403 research outputs found
Monitoring intracellular degradation of exogenous DNA using diffusion properties
No full textArtificial nonviral gene vectors have the potential to improve the safety of gene therapy; however, such artificial vectors are less efficient for gene expression due to the existence of various barriers to delivery of exogenous DNAs to the nucleus in the living cell. Here we describe the degradation activities of cytoplasmic nucleases, which are involved as a barrier to efficient exogenous gene expression. The size and structure of degraded DNA were monitored by fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) in solution and in the cytoplasm of living cells. Differences in degradation by endo- and exonucleases were confirmed by differences in the size and structure of DNA. Moreover, we confirmed the influence of the exonuclease degradation in cytoplasm on the expression rate of DNA transfection with a cationic lipid. Based on a comparison of in vitro and cell measurements, we conclude that cytoplasmic degradation by exonucleases can be a considerable barrier against efficient gene delivery
Frontmatter, Table of Contents, Preface, List of Authors
No full textFrontmatter, Table of Contents, Preface, List of Author
Microenvironment and Effect of Energy Depletion in the Nucleus Analyzed by Mobility of Multiple Oligomeric EGFPs
No full textFour different tandem EGFPs were constructed to elucidate the nuclear microenvironment by quantifying its diffusional properties in both aqueous solution and the nuclei of living cells. Diffusion of tandem EGFP was dependent on the length of the protein as a rod-like molecule or molecular ruler in solution. On the other hand, we found two kinds of mobility, fast diffusional mobility and much slower diffusional mobility depending on cellular compartments in living cells. Diffusion in the cytoplasm and the nucleoplasm was mainly measured as fast diffusional mobility. In contrast, diffusion in the nucleolus was complex and mainly much slower diffusional mobility, although both the fast and the slow diffusional mobilities were dependent on the protein length. Interestingly, we found that diffusion in the nucleolus was clearly changed by energy depletion, even though the diffusion in the cytoplasm and the nucleoplasm was not changed. Our results suggest that the nucleolar microenvironment is sensitive to energy depletion and very different from the nucleoplasm
Size determination of optineurin foci
No full textOptineurin (OPTN) plays an important role in membrane trafficking processes such as exocytosis and autophagy. The sizes and rate of formation of accumulated structures comprising OPTN, such as foci or inclusion bodies (IBs), are often disrupted by amyotrophic lateral sclerosis (ALS) and glaucoma-associated mutants of OPTN. Therefore, methods for the quantitative measurement of the size of the accumulated structure are necessary. Here, we show that, using spatial image correlation spectroscopy (ICS), the average diameter of accumulated structures of the wild-type and disease-associated mutants in living cells may he easily determined. Although OPTN was found to frequently form foci in the cytoplasm, regardless of ALS- and glaucoma-associated mutation, the diameter of OPTN foci decreased in an ALS-associated mutant and increased in a glaucoma-associated mutant. However, a portion of cells carried IBs of the AM-associated mutant that were larger than micrometre and ellipse-like shape, suggesting that this mutant accumulates non-uniformly in the IBs. The findings suggest that changes in their accumulation, determined via quantitative comparison of the OPTN foci and IBs in the cells, are involved in pathological features of ALS. In addition, this method enables rapid comparison of the average sizes of various other intracellular structures such as granules
Two-detector number and brightness analysis reveals spatio-temporal oligomerization of proteins in living cells
No full textNumber and brightness analysis (N&B) is a useful tool for the simultaneous visualization of protein oligomers and their localization, with single-molecule sensitivity. N&B determines particle brightness (fluorescence intensity per particle) and maps the spatial distribution of fluorescently labeled proteins by performing statistical analyses of the image series obtained using laser scanning microscopy. The brightness map reveals presence of the oligomers of the targeted protein and their distribution in living cells. However, even when corrections are applied, conventional N&B is affected by afterpulsing, shot noise, thermal noise, dead time, and overestimation of particle brightness when the concentration of the fluorescent particles changes during measurement. The drawbacks of conventional N&B can be circumvented by using two detectors, a novel approach that we henceforth call two-detector number and brightness analysis (TD-N&B), and introducing a linear regression of fluorescence intensity. This statistically eliminates the effect of noise from the detectors, and ensures that the correct particle brightness is obtained. Our method was theoretically assessed by numerical simulations and experimentally validated using a dilution series of purified enhanced green fluorescent protein (EGFP), EGFP tandem oligomers in cell lysate, and EGFP tandem oligomers in living cells. Furthermore, this method was used to characterize the complex process of ligand-induced glucocorticoid receptor dimerization and their translocation to the cell nucleus in live cells. Our method can be applied to other oligomer-forming proteins in cell signaling, or to aggregations of proteins such as those that cause neurodegenerative diseases. (C) 2018 Elsevier Inc. All rights reserved
Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate
No full textFluorescence cross-correlation spectroscopy (FCCS) reveals information about the spatiotemporal coincidence of two spectrally well-defined fluorescent molecules in a small observation area at the level of single-molecule sensitivity. To simultaneously evaluate the activities of caspase-3 and caspase-9, we constructed a chimeral protein that consisted of tandemly fused enhanced cyan fluorescent protein (ECFP), monomeric red fluorescent protein (mCherry) and monomeric yellow fluorescent protein (Venus). In HeLa cell lysates, a combination of tumor necrosis factor-α (TNF-α)- and cycloheximide (CHX-)-induced apoptosis was monitored. In this, decreases of cross-correlation amplitudes were observed between ECFP and mCherry and between mCherry and Venus. Moreover, time-dependent monitoring of single cells revealed decreases in the cross-correlation amplitudes between ECFP and mCherry and between mCherry and Venus before morphologic changes were observed by laser scanning fluorescence microscopy (LSM). Thus, our method could predict the fate of the cell in the early apoptotic stage
Empirical Bayes method using surrounding pixel information for number and brightness analysis
No full textNumber and brightness (N&B) analysis is useful for monitoring the spatial distribution of the concentration and oligomeric state of fluorescently labeled proteins in cells. N&B analysis is based on the statistical analysis of fluorescence images by using the method of moments (MoM). Furthermore, N&B analysis can determine the particle number and particle brightness, which indicate the concentration and oligomeric state, respectively. However, the statistical accuracy and precision are limited in actual experiments with fluorescent proteins, owing to low excitation and the limited number of images. In this study, we applied maximum likelihood (ML) estimation and maximum a posteriori (MAP) estimation coupled with the empirical Bayes (EB) method (referred to as EB-MAP). In EB-MAP, we constructed a simple prior distribution for a pixel to utilize the information of the surrounding pixels. To evaluate the accuracy and precision of our method, we conducted simulations and experiments and compared the results of MoM, ML, and EB-MAP. The results showed that MoM estimated the particle number with many outliers. The outliers hampered the visibility of the spatial distribution and cellular structure. In contrast, EB-MAP suppressed the number of outliers and improved the visibility notably. The precision of EB-MAP was better by an order of magnitude in terms of particle number and 1.5 times better in terms of particle brightness compared with those of MoM. The proposed method (EB-MAP-N&B) is applicable to studies on fluorescence imaging and would aid in accurately recognizing changes in the concentration and oligomeric state in cells. Our results hold significant importance because quantifying the concentration and oligomeric state would contribute to the understanding of dynamic processes in molecular mechanism in cells
Conformational stabilization of optineurin by the dynamic interaction of linear polyubiquitin
No full textOptineurin produces intracellular multi-functions involving autophagy, vesicular trafficking, and negative regulation of inflammation signaling through interaction with various proteins such as ATG8/LC3, Rab8, and polyubiquitin. Optineurin is a component of cytoplasmic inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis (ALS), and its mutation E478G, has been identified in patients with ALS. However, the mechanism by which polyubiquitin binding modulates the interaction partners of OPTN and ALS-associated IB formation is still unclear. To address this issue, we analyzed the interaction of Optineurin with Rab8 and LC3 in the absence and presence of linear polyubiquitin chains using fluorescence cross-correlation spectroscopy and IB formation efficiency of the E478G mutant of Optineurin during Rab8 depletion using fluorescence microscopy. Here, we hypothesize that linear polyubiquitin binding to Optineurin dynamically induces LC3 association and Rab8 dissociation, likely through a conformational change of Optineurin, and the dynamic conformational change may prevent the aggregate formation of mutant Optineurin
Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding
No full textClassical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS(SV40)) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS(SV40) in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS(SV40) formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS(SV40) likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS(SV40) can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells
Molecular chaperone HSP70 prevents formation of inclusion bodies of the 25-kDa C-terminal fragment of TDP-43 by preventing aggregate accumulation
No full textTransactive response DNA/RNA-binding protein 43-kDa (TDP-43) C-terminal fragments, such as a 25-kDa fragment (TDP-25), have been identified as a ubiquitinated and phosphorylated components of inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis patients. Cells contain proteins that function as molecular chaperones and prevent aggregate formation of misfolded and aggregation-prone proteins. Recently, we reported that heat shock protein (HSP)70, an abundant molecular chaperone, binds to TDP-25 in an ATP-dependent manner; however, whether HSP70 can prevent the formation of TDP-25-related IBs remains unknown. Here, we showed that HSP70 prevented TDP-25 aggregation according to green fluorescent protein-tagged TDP-25 (G-TDP-25) colocalization in the cytoplasm with mCherry-tagged HSP70 (HSP70-R). The mobile fraction of HSP70-R in the cytoplasmic IBs associated with G-TDP-25 increased relative to that of G-TDP-25, suggesting that HSP70 strongly bound to G-TDP-25 in the IBs, whereas a portion remained dissociated from the IBs. Importantly, the proportion of G-TDP-25 IBs was significantly decreased by HSP70-R overexpression; however, G-TDP-25 levels in the insoluble fraction remained unchanged by HSP70-R overexpression, suggesting that G-TDP-25 formed aggregated species that cannot be dissolved, even in the presence of strong detergents. These results indicated that HSP70 prevented the accumulation of G-TDP-25 aggregates in cytoplasmic IBs, but was insufficient for G-TDP-25 disassembly and solubilization
- …
