Biphasic pattern of gelsolin expression and variations in gelsolin-actin interactions during myogenesis

Scholz A, Hinssen H. Biphasic pattern of gelsolin expression and variations in gelsolin-actin interactions during myogenesis. Exp. Cell Res. 1995;219(2):384-391

Electron-microscopical localization of gelsolin in various crustacean muscles

Unger A, Hinssen H. Electron-microscopical localization of gelsolin in various crustacean muscles. CELL AND TISSUE RESEARCH. 2010;341(2):313-323.Gelsolin was localized by immunoelectron microscopy in fast and slow cross-striated muscles of the lobster Homarus americanus. When ultrathin sections of the muscles were labelled with anti-gelsolin and a gold-conjugated second antibody, 90% of all gold particles in the myoplasm were detected on myofibrils, preferentially in the I-band and AI-region of the sarcomeres. Both the region of the H-zone (lacking thin filaments) and the Z-disc contained no or little gold label. Under physiological conditions, a close association of gelsolin with the thin filaments was observed for both muscle types. The preferential localization of particles in the I- and AI-region indicated that gelsolin was distributed randomly over the whole length of the thin filaments. Preincubation of muscle strips with Ringer solution containing 0.5 mM EGTA resulted in a significantly different distribution pattern; gold particles were now localized preferentially in the cell periphery close to the sarcolemma, with significantly decreased abundance in the centre of the cell. Compared with the muscle under physiological conditions, the number of gold particles over sarcomeric structures was significantly reduced. Thus, binding of gelsolin to the thin filaments is apparently reversible in vivo and depends on the presence of calcium ions. We assume a functional role for gelsolin in the actin turnover processes in invertebrate muscle systems

Difference in polymerization and steady-state dynamics of free and gelsolin-capped filaments formed by alpha- and beta-isoactins

Khaitlina S, Hinssen H. Difference in polymerization and steady-state dynamics of free and gelsolin-capped filaments formed by alpha- and beta-isoactins. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS. 2008;477(2):279-284.The polymerization of scallop p-like actin is significantly slower than that of skeletal muscle alpha-actin. To reveal which steps of polymerization contribute to this difference, we estimated the efficiency of nucleation of the two actins, the rates of filament elongation at spontaneous and gelsolin-nucleated polymerization and the turnover rates of the filament Subunits at steady-state. Scallop actin nucleated nearly twice less efficient than rabbit actin. In actin filaments with free ends, when dynamics at the barbed ends overrides that at the pointed ends, the relative association rate constants of alpha- and beta-actin were similar, whereas the relative dissociation rate constant of beta-ATP-actin subunits was 2- to 3-fold higher than that of alpha-actin. The 2- to 3-fold faster polymerization of skeletal muscle versus scallop Ca-actin was preserved with gelsolin-capped actin filaments when only polymerization at the pointed end is possible. With gelsolin-induced polymerization, the rate constants of dissociation of ATP-actin subunits from the pointed ends were similar, while the association rate constant of beta-actin to the pointed filament ends was twice lower than that of alpha-actin. This difference may be of physiological relevance for functional intracellular sorting of actin isoforms. (C) 2008 Elsevier Inc. All rights reserved

Identification and localisation of nebulin as a thin filament component of invertebrate chordate muscles

Fock U, Hinssen H. Identification and localisation of nebulin as a thin filament component of invertebrate chordate muscles. JOURNAL OF COMPARATIVE PHYSIOLOGY B-BIOCHEMICAL SYSTEMIC AND ENVIRONMENTAL PHYSIOLOGY. 1999;169(8):555-560.The giant actin-binding protein nebulin is regarded as a component of the thin filaments in vertebrate skeletal muscles, whereas the existence of nebulin in invertebrate muscles has not yet been demonstrated. Using the cross-reactivities of polyclonal antibodies raised against nebulin from muscles of trout and lamprey. we were able to identify nebulin in the myofibrils of the cephalochordate Branchiostoma lanceolatum (lancelet) by immunoblot and immunofluorescence techniques. The similar to 720-kDa protein is localised in the I-bands of the sarcomere, where vertebrate nebulin has previously also been shown to br localised. Since lancelets have a phylogenetically key position at the vertebrate/invertebrate boundary, the detection of a high-molecular-weight nebulin indicates that nebulin-like proteins may be common to striated muscles in all chordates and increases the probability that non-chordate invertebrates also possess nebulin-related proteins

Nebulin is a thin filament protein of the cardiac muscle of the agnathans

Fock U, Hinssen H. Nebulin is a thin filament protein of the cardiac muscle of the agnathans. JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY. 2002;23(3):205-213.Nebulin is an integral protein of skeletal muscle thin laments and probably acts as a ruler for the thin lament length. Cardiac muscles of higher vertebrates have been shown earlier to lack nebulin. Instead in human and chicken cardiac muscle the much smaller protein nebulette replaces nebulin. Since nebulette is confined to the Z-disc region of the sarcomere and does not span the whole thin lament length, it must have functions significantly different from those assumed for nebulin. We have investigated nebulin in skeletal and cardiac muscles of the agnathans (lamprey, hagfish), elasmobranchs (shark), chondrosts (sturgeon) and teleosts (trout, eel) by SDS-PAGE and immunodetection methods. Among these, lamprey and hagfish cardiac muscles are unique in that both contain full-length nebulin identical in molecular mass to the nebulin of the respective body muscle. Using immuno fluorescence microscopy, lamprey cardiac nebulin was localised in the I-band of the sarcomere, the same as for nebulin in skeletal muscle. In contrast to this, all gnathostome species investigated lacked nebulin in cardiac muscles, while it was present in the respective skeletal muscles. This clearly shows that nebulin is not exclusively present in skeletal muscles of chordates. The findings also demonstrate a rare case of dramatic size reduction of a protein during evolution

Ca-dependent binding of actin to gelsolin

Khaitlina S, Hinssen H. Ca-dependent binding of actin to gelsolin. FEBS LETTERS. 2002;521(1-3):14-18.Ca2+ of 0.3-1.0 muM induces both the exposure of tryptic cleavage sites within the gelsolin molecule inaccessible in the Ca-free conformation, and binding of one actin monomer to the N-terminal half of gelsolin. On the other hand, gelsolin-induced enhancement of pyrene actin fluorescence was observed only above 50 muM Ca2+, and a ternary actin/gelsolin complex preformed in 200 muM Ca2+ was stable only above 30 muM Ca2+. These results provide direct evidence for a Ca-induced transitions from closed to open conformation of the gelsolin molecule in the range of 3 x 10(-7) to 10(-6) M Ca2+. They also suggest that Ca2+ > 10(-5) M is required to stabilize actin-actin contacts in the 2:1 actin/gelsolin complex. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved

IMMUNOCYTOCHEMICAL LOCALIZATION OF GELSOLIN IN FIBROBLASTS, MYOGENIC CELLS, AND ISOLATED MYOFIBRILS

DISSMANN E, Hinssen H. IMMUNOCYTOCHEMICAL LOCALIZATION OF GELSOLIN IN FIBROBLASTS, MYOGENIC CELLS, AND ISOLATED MYOFIBRILS. EUROPEAN JOURNAL OF CELL BIOLOGY. 1994;63(2):336-344.Gelsolin was localized by immunofluorescence in fibroblasts and skeletal muscle cells using antibodies which eliminated the risk of detecting xenogenic plasma gelsolin. Gelsolin was consistently found to be closely associated with the elements of the microfilament system: In fibroblasts, a preferential labeling of the stress fibers was observed, whereas with myogenic cells and myofibrils isolated from skeletal muscle, a specific staining of the I-Z-I region in the sarcomeres was found. From double labeling of gelsolin and actin it became evident that the staining patterns for both proteins were practically coincident: The width and location of the fluorescent bands varied with the degree of contraction of the myofibrils. The region of cross-bridges in the A-zone, where thick and thin filaments overlap, remained unstained. The gelsolin staining of myofibrils was EGTA-resistant; it persisted after glycerol extraction and extensive washing. The presence of gelsolin in myofibrils after this treatment was also confirmed by immunoblotting. From these observations it was concluded that a significant part of the total gelsolin in skeletal muscle cells is tightly associated with the thin filaments, and is an integral part of the myofibrils even at low Ca++-concentrations. Fmom the coincidence of actin and gelsolin staining in myofibrils it was concluded that gelsolin is localized along the whole length of the thin filaments in the sarcomere. Such a mode of association with filamentous actin is not fully explained by the current models of interaction of gelsolin with F-actin which involve either a filament severing or a capping of the ends of actin filaments rather than a stable lateral binding to the filaments without severing. It is assumed that, in myofibrils, actin-binding proteins like tropomyosin and nebulin inhibit the severing process but not the binding of gelsolin

Isoforms of gelsolin from lobster striated muscles differ in Calcium-dependence

Unger A, Brunne B, Hinssen H. Isoforms of gelsolin from lobster striated muscles differ in Calcium-dependence. Archives Of Biochemistry And Biophysics. 2013;536(1):38-45.Two distinct isoforms of the Ca-dependent actin filament severing protein gelsolin were identified in cross-striated muscles of the American lobster. The variants (termed LG1 and LG2) differ by an extension of 18 AA at the C-terminus of LG1, and by two substitutions at AA735 and AA736, the two C-terminal amino acids of LG2. Functional comparison of the isolated and purified proteins revealed gelsolin-typical properties for both with differences in Ca2+-sensitivity, LG2 being activated at significant lower Ca-concentration than LG1: Half maximal activation for both filament severing and G-actin binding was similar to 4 x 10(-7) M Ca2+ for LG2 vs. similar to 2 x 10(-6) M Ca2+ for LG1. This indicates a differential activation for the two isoproteins in vivo where they are present in almost equal amounts in the muscle cell. Structure prediction modeling on the basis of the known structure of mammalian gelsolin shows that LG2 lacks the C-terminal alpha-helix which is involved in contact formation between domains G6 and G2. In both mammalian gelsolin and LG1, this "latch bridge" is assumed to play a critical role in Ca2+-activation by keeping gelsolin in a closed, inactive conformation at low [Ca2+]. In LG2, the reduced contact between G6 and G2 may be responsible for its activation at low Ca2+-concentration. (C) 2013 Elsevier Inc. All rights reserved