title: Colocalization properties of elementary Ca(2+) release signals with structures specific to the contractile filaments and the tubular system of intact mouse skeletal muscle fibers.
abstract: Ca(2+) regulates several important intracellular processes. We combined second harmonic generation (SHG) and two photon excited fluorescence microscopy (2PFM) to simultaneously record the SHG signal of the myosin filaments and localized elementary Ca(2+) release signals (LCSs). We found LCSs associated with Y-shaped structures of the myosin filament pattern (YMs), so called verniers, in intact mouse skeletal muscle fibers under hypertonic treatment. Ion channels crucial for the Ca(2+) regulation are located in the tubular system, a system that is important for Ca(2+) regulation and excitation-contraction coupling. We investigated the tubular system of intact, living mouse skeletal muscle fibers using 2PFM and the fluorescent Ca(2+) indicator Fluo-4 dissolved in the external solution or the membrane dye di-8-ANEPPS. We simultaneously measured the SHG signal from the myosin filaments of the skeletal muscle fibers. We found that at least a subset of the YMs observed in SHG images are closely juxtaposed with Y-shaped structures of the transverse tubules (YTs). The distances of corresponding YMs and YTs yield values between 1.3 μm and 4.1 μm including pixel uncertainty with a mean distance of 2.52±0.10 μm (S.E.M., n=41). Additionally, we observed that some of the linear-shaped areas in the tubular system are colocalized with linear-shaped areas in the SHG images.
authors.affiliation: Medical Biophysics, Institute of Physiology and Pathophysiology, Ruprecht Karls Universität, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany. Electronic address: tihomir.georgiev@physiologie.uni-heidelberg.de.
authors.forename: Tihomir
authors.initials: T
authors.lastname: Georgiev
authors.affiliation: Medical Biophysics, Institute of Physiology and Pathophysiology, Ruprecht Karls Universität, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany; Max Planck Research Unit for Neurogenetics, 60438 Frankfurt am Main, Germany.
authors.forename: Bolek
authors.initials: B
authors.lastname: Zapiec
authors.affiliation: Medical Biophysics, Institute of Physiology and Pathophysiology, Ruprecht Karls Universität, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Förderer
authors.affiliation: Medical Biophysics, Institute of Physiology and Pathophysiology, Ruprecht Karls Universität, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany.
authors.forename: Rainer H A
authors.initials: RHA
authors.lastname: Fink
authors.affiliation: Medical Biophysics, Institute of Physiology and Pathophysiology, Ruprecht Karls Universität, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany; Max Planck Research Unit for Neurogenetics, 60438 Frankfurt am Main, Germany. Electronic address: martin.vogel@biophys.mpg.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Vogel
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008810:Mice, Inbred C57BL; D036641:Microscopy, Fluorescence, Multiphoton; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D015220:Calcium Channels; D009218:Myosins; D002118:Calcium
keywords: Longitudinal tubules; Second harmonic generation microscopy; Verniers; Z tubule