GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaerical protein was targeted to the plasma membrane through a Sec6-dependent process; on treatment with glucose, it was endocytosed through END3 and targeted for degradation in the vacuole. Gup1p belongs to the list of yeast proteins rapidly down-regulated by changing the carbon source in the culture medium, in agreement with the concept that post-translational modifications triggered by glucose affect proteins of peripheral functions. The immunoelectron microscopy assays of cells expressing either Gup1–GFP or GFP–Gup1 fusions suggested the Gup1p membrane topology: the N-terminus lies in the periplasmic space, whereas its C-terminal tail has an intracellular location. An extra cytosolic location of the N-terminal tail is not generally predicted or determined in yeast membrane transporters.
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Bolte, Susanne, Elodie Marcon, Mélanie Jaunario, Lucas Moyet, Maité Paternostre, Marcel Kuntz, and Anja Krieger-Liszkay. "Dynamics of the localization of the plastid terminal oxidase inside the chloroplast." Journal of Experimental Botany 71, no. 9 (February 15, 2020): 2661–69. http://dx.doi.org/10.1093/jxb/eraa074.
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Abstract The plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOXs). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration, and setting the redox poise for cyclic electron transport. PTOX activity has been previously shown to depend on its localization at the thylakoid membrane. Here we investigate the dynamics of PTOX localization dependent on the proton motive force. Infiltrating illuminated leaves with uncouplers led to a partial dissociation of PTOX from the thylakoid membrane. In vitro reconstitution experiments showed that the attachment of purified recombinant maltose-binding protein (MBP)–OsPTOX to liposomes and isolated thylakoid membranes was strongest at slightly alkaline pH values in the presence of lower millimolar concentrations of KCl or MgCl2. In Arabidopsis thaliana overexpressing green fluorescent protein (GFP)–PTOX, confocal microscopy images showed that PTOX formed distinct spots in chloroplasts of dark-adapted or uncoupler-treated leaves, while the protein was more equally distributed in a network-like structure in the light. We propose a dynamic PTOX association with the thylakoid membrane depending on the presence of a proton motive force.
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Chen, Chaoping, Ora A. Weisz, Donna B. Stolz, Simon C. Watkins, and Ronald C. Montelaro. "Differential Effects of Actin Cytoskeleton Dynamics on Equine Infectious Anemia Virus Particle Production." Journal of Virology 78, no. 2 (January 15, 2004): 882–91. http://dx.doi.org/10.1128/jvi.78.2.882-891.2004.
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ABSTRACT Retrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. Previous studies have shown that retroviral Gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. Using equine infectious anemia virus (EIAV), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. First, virion production was reduced when EIAV-infected cells were treated with phallacidin, a cell-permeable reagent that stabilizes actin filaments by slowing down their depolymerization. Confocal microscopy confirmed that the inhibition of EIAV production correlated temporally over several days with the incorporation dynamics of phallacidin into the actin cytoskeleton. Although the overall structure of the actin cytoskeleton and expression of viral protein appeared to be unaffected, phallacidin treatment dramatically reduced the amount of full-length Gag protein associated with the actin cytoskeleton. These data suggest that an association of full-length Gag proteins with de novo actin filaments might contribute to Gag assembly and budding. On the other hand, virion production was enhanced when EIAV-infected cells were incubated briefly (2 h) with the actin-depolymerizing drugs cytochalasin D and latrunculin B. Interestingly, the enhanced virion production induced by cytochalasin D required a functional late (L) domain, either the EIAV YPDL L-domain or the proline-rich L domains derived from human immunodeficiency virus type 1 or Rous sarcoma virus, respectively. Thus, depolymerization of actin filaments may be a common function mediated by retrovirus L domains during late stages of viral budding. Taken together, these observations indicate that dynamic actin polymerization and depolymerization may be associated with different stages of viral production.
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Fiore, Antonio, and Giuliano Scarcelli. "Opto-mechanical material characterization via dual-geometry Brillouin confocal microscopy." Journal of the Acoustical Society of America 152, no. 4 (October 2022): A225. http://dx.doi.org/10.1121/10.0016087.
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Brillouin scattering is a light-matter interaction that induces a frequency shift in the scattered photons. Such shift directly depends on the optical and mechanical properties of the material itself. In the last decade, Brillouin frequency shift has become a reliable proxy for mechanical stiffness in biological samples such as cells and tissues, with the subsequent development of Brillouin confocal microscopy; however, such correlation does not apply to samples with high degree of inhomogeneity. Recently, we showed that a dual geometry Brillouin microscopy enables the direct measurement of refractive index and speed of sound. We now report an improved dual-geometry microscopy technique that allows measurement of optoacoustical properties within a confocal volume. This goal has been achieved through the measurement of frequency, linewidth, and intensity of Brillouin components of the scattered light spectrum. Finally, we proved our three-dimensional imaging capability by mapping, with micron-scale resolution, the refractive index, density, and viscoelastic properties of a liquid-liquid phase separation system. This technique can be applied to a multitude of biological heterogenous scenarios such as nucleolus characterization, high concentration lipid sites, dynamic protein aggregates and more.
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Husakova, Marketa, and Karolina Ditrychova. "Application of confocal microscopy methods for estimation of lipid and proteins dynamics in thylakoid membranes." New Biotechnology 31 (July 2014): S152. http://dx.doi.org/10.1016/j.nbt.2014.05.1997.
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Nobile, Cinzia, Dominika Rudnicka, Milena Hasan, Nathalie Aulner, Françoise Porrot, Christophe Machu, Olivier Renaud, et al. "HIV-1 Nef Inhibits Ruffles, Induces Filopodia, and Modulates Migration of Infected Lymphocytes." Journal of Virology 84, no. 5 (December 16, 2009): 2282–93. http://dx.doi.org/10.1128/jvi.02230-09.
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ABSTRACT The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal “axial tomography,” this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.
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Nicoziani, Paolo, Frederik Vilhardt, Alicia Llorente, Leila Hilout, Pierre J. Courtoy, Kirsten Sandvig, and Bo van Deurs. "Role for Dynamin in Late Endosome Dynamics and Trafficking of the Cation-independent Mannose 6-Phosphate Receptor." Molecular Biology of the Cell 11, no. 2 (February 2000): 481–95. http://dx.doi.org/10.1091/mbc.11.2.481.
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It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)–containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules.
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Cerecedo, Doris, Dalila Martínez-Rojas, Oscar Chávez, Francisco Martínez-Pérez, Francisco García-Sierra, Álvaro Rendon, Dominique Mornet, and Ricardo Mondragón. "Platelet adhesion: Structural and functional diversity of short dystrophin and utrophins in the formation of dystrophinassociated-protein complexes related to actin dynamics." Thrombosis and Haemostasis 94, no. 12 (2005): 1203–12. http://dx.doi.org/10.1160/th04-11-0765.
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SummaryPlatelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71d, as well as the Up71 isoform and the dystrophin-associated proteins, α and β-dystrobrevins. Distribution of Dp71d/Dp71Δ110 m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71Δ110 m ~DAPC and Up400/Up71~DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71Δ110 m ~DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71Δ110 m ~DAPC and Up400/Up71~DAPC in the biological roles of the platelets is discussed.
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STEPHENS, David J., and George BANTING. "In vivo dynamics of the F-actin-binding protein neurabin-II." Biochemical Journal 345, no. 2 (January 10, 2000): 185–94. http://dx.doi.org/10.1042/bj3450185.
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Neurabin-II (spinophilin) is a ubiquitously expressed F-actin-binding protein containing an N-terminal actin-binding domain, a PDZ (PSD95/discs large/ZO-1) domain and a C-terminal domain predicted to form a coiled-coil structure. We have stably expressed a green fluorescent protein (GFP)-tagged version of neurabin-II in PC12 cells, and characterized the in vivo dynamics of this actin-binding protein using confocal fluorescence microscopy. We show that GFP-neurabin-II localizes to actin filaments, especially at cortical sites and areas underlying sites of active membrane remodelling. GFP-neurabin-II labels only a subset of F-actin within these cells, as indicated by rhodamine-phalloidin staining. Both actin filaments and small, highly motile structures within the cell body are seen. Photobleaching experiments show that GFP-neurabin-II also exhibits highly dynamic behaviour when bound to actin filaments. Latrunculin B treatment results in rapid relocalization of GFP-neurabin-II to the cytosol, whereas cytochalasin D treatment causes the collapse of GFP-neurabin-II fluorescence to intensely fluorescent foci of F-actin within the cell body. This collapse is reversed on cytochalasin D removal, recovery from which is greatly accelerated by stimulation of cells with epidermal growth factor (EGF). Furthermore, we show that this EGF-induced relocalization of GFP-neurabin-II is dependent on the activity of the small GTPase Rac1 but not the activity of ADP-ribosylation factor 6.
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Malcuit, Isabelle, María Rosa Marano, Tony A. Kavanagh, Walter De Jong, Alec Forsyth, and David C. Baulcombe. "The 25-kDa Movement Protein of PVX Elicits Nb-Mediated Hypersensitive Cell Death in Potato." Molecular Plant-Microbe Interactions® 12, no. 6 (June 1999): 536–43. http://dx.doi.org/10.1094/mpmi.1999.12.6.536.
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The potato gene Nb confers hypersensitive resistance to potato virus X (PVX). To characterize the viral elicitor of this resistance we introduced modifications into the genome of avirulent strains (ROTH1 and CP2) and virulent strains (UK3 and CP4) of PVX. From the analysis of the modified viral genomes, the Nb avirulence determinant was mapped in the PVX 25K gene coding for the 25-kDa movement protein. Furthermore, we showed that the isoleucine residue at position 6 of this protein was required for activation of the Nb response. Transient co-expression of the avirulent 25K gene with the β-glucuronidase (GUS) reporter gene introduced by particle bombardment in resistant and susceptible potato cells confirmed that the elicitor activity provided by the 25-kDa protein did not require other PVX-encoded proteins. To study cellular events associated with the Nb response, the 25-kDa proteins of PVX strains ROTH1 and UK3 were tagged with the green fluorescent protein (GFP) so that the dynamics of the subcellular distribution of the 25KGFP fusion proteins could be followed in living potato epidermal cells by laser scanning confocal microscopy. Using this method, we showed that the Nb-mediated response is associated with degradation of subcellular structures.
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Kim, Lee, Fujii, Lee, Lee, Park, Kim, Lee, and Pack. "Mitotic Chromosomes in Live Cells Characterized Using High-Speed and Label-Free Optical Diffraction Tomography." Cells 8, no. 11 (October 31, 2019): 1368. http://dx.doi.org/10.3390/cells8111368.
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The cell nucleus is a three-dimensional, dynamic organelle organized into subnuclear compartments such as chromatin and nucleoli. The structure and function of these compartments are maintained by diffusion and interactions between related factors as well as by dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in heterochromatin and in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome during cell division are not fully understood. In the present study, characteristic properties such as the refractive index (RI), volume of the mitotic chromosomes, and diffusion coefficient (D) of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser-scanning microscopy and fluorescence correlation spectroscopy. Variations in these parameters correlated with osmotic conditions, suggesting that changes in RI are consistent with those of the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobilities of fluorescent proteins.
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OATEY, B. Paru, David H. J. VAN WEERING, P. Stephen DOBSON, W. Gwyn GOULD, and Jeremy M. TAVARÉ. "GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein." Biochemical Journal 327, no. 3 (November 1, 1997): 637–42. http://dx.doi.org/10.1042/bj3270637.
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Insulin stimulates glucose uptake into its target cells by a process which involves the translocation of the GLUT4 isoform of glucose transporter from an intracellular vesicular compartment(s) to the plasma membrane. The step(s) at which insulin acts in the vesicle trafficking pathway (e.g. vesicle movement or fusion with the plasma membrane) is not known. We expressed a green-fluorescent protein-GLUT4 (GFP-GLUT4) chimaera in 3T3 L1 adipocytes. The chimaera was expressed in vesicles located throughout the cytoplasm and also close to the plasma membrane. Insulin promoted a substantial translocation of GFP-GLUT4 to the plasma membrane. Time-lapse confocal microscopy demonstrated that the majority of GFP-GLUT4-containing vesicles in the basal state were relatively static, as if tethered (or attached) to an intracellular structure. A proportion (approx. 5%) of the vesicles spontaneously lost their tether, and were observed to move rapidly within the cell. Other vesicles appear to be tethered only on one edge and were observed in a rapid stretching motion. The data support a model in which GLUT4-containing vesicles are tightly tethered to an intracellular structure(s), and indicate that a primary site of insulin action must be to release these vesicles, allowing them to then translocate to and fuse with the plasma membrane.
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Costa, Roberta, Maria Teresa Rodia, Nicoletta Zini, Valentina Pegoraro, Roberta Marozzo, Cristina Capanni, Corrado Angelini, Giovanna Lattanzi, Spartaco Santi, and Giovanna Cenacchi. "Morphological study of TNPO3 and SRSF1 interaction during myogenesis by combining confocal, structured illumination and electron microscopy analysis." Molecular and Cellular Biochemistry 476, no. 4 (January 15, 2021): 1797–811. http://dx.doi.org/10.1007/s11010-020-04023-y.
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AbstractTransportin3 (TNPO3) shuttles the SR proteins from the cytoplasm to the nucleus. The SR family includes essential splicing factors, such as SRSF1, that influence alternative splicing, controlling protein diversity in muscle and satellite cell differentiation. Given the importance of alternative splicing in the myogenic process and in the maintenance of healthy muscle, alterations in the splicing mechanism might contribute to the development of muscle disorders. Combining confocal, structured illumination and electron microscopy, we investigated the expression of TNPO3 and SRSF1 during myogenesis, looking at nuclear and cytoplasmic compartments. We investigated TNPO3 and its interaction with SRSF1 and we observed that SRSF1 remained mainly localized in the nucleus, while TNPO3 decreased in the cytoplasm and was strongly clustered in the nuclei of differentiated myotubes. In conclusion, combining different imaging techniques led us to describe the behavior of TNPO3 and SRSF1 during myogenesis, showing that their dynamics follow the myogenic process and could influence the proteomic network necessary during myogenesis. The combination of different high-, super- and ultra-resolution imaging techniques led us to describe the behavior of TNPO3 and its interaction with SRSF1, looking at nuclear and cytoplasmic compartments. These observations represent a first step in understanding the role of TNPO3 and SRFSF1 in complex mechanisms, such as myogenesis.
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Kreshchenko, Skavulyak, Bondarenko, and Ermakov. "MELATONIN MODULATES DYNAMICS OF PLANARIAN STEM CELL PROLIFERATION." THEORY AND PRACTICE OF PARASITIC DISEASE CONTROL, no. 21 (May 29, 2020): 157–63. http://dx.doi.org/10.31016/978-5-9902341-5-4.2020.21.157-163.
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Melatonin is a derivate of biogenic amine of serotonin identified in all classes of animals including flatworms. Melatonin demonstrates different physiological functions the main of which is circadian rhythm regulation. Via specific G-protein coupled receptors, melatonin affects the target cells changing the levels of other hormones. On early stages of embryonic development, biogenic amines as well as melatonin play a role of specific signal cell molecules that regulate processes of cellular renewal. This work has studied physiological function of melatonin in free-living flatworms, planarian Schmidtea mediterranea. The influence of melatonin on diurnal dynamics of stem cells proliferation was investigated using an immunocytochemical method and confocal laser scanning microscopy. The specific antibodies against H3 phosphohistones were applied for immunocytochemical identification of proliferative cells. It was shown that melatonin (1 µМ) decreased the total number of proliferative cells in planarians. It was also found that the diurnal dynamics of cells proliferation in planarians was changed by melatonin: regular rhythmic oscillations observed in the control group of animals were smoothening. Further researches are required to clarify mechanisms of melatonin actions.
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Temme, Achim, Michael Rieger, Friedemann Reber, Dirk Lindemann, Bernd Weigle, Petra Diestelkoetter-Bachert, Gerhard Ehninger, Masaaki Tatsuka, Yasuhiko Terada, and Ernst Peter Rieber. "Localization, Dynamics, and Function of Survivin Revealed by Expression of Functional SurvivinDsRed Fusion Proteins in the Living Cell." Molecular Biology of the Cell 14, no. 1 (January 2003): 78–92. http://dx.doi.org/10.1091/mbc.e02-04-0182.
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Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.
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Nagy, Péter, László Mátyus, Attila Jenei, György Panyi, Sándor Varga, János Matkó, János Szöllősi, Rezső Gáspár, Thomas M. Jovin, and Sándor Damjanovich. "Cell fusion experiments reveal distinctly different association characteristics of cell-surface receptors." Journal of Cell Science 114, no. 22 (November 15, 2001): 4063–71. http://dx.doi.org/10.1242/jcs.114.22.4063.
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The existence of small- and large-scale membrane protein clusters, containing dimers, oligomers and hundreds of proteins, respectively, has become widely accepted. However, it is largely unknown whether the internal structure of these formations is dynamic or static. Cell fusion was used to perturb the distribution of existing membrane protein clusters, and to investigate their mobility and associations. Scanning near-field optical microscopy, confocal and electron microscopy were applied to detect the exchange of proteins between large-scale protein clusters, whereas photobleaching fluorescence energy transfer was used to image the redistribution of existing small-scale membrane protein clusters. Large-scale clusters of major histocompatibility complex (MHC)-I exchanged proteins with each other and with MHC-II clusters. Similarly to MHC-I, large-scale MHC-II clusters were also dynamic. Exchange of components between small-scale protein clusters was not universal: intermixing did not take place in the case of MHC-II homoclusters; however, it was observed for homoclusters of MHC-I and for heteroclusters of MHC-I and MHC-II. These processes required a fluid state of the plasma membrane, and did not depend on endocytosis-mediated recycling of proteins. The redistribution of large-scale MHC-I clusters precedes the intermixing of small-scale clusters of MHC-I indicating a hierarchy in protein association. Investigation of a set of other proteins (α subunit of the interleukin 2 receptor, CD48 and transferrin receptor) suggested that a large-scale protein cluster usually exchanges components with the same type of clusters. These results offer new insight into processes requiring time-dependent changes in membrane protein interactions.
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Twaroski, Danielle M., Yasheng Yan, Ivan Zaja, Eric Clark, Zeljko J. Bosnjak, and Xiaowen Bai. "Altered Mitochondrial Dynamics Contributes to Propofol-induced Cell Death in Human Stem Cell–derived Neurons." Anesthesiology 123, no. 5 (November 1, 2015): 1067–83. http://dx.doi.org/10.1097/aln.0000000000000857.
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Abstract Background Studies in developing animals have shown that anesthetic agents can lead to neuronal cell death and learning disabilities when administered early in life. Development of human embryonic stem cell–derived neurons has provided a valuable tool for understanding the effects of anesthetics on developing human neurons. Unbalanced mitochondrial fusion and fission lead to various pathological conditions including neurodegeneration. The aim of this study was to dissect the role of mitochondrial dynamics in propofol-induced neurotoxicity. Methods Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate in situ nick-end labeling staining was used to assess cell death in human embryonic stem cell–derived neurons. Mitochondrial fission was assessed using TOM20 staining and electron microscopy. Expression of mitochondrial fission-related proteins was assessed by Western blot, and confocal microscopy was used to assess opening time of the mitochondrial permeability transition pore (mPTP). Results Exposure to 6 h of 20 μg/ml propofol increased cell death from 3.18 ± 0.17% in the control-treated group to 9.6 ± 0.95% and led to detrimental increases in mitochondrial fission (n = 5 coverslips per group) accompanied by increased expression of activated dynamin-related protein 1 and cyclin-dependent kinase 1, key proteins responsible for mitochondrial fission. Propofol exposure also induced earlier opening of the mPTP from 118.9 ± 3.1 s in the control-treated group to 73.3 ± 1.6 s. Pretreatment of the cells with mdivi-1, a mitochondrial fission blocker rescued the propofol-induced toxicity, mitochondrial fission, and mPTP opening time (n = 75 cells per group). Inhibiting cyclin-dependent kinase 1 attenuated the increase in cell death and fission and the increase in expression of activated dynamin-related protein 1. Conclusion These data demonstrate for the first time that propofol-induced neurotoxicity occurs through a mitochondrial fission/mPTP-mediated pathway.
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Díaz-Hernández, Mitzi, Rosario Javier-Reyna, Diana Martínez-Valencia, Sarita Montaño, and Esther Orozco. "Dynamic Association of ESCRT-II Proteins with ESCRT-I and ESCRT-III Complexes during Phagocytosis of Entamoeba histolytica." International Journal of Molecular Sciences 24, no. 6 (March 9, 2023): 5267. http://dx.doi.org/10.3390/ijms24065267.
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By their active movement and voraux phagocytosis, the trophozoites of Entamoeba histolytica constitute an excellent system to investigate the dynamics of the Endosomal Sorting Complex Required for Transport (ESCRT) protein interactions through phagocytosis. Here, we studied the proteins forming the E. histolytica ESCRT-II complex and their relationship with other phagocytosis-involved molecules. Bioinformatics analysis predicted that EhVps22, EhVps25, and EhVps36 are E. histolytica bona fide orthologues of the ESCRT-II protein families. Recombinant proteins and specific antibodies revealed that ESCRT-II proteins interact with each other, with other ESCRT proteins, and phagocytosis-involved molecules, such as the adhesin (EhADH). Laser confocal microscopy, pull-down assays, and mass spectrometry analysis disclosed that during phagocytosis, ESCRT-II accompanies the red blood cells (RBCs) from their attachment to the trophozoites until their arrival to multivesicular bodies (MVBs), changing their interactive patterns according to the time and place of the process. Knocked-down trophozoites in the Ehvps25 gene presented a 50% lower rate of phagocytosis than the controls and lower efficiency to adhere RBCs. In conclusion, ESCRT-II interacts with other molecules during prey contact and conduction throughout the phagocytic channel and trophozoites membranous system. ESCRT-II proteins are members of the protein chain during vesicle trafficking and are fundamental for the continuity and efficiency of phagocytosis.
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Subach, Oksana M., Anna V. Vlaskina, Yuliya K. Agapova, Pavel V. Dorovatovskii, Alena Y. Nikolaeva, Olga I. Ivashkina, Vladimir O. Popov, et al. "LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift." International Journal of Molecular Sciences 22, no. 23 (November 28, 2021): 12887. http://dx.doi.org/10.3390/ijms222312887.
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Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.
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Prasad, Ankush, Renuka Ramalingam Manoharan, Michaela Sedlářová, and Pavel Pospíšil. "Free Radical-Mediated Protein Radical Formation in Differentiating Monocytes." International Journal of Molecular Sciences 22, no. 18 (September 15, 2021): 9963. http://dx.doi.org/10.3390/ijms22189963.
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Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.
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Kaufmann, Tanja, Sébastien Herbert, Benjamin Hackl, Johanna Maria Besold, Christopher Schramek, Josef Gotzmann, Kareem Elsayad, and Dea Slade. "Direct measurement of protein–protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells." Nucleic Acids Research 48, no. 21 (October 14, 2020): e122-e122. http://dx.doi.org/10.1093/nar/gkaa859.
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Abstract Protein–protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein–protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein–protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein–protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein–protein interactions at DNA damage sites.
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Blacutt, Jacob, Ziyang Lan, Elizabeth M. Cosgriff-Hernandez, and Vernita D. Gordon. "Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels." Royal Society Open Science 8, no. 1 (January 2021): 201453. http://dx.doi.org/10.1098/rsos.201453.
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The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure. Biofilm development initiates when bacteria attach to and sense a surface. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, the transition to the biofilm phenotype is controlled by the intracellular signal and second messenger cyclic-di-GMP (c-di-GMP). It is not known how biomedical materials might be adjusted to impede c-di-GMP signalling, and there are few extant methods for conducting such studies. Here, we develop such a method. We allowed P. aeruginosa to attach to the surfaces of poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These bacteria contained a plasmid for a green fluorescent protein (GFP) reporter for c-di-GMP. We used laser-scanning confocal microscopy to measure the dynamics of the GFP reporter for 3 h, beginning 1 h after introducing bacteria to the hydrogel. We controlled for the effects of changes in bacterial metabolism using a promoterless plasmid for GFP, and for the effects of light passing through different hydrogels being differently attenuated by using fluorescent plastic beads as ‘standard candles’ for calibration. We demonstrate that this method can measure statistically significant differences in c-di-GMP signalling associated with different PEGDA gel types and with the surface-exposed protein PilY1.
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Schweizer, A., M. Ericsson, T. Bachi, G. Griffiths, and H. P. Hauri. "Characterization of a novel 63 kDa membrane protein. Implications for the organization of the ER-to-Golgi pathway." Journal of Cell Science 104, no. 3 (March 1, 1993): 671–83. http://dx.doi.org/10.1242/jcs.104.3.671.
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Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15 degrees C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.
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Zhang, Bodi, Kostas Alysandratos, Asimenia Angelidou, Duraisamy Kempuraj, Michael Tagen, Magdalini Vasiadi, Shahrzad Asadi, and Theoharis Theoharides. "TNF secretion from human mast cells is regulated by mitochondrial dynamics and mitochondrial uncoupling protein 2 (UCP2) (135.11)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 135.11. http://dx.doi.org/10.4049/jimmunol.184.supp.135.11.
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Abstract Tumor necrosis factor (TNF) is involved in T cell-dependent diseases such as psoriasis, and in T cell activation by mast cells, which can both store and de novo synthesize TNF. Yet, regulation of TNF secretion is unknown hampering our understanding of how mast cells regulate allergic, immune and inflammatory processes. Here we show that stimulated human LAD2 leukemic and umbilical cord-derived cultured mast cell (hCBMCs) by SP or IgE/anti-IgE induces degranulation and prestored TNF release, which is accompanied by mitochondrial fission. Images of Confocal microscopy and electronic transmitted microscopy show fragmented mitochondria and relocation from a perinuclear distribution to the cell surface. Moreover, treatment with a mitochondrial fission blocker inhibits degranulation, indicating the necessarily of mitochondria fission in mast cell secretion. Besides mitochondria morphological changes, SP stimulation of LAD2 and hCBMCs increased expression (8 hr) of the mitochondrial uncoupling protein 2 (UCP2), which regulates production of ROS and intracellular calcium, leading to inhibition of subsequent re-stimulation by SP, when is enough for mast cells to recover. Moreover, UCP2 gene expression was reduced, as compared to control normal skin, in affected psoriatic skin, an inflammatory diseases in which mast cells play important role. These findings indicate that mitochondria play a key regulatory role in mast cell TNF secretion and in psoriasis.
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Cao, Yangyang, Qizouhong He, Zengxing Qi, Yan Zhang, Liang Lu, Jingyuan Xue, Junling Li, and Ruili Li. "Dynamics and Endocytosis of Flot1 in Arabidopsis Require CPI1 Function." International Journal of Molecular Sciences 21, no. 5 (February 25, 2020): 1552. http://dx.doi.org/10.3390/ijms21051552.
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Membrane microdomains are nano-scale domains (10–200 nm) enriched in sterols and sphingolipids. They have many important biological functions, including vesicle transport, endocytosis, and pathogen invasion. A previous study reported that the membrane microdomain-associated protein Flotillin1 (Flot1) was involved in plant development in Arabidopsis thaliana; however, whether sterols affect the plant immunity conveyed by Flot1 is unknown. Here, we showed that the root length in sterol-deficient cyclopropylsterol isomerase 1 (cpi1-1) mutants expressing Flot1 was significantly shorter than in control seedlings. The cotyledon epidermal cells in cpi1-1 mutants expressing Flot1 were smaller than in controls. Moreover, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) and single-particle tracking (SPT) analysis demonstrated that the long-distance Flot1-GFP movement was decreased significantly in cpi1-1 mutants compared with the control seedlings. Meanwhile, the value of the diffusion coefficient Ĝ was dramatically decreased in cpi1-1 mutants after flagelin22 (flg22) treatment compared with the control seedlings, indicating that sterols affect the lateral mobility of Flot1-GFP within the plasma membrane. Importantly, using confocal microscopy, we determined that the endocytosis of Flot1-GFP was decreased in cpi1-1 mutants, which was confirmed by fluorescence cross spectroscopy (FCS) analysis. Hence, these results demonstrate that sterol composition plays a critical role in the plant defense responses of Flot1.
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Mortati, Leonardo, Barbara Pergolizzi, Cristina Panuzzo, and Enrico Bracco. "Present and Future Opportunities in Imaging the Ubiquitin System (Ub-System)." Biophysica 2, no. 3 (July 28, 2022): 174–83. http://dx.doi.org/10.3390/biophysica2030018.
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From yeast to mammalian cells, ubiquitination is one of the most conserved, and reversible, eukaryotic post-translational modifications (PTMs) responsible for controlling nearly all cellular processes. Potentially, every single eukaryotic cell can accomplish different ubiquitination processes at once, which in turn control the execution of specific cellular events in time and space with different biological significance (e.g., protein degradation or protein–protein interaction). Overall, all these signals are highly dynamic and need to be finely integrated to achieve a proper cellular response. Altogether, ubiquitination appears to be an extremely complex process, likely more than any other PTMs. Until a few years ago, the prevailing experimental approaches to investigate the different aspects of the ubiquitin system entailed genetic and biochemical analysis. However, recently, reagents and technologies have been developed enabling microscopy-based imaging of ubiquitination to enter the scene. In this paper, we discuss the progress made with conventional (confocal fluorescence microscopy) and non-conventional non-linear microscopy (Atomic Force Microscopy—AFM, Coherent Anti-Stokes Raman Scattering—CARS, Stimulated Raman Scattering—SRS) and we speculate on future developments.