709 research outputs found
The role of cytoskeleton networks on lipid-mediated delivery of DNA
No full textBackground: 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
Development of an image Mean Square Displacement (iMSD)-based method as a novel approach to study the intracellular trafficking of nanoparticles
No full textFluorescence 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
Changes in Chromatin Compaction During the Cell Cycle Revealed by Micrometer-Scale Measurement of Molecular Flow in the Nucleus
No full textAbstractWe 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
In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow
No full textNo 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
Interlaced biophysical methods to unveil membrane receptor organization
No full textFrom the introduction: "In my thesis I set out to address these issues by a toolbox of fluorescence imaging techniques such as Förster Resonance Energy Transfer, Fluorescence Anisotropy Imaging, and Spatio-temporal image correlation spectroscopy. I investigated receptor properties such as membrane mobility, microtubule and caveolin-1 binding, and TRPV1 oligomerization status. My experimental strategies benefited from the use of genetically-encodable fluorescent reporters belonging to the green fluorescent protein family.
Pair correlation microscopy of intracellular molecular transport
No full textPair 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
Intracellular trafficking of cationic liposome–DNA complexes in living cells
No full textThree-dimensional single-particle tracking (SPT) was used to calculate the mean square displacement (MSD) and the diffusion coefficients of multicomponent cationic liposome–DNA complexes (lipoplexes) in CHO-K1 living cells. In untreated (NT) control cells, we found that the intracellular lipoplex motion was either directed or Brownian with active transportation being definitely more frequent (more than 70%) than Brownian diffusion. The MSD analysis was supported by the calculation of the three-dimensional asphericity, A3, which was close to unity, denoting the preponderant occurrence of movement along a direction. To elucidate the role of the cytoskeleton structure in the lipoplex trafficking, cells were treated with cytoskeleton (actin microfilaments and microtubules) polymerization inhibitors (latrunculin B and nocodazole, respectively). When cells were treated with inhibitors, the lipoplex movement tended towards a random walk at the expense of directed motion. The disassembly of microtubules had a stronger effect on the reduction of directional movement than that of actin microfilaments. Relevance of the results for enhanced gene delivery is discussed
The Impact of Mitotic versus Interphase Chromatin Architecture on the Molecular Flow of EGFP by Pair Correlation Analysis
No full textAbstractHere 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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Multiphoton imaging and phasor approach to identify new biomarkers in Huntington Disease
No full textNeurodegenerative diseases occur when brain cells (neurons) start to deteriorate. Changes in these cells will lead to dysfunction and eventual cell death. This will lead to mild symptoms like problems with coordination, psychiatric disorders, or memory loss; and as more neurons die the symptoms also progressively worsen. World Health Organization (WHO) indicates up to 1 billion people worldwide are affected by various types of neurodegenerative diseases. In this study, I focused on Huntington disease (HD), a model to study neurodegeneration that is caused by a glitch in a single gene called huntingtin gene (HTT). Huntington disease is an autosomal dominant inherited neurodegenerative disease characterized by movement, cognitive and emotional disorders. We all carry HTT; however, the normal length of DNA trinucleotide, CAG, that codes for glutamine are between 10-35. The expanded repeats of above 40 or more will lead to HD. Using advanced functional imaging technique called Two-Photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM), and spectral and temporal phasor approach, spectro-temporal phasor map in living mammalian cells and animal tissue was obtained. Using this sophisticated imaging technique, I have developed new methods and identified novel biomarkers that can help detect Huntington disease early on. This can also help for evaluating the efficacy of treatment. The novel method established in this work is noninvasive and can be performed at the single cell level. Phasor transformation used here simplifies the FLIM and spectral measurements by providing a graphical global view of the process at each pixel and avoids some of the complexity of the multi-exponential analysis. In this way, using a fit free approach that can be applied to both time and frequency domain measurements, Fluorescence Lifetime and spectral emission can be analyzed. It is hoped that this work shed a light on understanding the mechanism of Huntington disease and for new drug discovery and early diagnosis of the disease. The approach introduced in this work can also be applied as a method for understating similar neurodegenerative diseases. This work is supported in part by NIH grant P41 GM103540, NSF BEST IGERT and UC PDY grant
Growth performance of Penaeus monodon in lablab, lumut, and digman ponds under various farm practices
No full textThe influence of lablab, lumut (filamentous algae), and digmaan (Najas graminea) on the growth of prawns as associates of natural food biomass in brackishwater ponds was investigated. Ponds, each grown with the above macrophytes species and with no supplementary feed used, were selected as sampling areas. Sampling was conducted on a biweekly basis. Results showed that although the specific growth rate of prawns reared in the lablab pond was significantly higher than that of prawns reared in the lumut and digman ponds (p<0.01), absolute values in growth showed otherwise. Prawns reared in the digman pond attained highest growth which was significant (p<0.01). The digman pond was able to support a much higher density and produce a higher growth, coupled with a substantial survival. The digman pond yielded 606 kg of prawns, while the lablab pond yielded 569.5 kg and the lumut pond 305 kg. At value of $ 10 per kg, the rearing of prawns in the digman pond would be more profitable than the other treatments
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