We examined the effect of the differences in spatial arrangement of actin in contracting muscles from the left (LV) and right (RV) ventricles of rabbit heart. Spatial arrangement is a fundamental property of muscle because it reflects the interaction of individual actin molecules with myosin cross-bridges. Macroscopic parameters of contraction, such as maximum tension, speed of shortening or ATPase activity, are unlikely to reveal differences between the ventricles because they are made on whole organs containing trillions of actin and myosin molecules. Averaging the data collected from such a large assembly is likely to conceal small differences. To eliminate complications arising from the differences in the basic myocardial architecture and fiber structures of the ventricles, and to ensure that the molecular crowding influences contraction in the same way as under the in-vivo conditions, we collected data from isolated myofibrils ex-vivo. Control anisotropy experiments revealed that the orientation of actin reflected the orientation of myosin cross-bridges. The results showed that the changes in the distribution of actin induced by the interaction of actin filaments with myosin heads were demonstrated only in LVs, not in RVs. This suggests that both ventricles interact differently with myosin cross-bridges. We think that the stress induced in the thin filaments of LV causes loosening of bonds between actin monomers which leads to greater mobility of actin monomers.
Change in metabolic pathways is an important indicator of modifications in physiological, pathological and/or differentiation state of the cell. Its non-invasive investigation can significantly improve medical practice, namely the determination of the suspected tissue during surgery, including fast biopsy used for pre-operative tissue diagnostics, and/or resection guidance during surgical operations. Early detection of these changes is a prerequisite of fast and precise diagnostics directly in living cells and tissues and is thus crucial for the patient's survival and/or his/hers better quality of life. In this contribution, we review time-resolved fluorescence measurements of endogenous NAD(P)H in cells and tissues aimed at separation of specific metabolic pathways (glycolysis vs. oxidative phosphorylation – OXPHOS). Other complex situations, including the presence of oxidative stress, are also discussed. Current state of knowledge on non-invasive diagnostics of pathophysiological state in living cells and tissues, based on time-resolved autofluorescence, is presented.
Many cellular processes are orchestrated by protein–protein interactions that allow the formation of protein networks involved in subcellular compartmentalization, communication, and signalling. Postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase protein (MAGuK) family, is a central scaffold protein of the postsynaptic density (PSD) of excitatory synapses in the mammalian central nervous system. PSD-95 serves as a matrix for targeting and accumulation of yet other PSD proteins including ion channels and receptors via its three N-terminal PDZ (PSD-95, discs large, zonula occludens-1) domains. However, the stoichiometry of PSD-95 and its binding partners in such complexes and the dynamic regulation of their interactions remain elusive. Here, we have investigated the protein–protein interaction between PSD-95 and the inward rectifier potassium channel Kir2.1, which we consider as a model for other PDZ domain based interactions at synapses. By using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), we show that PSD-95 and Kir2.1 directly interact within clusters which are formed at the plasma membrane of culture cells. Our in vivo FRET data indicate that Kir2.1 binds to more than one PDZ domain of PSD-95, suggesting a structural model of synergistic target binding by the first two PDZ domains of the scaffold protein. We show that the cluster formation is induced by the channel whereas PSD-95 alone does not form clusters. The interaction of PSD-95 and Kir2.1 is dynamically regulated by protein kinase A (PKA) mediated phosphorylation, which is directly visualized and quantified in living cells and in real time.
Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several features, such as high photon efficiency, high lifetime accuracy, resolution of multi-exponential decay profiles, simultaneous recording in several wavelength intervals and optical sectioning capability. The combination of multi-dimensional time-correlated single photon counting (TCSPC) with confocal or two-photon laser scanning meets these requirements almost ideally. Multi-dimensional TCSPC is based on the excitation of the sample by a high repetition rate laser and the detection of single photons of the fluorescence signal. Each photon is characterised by its arrival time with respect to the laser pulse and the coordinates of the laser beam in the scanning area. The recording process builds up a photon distribution over these parameters. The result can be interpreted as an array of pixels, each containing a full fluorescence decay curve. More parameters can be added to the photon distribution, such as the wavelength of the photons, the time from a stimulation of the sample, or the time with respect to an additional modulation of the laser. In this review, the application of the technique will be described for the measurement of molecular environment parameters within a sample, protein interaction experiments by Förster resonance energy transfer (FRET), autofluorescence measurements of cells and tissue, and in-vivo imaging of human skin and the fundus of the human eye.
