Ryoki Ishikawa

Video imaging of walking myosin V by high-speed atomic force microscopy.

The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative ‘visual evidence’ for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a powerful new approach to studying the structure and dynamics of biomolecules in action.

Inhibition by drebrin of the actin-bundling activity of brain fascin, a protein localized in filopodia of growth cones

Abstract: The purification of drebrin, an actin‐binding protein that is specifically expressed in embryonic rat brain, was described previously. During the purification of drebrin, we found that an actin‐binding protein of 54 kDa was also expressed at high levels in embryonic brain, and this protein was identified by immunoblotting as fascin. To explore the roles of fascin in brain development, we purified fascin from brains of infant rats and characterized it. We found that the actin‐binding activity of fascin was strongly inhibited by drebrin. Fascin caused formation of actin bundles, a process that was inhibited in the presence of drebrin, as confirmed by electron microscopy and a low‐speed centrifugation assay. In PC12 cells, fascin was localized in the filopodia of growth cones, whereas drebrin was localized in the basal region of growth cones. Our results suggest that fascin might play an important role in the organization of actin in filopodia and that this organization might be regulated by drebrin.

Polarized actin bundles formed by human fascin-1: their sliding and disassembly on myosin II and myosin V in vitro

Fascin‐1 is a putative bundling factor of actin filaments in the filopodia of neuronal growth cones. Here, we examined the structure of the actin bundle formed by human fascin‐1 (actin/fascin bundle), and its mode of interaction with myosin in vitro. The distance between cross‐linked filaments in the actin/bundle was 8–9 nm, and the bundle showed the transverse periodicity of 36 nm perpendicular to the bundle axis, which was confirmed by electron microscopy. Decoration of the actin/fascin bundle with heavy meromyosin revealed that the arrowheads of filaments in the bundle pointed in the same direction, indicating that the bundle has polarity. This result suggested that fascin‐1 plays an essential role in polarity of actin bundles in filopodia. In the in vitro motility assay, actin/fascin bundles slid as fast as single actin filaments on myosin II and myosin V. When myosin was attached to the surface at high density, the actin/fascin bundle disassembled to single filaments at the pointed end of the bundle during sliding. These results suggest that myosins may drive filopodial actin bundles backward by interacting with actin filaments on the surface, and may induce disassembly of the bundle at the basal region of filopodia.

The fastest-actin-based motor protein from the green algae, Chara, and its distinct mode of interaction with actin

The endoplasmic streaming in Characean cells is an actin‐dependent movement. The motor protein responsible for the streaming was partially purified and characterized. It was soluble at low ionic strength, an ATPase of a molecular mass of 225 kDa and activated more than 100 times by muscle F‐actin. Surprisingly, in an in vitro motility assay, the motor protein moved muscle F‐actin at 60 μm/s, which is similar to the velocity of streaming in a living cell and 10 times faster than muscle myosin. Proteolytic cleavage of actin impaired movement crucially on muscle myosin, but did not affect movement at all on the Chara motor protein, suggesting that the Chara motor protein would interact with actin via a set of sites different from those of muscle myosin.

Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase (PDE1) Is a Pharmacological Target of Differentiation-Inducing Factor-1, an Antitumor Agent Isolated from Dictyostelium

The differentiation-inducing factor-1 (DIF-1) isolated from Dictyostelium discoideum is a potent antiproliferative agent that induces growth arrest and differentiation in mammalian cells in vitro. However, the specific target molecule(s) of DIF-1 has not been identified. In this study, we have tried to identify the target molecule(s) of DIF-1 in mammalian cells, examining the effects of DIF-1 and its analogs on the activity of some candidate enzymes. DIF-1 at 10–40 μm dose-dependently suppressed cell growth and increased the intracellular cyclic AMP concentration in K562 leukemia cells. It was then found that DIF-1 at 0.5–20 μm inhibited the calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) in vitro in a dose-dependent manner. Kinetic analysis revealed that DIF-1 acted as a competitive inhibitor of PDE1 versus the substrate cyclic AMP. Because DIF-1 did not significantly affect the activity of other PDEs or CaM-dependent enzymes and, in addition, an isomer of DIF-1 was a less potent inhibitor, we have concluded that PDE1 is a pharmacological and specific target of DIF-1.

Plectin 1 links intermediate filaments to costameric sarcolemma through β-synemin, α-dystrobrevin and actin

In skeletal muscles, the sarcolemma is possibly stabilized and protected against contraction-imposed stress by intermediate filaments (IFs) tethered to costameric sarcolemma. Although there is emerging evidence that plectin links IFs to costameres through dystrophin-glycoprotein complexes (DGC), the molecular organization from plectin to costameres still remains unclear. Here, we show that plectin 1, a plectin isoform expressed in skeletal muscle, can interact with β-synemin, actin and a DGC component, α-dystrobrevin, in vitro. Ultrastructurally, β-synemin molecules appear to be incorporated into costameric dense plaques, where they seem to serve as actin-associated proteins rather than IF proteins. In fact, they can bind actin and α-dystrobrevin in vitro. Moreover, in vivo immunoprecipitation analyses demonstrated that β-synemin- and plectin-immune complexes from lysates of muscle light microsomes contained α-dystrobrevin, dystrophin, nonmuscle actin, metavinculin, plectin and β-synemin. These findings suggest a model in which plectin 1 interacts with DGC and integrin complexes directly, or indirectly through nonmuscle actin and β-synemin within costameres. The DGC and integrin complexes would cooperate to stabilize and fortify the sarcolemma by linking the basement membrane to IFs through plectin 1, β-synemin and actin. Besides, the two complexes, together with plectin and IFs, might have their own functions as platforms for distinct signal transduction.

Zinc inhibits calcineurin activity in vitro by competing with nickel

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN.

Partial purification of myosin from lily pollen tubes by monitoring with in vitro motility assay

SummaryMyosin in pollen tubes ofLilium longiflorum was partially purified, using an in vitro motility assay as a monitor. The main components in the partially purified preparation had molecular masses of 110, 120, and 140 kDa in SDS-PAGE. They became bound to actin filaments in an ATP-dependent manner. Among the components, only that of 120 kDa became bound to ATP and was concluded to be the heavy chain of pollen tube myosin.

A novel regulatory effect of myosin light chain kinase from smooth muscle on the ATP-dependent interaction between actin and myosin.

The actin-binding activity of myosin light chain kinase (MLCK) from smooth muscle was studied with special reference to the ATP-dependent interaction between actin and myosin. MLCK in the presence of calmodulin endowed sensitivity to Ca2+ on the movement of actin filaments on phosphorylated myosin from smooth muscle that was fixed on a coverslip. This regulatory effect was not attributable to the kinase activity of MLCK but could be explained by its actin-binding activity. The importance of the actin-binding activity was further substantiated by results of an experiment with Nitellopsis actin-cables in which MLCK regulated the interaction under conditions where MLCK was exclusively associated with the actin-cables.

Utilization of myosin and actin bundles for the transport of molecular cargo.

The utilization of motor proteins for the movement and assembly of synthetic components is currently a goal of nanoengineering research. Application of the myosin actin motor system for nanotechnological uses has been hampered due to the low flexural rigidity of individual F-actin filaments. Here it is demonstrated how actin bundling can be used to affect the translational behavior of myosin-propelled filaments, transport molecules across a motor-patterned surface, and that the movement of bundled actin can be regulated photonically. These data suggest that actin bundling may significantly improve the applicability of the myosin motor for future nanotechnological applications.