Dynamics of Thin-Filament Activation in Rabbit Skeletal Muscle Fibers Examined by Time-Resolved X-Ray Diffraction

By using skinned-rabbit skeletal muscle fibers, the time courses of changes of thin filament-based x-ray reflections were followed at a 3.4-ms time resolution during thin-filament activation. To discriminate between the effects of calcium binding and myosin binding on thin-filament activity, measurements were performed after caged-calcium photolysis in fibers with full-filament or no-filament overlap, or during force recovery after a quick release. All three reflections examined, i.e., the second actin layer line (second ALL, reporting the tropomyosin movement), the sixth ALL (reporting actin structural change), and the meridional troponin reflections, exhibited calcium-induced and myosin-induced components, but their rate constants and polarities were different. Generally, calcium-induced components exhibited fast rate constants (>100 s⁻¹). The myosin-induced components of the second ALL had a rate constant similar to that of the force (7-10 s⁻¹), but that of the sixth ALL was apparently faster. The myosin-induced component of troponin reflection was the only one with negative polarity, and was too slow to be analyzed with this protocol. The results suggest that the three regulation-related proteins change their structures with different rate constants, and the significance of these findings is discussed in the context of a cooperative thin-filament activation mechanism.     

Homodimerization of Amyloid Precursor Protein at the Plasma Membrane: A homoFRET Study by Time-Resolved Fluorescence Anisotropy Imaging

Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer’s disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions.     

Reaction Dynamics of Halorhodopsin Studied by Time-Resolved Diffusion

Reaction dynamics of a chloride ion pump protein, halorhodopsin (HR), from Natronomonas pharaonis (N. pharaonis) (NpHR) was studied by the pulsed-laser-induced transient grating (TG) method. A detailed investigation of the TG signal revealed that there is a spectrally silent diffusion process besides the absorption-observable reaction dynamics. We interpreted these dynamics in terms of release, diffusion, and uptake of the Cl⁻ ion. From a quantitative global analysis of the signals at various grating wavenumbers, it was concluded that the release of the Cl⁻ ion is associated with the L2 → (L2 (or N) ⇆ O) process, and uptake of Cl⁻ occurs with the (L2 (or N) ⇆ O) → NpHR′ process. The diffusion coefficient of NpHR solubilized in a detergent did not change during the cyclic reaction. This result contrasts the behavior of many photosensor proteins and implies that the change in the H-bond network from intra- to intermolecular is not significant for the activity of this protein pump.     

Activation of Nuclear Calcium Dynamics by Synaptic Stimulation in Cultured Cortical Neurons

L-type voltage-sensitive Ca2+ channels (VSCCs) are enriched on the neuronal soma and trigger gene expression during synaptic activity. To understand better how these channels regulate somatic and nuclear Ca2+ dynamics, we have investigated Ca2+ influx through L-type VSCCs following synaptic stimulation, using the long-wavelength Ca2+ indicator fluo-3 combined with laser scanning confocal microscopy. Single synaptic stimuli resulted in rapid Ca2+ transients in somatic cytoplasmic compartments (<5 ms rise time). Nuclear Ca2+ elevations lagged behind cytoplasmic levels by approximately 60 ms, consistent with a dependence on diffusion from a cytoplasmic source. Pharmacological experiments indicated that L-type VSCCs mediated approximately 50% of the nuclear and somatic (cytoplasmic) Ca2+ elevation in response to strong synaptic stimulation. In contrast, relatively weak excitatory postsynaptic potentials (EPSPs; approximately 15 mV) or single action potentials were much less effective at activating L-type VSCCs. Antagonist experiments indicated that activation of the NMDA-type glutamate receptor leads to a long-lasting somatic depolarization necessary to activate L-type VSCCs effectively during synaptic stimuli. Simulation of action potential and somatic EPSP depolarization using voltage-clamp pulses indicated that nuclear Ca2+ transients mediated by L-type VSCCs were produced by sustained depolarization positive to -25 mV. In the absence of synaptic stimulation, action potential stimulation alone led to elevations in nuclear Ca2+ mediated by predominantly non-L-type VSCCs. Our results suggest that action potentials, in combination with long-lived synaptic depolarizations, facilitate the activation of L-type VSCCs. This activity elevates somatic Ca2+ levels that spread to the nucleus.     

Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment.     

Fluorescence Anisotropy Reveals Order and Disorder of Protein Domains in the Nuclear Pore Complex

We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport.     

Dynamics of the Ligand Binding Domain Layer during AMPA Receptor Activation

Ionotropic glutamate receptors are postsynaptic tetrameric ligand-gated channels whose activity mediates fast excitatory transmission. Glutamate binding to clamshell-shaped ligand binding domains (LBDs) triggers opening of the integral ion channel, but how the four LBDs orchestrate receptor activation is unknown. Here, we present a high-resolution x-ray crystal structure displaying two tetrameric LBD arrangements fully bound to glutamate. Using a series of engineered metal ion trapping mutants, we showed that the more compact of the two assemblies corresponds to an arrangement populated during activation of full-length receptors. State-dependent cross-linking of the mutants identified zinc bridges between the canonical active LBD dimers that formed when the tetramer was either fully or partially bound by glutamate. These bridges also stabilized the resting state, consistent with the recently published full-length apo structure. Our results provide insight into the activation mechanism of glutamate receptors and the complex conformational space that the LBD layer can sample.     

雄激素受体对真核基因转录的调控

雄激素受体是典型的核受体,它对真核基因转录的调控作用受到日益广泛的重视。本文主要阐述了雄激素受体的分子结构,重点总结了雄激素受体介导真核基因转录起始的过程,概述了激素受体辅助使用因子及受体的核转运等受体功能的调控,这些是进一步研究真核基因表达调控机制及治疗雄激素相关疾病的理论基础。     

Optimizing Detection of Tissue Anisotropy by Fluorescence Recovery after Photobleaching

Fluorescence recovery after photobleaching (FRAP) has been widely used to measure fluid flow and diffusion in gels and tissues. It has not been widely used in detection of tissue anisotropy. This may be due to a lack of applicable theory, or due to inherent limitations of the method. We discuss theoretical aspects of the relationship between anisotropy of tissue structure and anisotropy of diffusion coefficients, with special regard to the size of the tracer molecule used. We derive a semi-mechanistic formula relating the fiber volume fraction and ratio of fiber and tracer molecule diameters to the expected anisotropy of the diffusion coefficients. This formula and others are tested on simulated random walks through random simulated and natural media. We determine bounds on the applicability of FRAP for detection of tissue anisotropy, and suggest minimum tracer sizes for detection of anisotropy in tissues of different composition (fiber volume fraction and fiber diameter). We find that it will be easier to detect anisotropy in monodisperse materials than in polydisperse materials. To detect mild anisotropy in a tissue, such as cartilage, which has a low fiber fraction would require a tracer molecule so large that it would be difficult to deliver to the tissue. We conclude that FRAP can be used to detect tissue anisotropy when the tracer molecule is sufficiently large relative to the fiber diameter, volume fraction, and degree of polydispersivity, and when the anisotropy is sufficiently pronounced.