A Sarcomere-Mimetic Gel: Gelation of Astral-Shaped Actin Filaments with Their Plus End Connected on Photopolymer Beads b
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A Sarcomere-Mimetic Gel: Gelation of Astral-Shaped Actin Filaments with Their Plus End Connected on Photopolymer Beads by Myosin Filaments Taiji Ikawa1, Masahito Shiozawa1, Makoto Mouri1, Mamiko Narita1 and Osamu Watanabe1 1
Toyota Central R&D Labs., Inc., Nagakute, Aichi, 480-1192, Japan
ABSTRACT We have developed a method of a stepwise construction of a gel consisting of (i) astralshaped actin filaments with their plus end connected on photo-responsive polymer beads and (ii) bipolar myosin filaments as linkers in order to mimic sarcomeric structure, the basic unit of a muscle. In the method, firstly, 4 ȝm diam. beads were prepared from an acrylate polymer containing azobenzene moiety by a good-solvent evaporation technique. Next, gelsolin, which servers and remains bound to the plus end of an actin filament, was adsorbed and then immobilized on the bead surface by exposure to light from blue light-emitting diodes, and then fluorescent actin filaments were mixed with the beads. Formation of star-like, astral actin filaments on the beads were observed in fluorescent microscopy. Finally, the beads with actin filaments were mixed with myosin mini filaments with ca. 1 ȝm in length. Dozens of the beads were observed to be assembled into a gel form in optical microscopy. After adding adenosine triphosphate to the gel solution, the gel was slowly contract up to 60% comparing with its original volume, suggesting that linker myosin filaments moved on the actin filaments toward the plus end on the beads. INTRODUCTION Motor proteins have a fascinating property that they bind to a cytoskeletal filament and move steadily along it [1]. They are powered by the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and convert chemical energy into mechanical work. Motor proteins cause cytoskeletal filaments to slide against each other, generating the force that drives cell division, ciliary beating and muscle contraction. The muscle contraction is caused by the ATP-driven sliding of highly organized arrays of cytoskeletal actin filaments and myosin II filaments in tiny contractile units called sarcomere. The actin filaments are attached at their plus end to a Z-disc, which represents the lateral boundary of the sarcomere and functions as a nodal point in transmission of force generated within the sarcomere. The actin filaments are overlapped with bipolar assembly of myosin II at the middle of the sarcomere. Force-producing head units stick out from the side of myosin II filaments walk toward the plus ends of two sets of actin filaments of opposite orientations, causing sarcomere shortening. In the late 1980s it has been demonstrated that actin filaments is glide on the glass slide on which the myosin head units are attached [2, 3]. Using the experimental technique, molecularscale mechanics of the motor protein has been studied. In the 2000s, directional control of the movement of the actin filaments was achieved on the nano patterned substrate based on the nanolithographic technology [4]. The previous methods,
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