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91.
Adam D. Hoppe Brandon L. Scott Timothy P. Welliver Samuel W. Straight Joel A. Swanson 《PloS one》2013,8(6)
Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells. 相似文献
92.
93.
Lukas Jaroslaw Motloch Robert Larbig Tina Gebing Sara Reda Astrid Schwaiger Johannes Leitner Martin Wolny Lars Eckardt Uta C. Hoppe 《PloS one》2016,11(2)
Introduction
The possible role of UCP2 in modulating mitochondrial Ca2+-uptake (mCa2+-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.Methods
Thus, we analyzed mCa2+-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca2+-transients from mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2-/- mice.Results
Isolated mitochondria showed a Ru360 sensitive mCa2+-uptake, which was significantly decreased in UCP2-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca2+-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2-/- cardiomyocytes (Ca2+)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca2+)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca2+-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa2+-uptake in WT mitoplasts and (Ca2+)m of cardiomyocytes leading to an increase of (Ca2+)c while no ATP dependent effect was observed in UCP2-/-.Conclusion
Our results indicate regulatory effects of UCP2 on mCa2+-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling. 相似文献94.
95.
Louise Robertson Travis Featherby Stuart Howell James Hughes Paul Thomas 《Genes, Brain & Behavior》2019,18(5)
Mutations in proline‐rich transmembrane protein 2 (PRRT2) cause a range of episodic disorders that include paroxysmal kinesigenic dyskinesia and benign familial infantile epilepsy. Mutations are generally loss of function and include the c649dupC frameshifting mutation that is present in around 80% of affected individuals. To investigate how Prrt2 loss of function mutations causes disease, we performed a phenotypic investigation of a transgenic Prrt2 knockout (Prrt2 KO) mouse. We observed spontaneous paroxysmal episodes with behavioural features of both seizure and movement disorders, as well as unexplained deaths in KO and HET animals. KO mice showed spatial learning deficits in the Morris water maze, as well as gait abnormalities in the quantitative Digigait analysis; both of which may be representative of the more severe phenotypes experienced by homozygous patients. These findings extend the described phenotypes of Prrt2 mutant mice, further confirming their utility for in vivo investigation of the role of Prrt2 mutations in episodic diseases. 相似文献
96.
Travis F. Henchie Ellen M. Gravallese Todd L. Bredbenner 《Computer methods in biomechanics and biomedical engineering》2019,22(10):942-952
Quantifying joint deformity in people with rheumatoid (RA) and psoriatic arthritis (PsA) remains challenging. Here, we demonstrate a new method to measure bone erosions and abnormal periosteal growths, based on the difference between a predicted healthy and actual diseased joint surface. We optimized the method by creating and measuring artificial bone erosions and growths. Then we measured 46 healthy and diseased patient surfaces. We found average sensitivity errors of ≤0.27?mm when measuring artificial erosions and growths. Patients had significantly more bone erosion than healthy subjects. Surface based outcomes are a novel way to interpret and quantify bone changes in PsA and RA. 相似文献
97.
The feedbacks from population density to demographic parameters, which drive population regulation, are the accumulated results of several ecological processes. The compensatory feedback from increased population density to fertility includes at least two distinct factors, the effects of decreases in per capita food level and increases in the social density (the number of interacting individuals). Because these effects have been studied separately, their relative importance is unknown. It is also unclear whether food limitation and social density combine additively to influence fertility. We investigated these questions with two factorial experiments on reproduction in the Least Killifish, Heterandria formosa. In one experiment, we crossed two levels of density with two levels of a total food ration that was distributed to all individuals. In the other experiment, we crossed two levels of density with two levels of per capita food. Whereas the first experiment suggested that the effects of variation in food level and density were synergistic, the second experiment indicated that they were not. The apparent synergism—the statistical interaction of food and density levels—was the result of confounding per capita food with social density in that design. In the second experiment, the effects of social density on reproductive rate were stronger than the effects of food level, whereas the effects of food level were stronger on offspring size at parturition than those of social density. The results suggest that the social stresses that emerge at higher densities play an important role in the compensatory response of fertility to density, a role, that is, at least as important as that of decreased per capita food levels. 相似文献
98.
Angela M. Mech Kathryn A. Thomas Travis D. Marsico Daniel A. Herms Craig R. Allen Matthew P. Ayres Kamal J. K. Gandhi Jessica Gurevitch Nathan P. Havill Ruth A. Hufbauer Andrew M. Liebhold Kenneth F. Raffa Ashley N. Schulz Daniel R. Uden Patrick C. Tobin 《Ecology and evolution》2019,9(21):12216-12230
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99.
Todi SV Laco MN Winborn BJ Travis SM Wen HM Paulson HL 《The Journal of biological chemistry》2007,282(40):29348-29358
Ataxin-3, a deubiquitinating enzyme, is the disease protein in spinocerebellar ataxia type 3, one of many neurodegenerative disorders caused by polyglutamine expansion. Little is known about the cellular regulation of ataxin-3. This is an important issue, since growing evidence links disease protein context to pathogenesis in polyglutamine disorders. Expanded ataxin-3, for example, is more neurotoxic in fruit fly models when its active site cysteine is mutated (1). We therefore sought to determine the influence of ataxin-3 enzymatic activity on various cellular properties. Here we present evidence that the catalytic activity of ataxin-3 regulates its cellular turnover, ubiquitination, and subcellular distribution. Cellular protein levels of catalytically inactive ataxin-3 were much higher than those of active ataxin-3, in part reflecting slower degradation. In vitro studies revealed that inactive ataxin-3 was more slowly degraded by the proteasome and that this degradation occurred independent of ubiquitination. Slower degradation of inactive ataxin-3 correlated with reduced interaction with the proteasome shuttle protein, VCP/p97. Enzymatically active ataxin-3 also showed a greater tendency to concentrate in the nucleus, where it colocalized with the proteasome in subnuclear foci. Taken together, these and other findings suggest that the catalytic activity of this disease-linked deubiquitinating enzyme regulates several of its cellular properties, which in turn may influence disease pathogenesis. 相似文献