Nonmuscle myosin-2: mix and match

Members of the nonmuscle myosin-2 (NM-2) family of actin-based molecular motors catalyze the conversion of chemical energy into directed movement and force thereby acting as central regulatory components of the eukaryotic cytoskeleton. By cyclically interacting with adenosine triphosphate and F-actin, NM-2 isoforms promote cytoskeletal force generation in established cellular processes like cell migration, shape changes, adhesion dynamics, endo- and exo-cytosis, and cytokinesis. Novel functions of the NM-2 family members in autophagy and viral infection are emerging, making NM-2 isoforms regulators of nearly all cellular processes that require the spatiotemporal organization of cytoskeletal scaffolding. Here, we assess current views about the role of NM-2 isoforms in these activities including the tight regulation of NM-2 assembly and activation through phosphorylation and how NM-2-mediated changes in cytoskeletal dynamics and mechanics affect cell physiological functions in health and disease.     

Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac β-myosin ATPase to only 50?% of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72?h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.     

Molecular mechanism of actomyosin-based motility

Sophisticated molecular genetic, biochemical and biophysical studies have been used to probe the molecular mechanism of actomyosin-based motility. Recent solution measurements, high-resolution structures of recombinant myosin motor domains, and lower resolution structures of the complex formed by filamentous actin and the myosin motor domain provide detailed insights into the mechanism of chemomechanical coupling in the actomyosin system. They show how small conformational changes are amplified by a lever-arm mechanism to a working stroke of several nanometres, explain the mechanism that governs the directionality of actin-based movement, and reveal a communication pathway between the nucleotide binding pocket and the actin-binding region that explains the reciprocal relationship between actin and nucleotide affinity. Here we focus on the interacting elements in the actomyosin system and the communication pathways in the myosin motor domain that respond to actin binding.Received 12 January 2005; received after revision 4 March 2005; accepted 23 March 2005     

Molecular engineering of a backwards-moving myosin motor

All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin- and nucleotide-binding sites and produces force and movement. The light-chain-binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm. Myosins move on polarized actin filaments either forwards to the barbed (+) or backwards to the pointed (-) end. Here, we describe the engineering of an artificial backwards-moving myosin from three pre-existing molecular building blocks. These blocks are: a forward-moving class I myosin motor domain, a directional inverter formed by a four-helix bundle segment of human guanylate-binding protein-1 and an artificial lever arm formed by two alpha-actinin repeats. Our results prove that reverse-direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180 degrees.