1,721,313 research outputs found
Changes in Chromatin Compaction During the Cell Cycle Revealed by Micrometer-Scale Measurement of Molecular Flow in the Nucleus
Full text linkAbstractWe present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca2+ intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of molecular diffusion may become relevant for many cellular processes
Development of an image Mean Square Displacement (iMSD)-based method as a novel approach to study the intracellular trafficking of nanoparticles
Full text linkFluorescence microscopy and spectroscopy techniques are commonly used to investigate complex and interacting biological systems (e.g. proteins and nanoparticles in living cells), since these techniques can explore intracellular dynamics with high time resolution at the nanoscale. Here we extended one of the Image Correlation Spectroscopy (ICS) methods, i.e. the image Mean Square Displacement, in order to study 2-dimensional diffusive and flow motion in confined systems, whose driving speed is uniformly distributed in a variable angular range. Although these conditions are not deeply investigated in the current literature, they can be commonly found in the intracellular trafficking of nanocarriers, which diffuse in the cytoplasm and/or may move along the cytoskeleton in different directions. The proposed approach could reveal the underlying system's symmetry using methods derived from fluorescence correlation concepts and could recover dynamic and geometric features which are commonly done by single particle analyses. Furthermore, it improves the characterization of low-speed flow motions, when compared to SpatioTemporal Image Correlation Spectroscopy (STICS). Although we present a specific example (lipoplexes in living cells), the emphasis is in the discussion of the method, its basic assumptions and its validation on numeric simulations. Statement of Significance Recent advances in nanoparticle-based drug and gene delivery systems have pointed out the interactions at cellular and subcellular levels as key-factors for the efficiency of the adopted biomaterials. Such biochemical and biophysical interactions drive and affect the intracellular dynamics, that is commonly characterized by means of fluorescence microscopy and spectroscopy techniques. Here we present a novel Image Correlation Spectroscopy (ICS) method as a promising tool to capture the intracellular behavior of nanoparticles with high resolution and low background's sensitivity. This study overcomes some of the approximations adopted so far, by decoupling the flow terms of the investigated dynamics and thus recovering ensemble's information from specific single particle behaviors. Finally, relevant implications for nanoparticle-based drug delivery are shown
The role of cytoskeleton networks on lipid-mediated delivery of DNA
Full text linkBackground: Lipid-mediated delivery of DNA is hindered by extracellular and intracellular barriers that significantly reduce the transfection efficiency of synthetic nonviral vectors. Results: In this study we investigated the role of the actin and microtubule networks on the uptake and cytoplasmic transport of multicomponent cationic liposome–DNA complexes in CHO-K1 live cells by means of confocal laser scanning microscopy and 3D single particle tracking. Treatment with actin (latrunculin B)- and microtubule-disrupting (nocodazole) reagents indicated that intracellular trafficking of complexes predominantly involves microtubule-dependent active transport. We found that the actin network has a major effect on the initial uptake of complexes, while the microtubule network is mainly responsible for the subsequent active transportation to the lysosomes. Conclusion: Collectively, a strategy to improve the efficiency of lipid gene vectors can be formulated. We could find a lipid formulation that allows the nanoparticles to avoid the microtubule pathway to lysosomes.Fil: Coppola, Stefano. Sapienza University of Rome. Dipartimento di Medicina Molecolare; ItaliaFil: Cardarelli, Francesco. Istituto Italiano di Tecnologia. Center for Nanotechnology Innovation at NEST; ItaliaFil: Pozzi, Daniela. Sapienza University of Rome. Dipartimento di Medicina Molecolare; ItaliaFil: Estrada, Laura Cecilia. University of California. Department of Biomedical Engineering. Laboratory for Fluorescence Dynamics; Estados Unidos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Electrónica Cuántica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; ArgentinaFil: Digman, Michelle. University of California. Department of Biomedical Engineering. Laboratory for Fluorescence Dynamics; Estados UnidosFil: Gratton, Enrico. University of California. Department of Biomedical Engineering. Laboratory for Fluorescence Dynamics; Estados UnidosFil: Bifone, Angelo . Istituto Italiano di Tecnologia. Center for Nanotechnology Innovation at NEST; ItaliaFil: Marianecci, Carlotta. Sapienza University of Rome. Dipartimento di Chimica e Tecnologie del Farmaco; ItaliaFil: Caracciolo, Giulio. Sapienza University of Rome. Dipartimento di Medicina Molecolare; Itali
In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow
Full text linkNo methods proposed thus far have the capability to measure overall molecular flow in the nucleus of living cells. Here, we apply the pair correlation function analysis (pCF) to measure molecular anisotropic diffusion in the interphase nucleus of live cells. In the pCF method, we cross-correlate fluctuations at several distances and locations within the nucleus, enabling us to define migration paths and barriers to diffusion. We use monomeric EGFP as a prototypical inert molecule and measure flow in and between different nuclear environments. Our results suggest that there are two disconnect molecular flows throughout the nucleus associated with high and low DNA density regions. We show that different density regions of DNA form a networked channel that allows EGFP to diffuse freely throughout, however with restricted ability to traverse the channel. We also observe rare and sudden bursts of molecules traveling across DNA density regions with characteristic time of ≈300 ms, suggesting intrinsic localized change in chromatin structure. This is a unique in vivo demonstration of the intricate chromatin network showing channel directed diffusion of an inert molecule with high spatial and temporal resolution.</jats:p
Pair correlation microscopy of intracellular molecular transport
Full text linkPair correlation microscopy is a unique approach to fluorescence correlation spectroscopy that can track the long-range diffusive route of a population of fluorescent molecules in live cells with respect to intracellular architecture. This method is based on the use of a pair correlation function (pCF) that, through spatiotemporal comparison of fluctuations in fluorescence intensity recorded throughout a microscope data acquisition, enables changes in a molecule's arrival time to be spatially mapped and statistically quantified. In this protocol, we present guidelines for the measurement and analysis of line scan pair correlation microscopy data acquired on a confocal laser scanning microscope (CLSM), which will enable users to extract a fluorescent molecule's transport pattern throughout a living cell, and then quantify the molecular accessibility of intracellular barriers encountered or the mode of diffusion governing a molecular trafficking event. Finally, we demonstrate how this protocol can be extended to a two-channel line scan acquisition that, when coupled with a cross pCF calculation, enables a fluorescent molecule's transport pattern to be selectively tracked as a function of complex formation with a spectrally distinct fluorescent ligand. For a skilled user of a CLSM, the line scan data acquisition and analysis described in this protocol will take ~1-2 d, depending on the sample and the number of experiments to be processed
The Impact of Mitotic versus Interphase Chromatin Architecture on the Molecular Flow of EGFP by Pair Correlation Analysis
Full text linkAbstractHere we address the impact nuclear architecture has on molecular flow within the mitotic nucleus of live cells as compared to interphase by the pair correlation function method. The mitotic chromatin is found to allow delayed but continuous molecular flow of EGFP in and out of a high chromatin density region, which, by pair correlation function analysis, is shown as a characteristic arc shape that appears upon entry and exit. This is in contrast to interphase chromatin, which regulates flow between different density chromatin regions by means of a mechanism which turns on and off intermittently, generating discrete bursts of EGFP. We show that the interphase bursts are maintained by metabolic energy, whereas the mitotic mechanism of regulation responsible for the arc is not sensitive to ATP depletion. These two distinct routes of molecular flow were concomitantly measured in the Caenorhabditis elegans germ line, which indicates a conservation of mechanism on a scale more widespread than cell type or organism
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Characterization of Local Conformational Structure and Mechanical Properties of Chromatin using Fluorescence Lifetime Imaging Microscopy and Phasor Analysis
Full text linkChromatin is a complex of macromolecules that plays a very important role in packaging the long strand of DNA inside the eukaryotic nucleus. Chromatin enables and regulates many DNA functions and interactions with other molecules including DNA transcription, replication, and repair. During the cell division, chromatin ensures proper division and transfer of the genetic material to daughter cells. To fulfill these roles, chromatin must constantly undergo reconfiguration and reorganization of its structure. Therefore, local conformational structure and mechanical properties of chromatin play a crucial role in creating the highly dynamic structure of chromatin. Furthermore, recent studies link mechanobiology of the nucleus, specifically nuclear stiffness and deformability, to cancer metastasis. Due to the fact that chromatin structure is highly compact and dynamic, visualizing and studying its structure, remodeling and mechanical properties is very difficult. In this research, we showed that Fluorescence Lifetime Imaging Microscopy (FLIM) and phasor analysis can be used as a powerful technique to study local conformational structure and mechanical properties of chromatin during mitotic cell division and in metastatic tumor cells. More specifically, the findings from the lifetime values revealed information about the size, stiffness, deformability, and accessibility of the minor grooves in chromatin. The findings from this study confirmed significant structural differences between metaphase and interphase chromatin, but they also revealed significant variations within chromatin at any stage. We postulated that these variations are largely sequence dependent. Two types of regions with distinct conformational and mechanical properties were identified in chromatin. One of them had a significantly longer lifetime which was indicative of more rigid and larger binding area. These regions were attributed to GC-rich minor grooves. The other regions with significantly shorter lifetime, indicating softer and smaller binding sites, were associated with AT-rich minor grooves. Moreover, the lifetime of metaphase and interphase chromatin differed more significantly in GC-associated grooves. Therefore, we concluded that there were far more AT-rich minor grooves accessible for binding in both metaphase and interphase chromatin to the extent that the effect of chromatin condensation during metaphase did not cause very significant differences in the lifetime values. Finally, the fluorescence lifetime analysis revealed that chromatin in highly aggressive metastatic tumors like MB231 and MFC7 was significantly softer and more deformable than non-tumorigenic cells like MCF10A. There was a correlation between softness and deformability of chromatin and the metastatic aggressiveness of the tumors
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Spatial-temporal dynamics and metabolic alterations of p53 upon cellular stress
Full text linkp53 is a tumor suppressor protein that plays a very important role in determining the fate of damaged cells. Depending on the extent of damage, p53 being a transcription factor, induces target genes that are involved in cell repair mechanisms and apoptosis. In doing so, it prevents proliferation of abnormal cells that could lead to tumorigenesis. Primarily existing in its monomeric or dimeric form, when activated, it binds to DNA as a tetramer. The localization of these tetramers in cells has never been mapped. Since p53 is mutated in 50% of human cancers, its ability to tetramerize efficiently and hence bind to the DNA is disrupted leading to tumor progression. Here we use the Number and Brightness (N&B) analysis, a powerful method to measure protein oligomerization pixel by pixel from raster scanned images thereby providing spatial maps of p53 aggregates. The research described here shows, for the first time, the oligomerization maps of the p53 protein and its mutant counterparts to establish the crucial role of p53 tetramers in tumor suppression. In addition, p53 also regulates the metabolism of the cell by modulating important metabolic pathways upon cellular stress. To determine whether this switch is indicative of the balance between apoptosis and DNA repair, the phasor approach to lifetime imaging microscopy (FLIM) was employed to detect the free and bound lifetime of reduced nicotinamide adenine dinucleotide (NADH). The shifts in lifetime are informative of the level of stress and give an indication whether the cell is undergoing cell cycle arrest or apoptosis. The ratios of free and bound NADH obtained from this data may be used as a marker of transcriptional activity. The N&B and FLIM results together provide an insight to p53 activation and this information can be further exploited to improve the field of cancer research
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Estrogen Receptor Alpha Dynamics and Function in Mammalian Cells
Full text linkThe role of estrogen receptors (ER) is highly dependent on their sub-cellular localization and concentration. Here, we propose an approach to detect molecular transport, diffusion and localization of the estrogen receptor by measuring the time cross-correlation between pairs of locations and the average number of molecules by means of fluorescence fluctuations in mammalian cells. From this data we find that there is concentration dependence for the localization of the estrogen receptor and that 17--Estradiol (E2) reduces the apparent diffusion of the receptor. In addition, we use fluorescence lifetime imaging, a label-free, non-invasive imaging method to demonstrate changes in the glucose metabolic pathway in ER-positive breast cancer cells. We observe a higher free to bound NADH ratio in high glucose conditions, reflecting and increased glycolysis/oxidative phosphorylation ratio. Furthermore, E2 is able to potentiate metabolic adaptation and cell viability depending on the glucose availability. Taking advantage of a wide array of available biophysical analysis techniques may provide additional useful information for estrogen receptors and in breast cancer research
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Acute Alcohol Exposure Shifts Metabolism of Breast Cancer Cells
Full text linkAlcohol consumption has been recognized as a risk factor for breast cancer. It is positively correlated with the progression of breast cancer. Ethanol is studied to shift the metabolism of breast cancer cells. According to the Warburg Effect, cancer cells predominantly produce their energy through a high rate of glycolysis. I hypothesized that with acute ethanol exposure the breast cancer cells will shift their metabolism from oxidative phosphorylation to glycolysis. The metabolic shift of cancer cells changes the ratio of free and protein bound NADH, which is an essential coenzyme in cellular metabolic pathway. As such, investigating the fluorescent lifetime of free and protein bound NADH of breast cancer cells through Fluorescence Lifetime Imaging Microscopy (FLIM), I report the preliminary results on the metabolic shifts of two breast cancer cell lines: MDA-MB-231 and MCF-7 due to acute alcohol exposure
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