Makito Miyazaki

Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.

Spatial confinement of active microtubule networks induces large-scale rotational cytoplasmic flow

Significance At the microscopic scale, collective behaviors of motile units can induce directed fluid flow on a larger length scale than individual units based on their hydrodynamic interactions. Here, we found that the motor-driven extensile behaviors of microtubule bundles in the cytoplasm induce rotational flow in a cell-sized confined space on length scale and timescale that were 10- to 100-fold longer than the vortex flows emerging in the bulk space. These scale differences were derived from mechanical force generation by microtubule bundle elongation near the physical boundary and the transmission of this force over the microtubule network. These findings suggest that the microtubule cytoskeleton utilizes not only hydrodynamic interactions but also mechanical interactions to induce large-scale cytoplasmic flow. Collective behaviors of motile units through hydrodynamic interactions induce directed fluid flow on a larger length scale than individual units. In cells, active cytoskeletal systems composed of polar filaments and molecular motors drive fluid flow, a process known as cytoplasmic streaming. The motor-driven elongation of microtubule bundles generates turbulent-like flow in purified systems; however, it remains unclear whether and how microtubule bundles induce large-scale directed flow like the cytoplasmic streaming observed in cells. Here, we adopted Xenopus egg extracts as a model system of the cytoplasm and found that microtubule bundle elongation induces directed flow for which the length scale and timescale depend on the existence of geometrical constraints. At the lower activity of dynein, kinesins bundle and slide microtubules, organizing extensile microtubule bundles. In bulk extracts, the extensile bundles connected with each other and formed a random network, and vortex flows with a length scale comparable to the bundle length continually emerged and persisted for 1 min at multiple places. When the extracts were encapsulated in droplets, the extensile bundles pushed the droplet boundary. This pushing force initiated symmetry breaking of the randomly oriented bundle network, leading to bundles aligning into a rotating vortex structure. This vortex induced rotational cytoplasmic flows on the length scale and timescale that were 10- to 100-fold longer than the vortex flows emerging in bulk extracts. Our results suggest that microtubule systems use not only hydrodynamic interactions but also mechanical interactions to induce large-scale temporally stable cytoplasmic flow.

Biphasic Effect of Profilin Impacts the Formin mDia1 Force-Sensing Mechanism in Actin Polymerization

Formins are force-sensing proteins that regulate actin polymerization dynamics. Here, we applied stretching tension to individual actin filaments under the regulation of formin mDia1 to investigate the mechanical responses in actin polymerization dynamics. We found that the elongation of an actin filament was accelerated to a greater degree by stretching tension for ADP-G-actin than that for ATP-G-actin. An apparent decrease in the critical concentration of G-actin was observed, especially in ADP-G-actin. These results on two types of G-actin were reproduced by a simple kinetic model, assuming the rapid equilibrium between pre- and posttranslocated states of the formin homology domain two dimer. In addition, profilin concentration dramatically altered the force-dependent acceleration of actin filament elongation, which ranged from twofold to an all-or-none response. Even under conditions in which actin depolymerization occurred, applications of a several-piconewton stretching tension triggered rapid actin filament elongation. This extremely high force-sensing mechanism of mDia1 and profilin could be explained by the force-dependent coordination of the biphasic effect of profilin; i.e., an acceleration effect masked by a depolymerization effect became dominant under stretching tension, negating the latter to rapidly enhance the elongation rate. Our findings demonstrate that the biphasic effect of profilin is controlled by mechanical force, thus expanding the function of mDia1 as a mechanosensitive regulator of actin polymerization.

Directional Bleb Formation in Spherical Cells under Temperature Gradient

Living cells sense absolute temperature and temporal changes in temperature using biological thermosensors such as ion channels. Here, we reveal, to our knowledge, a novel mechanism of sensing spatial temperature gradients within single cells. Spherical mitotic cells form directional membrane extensions (polar blebs) under sharp temperature gradients (≥∼0.065°C μm(-1); 1.3°C temperature difference within a cell), which are created by local heating with a focused 1455-nm laser beam under an optical microscope. On the other hand, multiple nondirectional blebs are formed under gradual temperature gradients or uniform heating. During heating, the distribution of actomyosin complexes becomes inhomogeneous due to a break in the symmetry of its contractile force, highlighting the role of the actomyosin complex as a sensor of local temperature gradients.

Dynamic properties of bio-motile systems with a liquid-crystalline structure

ABSTRACT Bio-motile systems have liquid-crystalline structures. This review first describes the contractile system of striated muscle having a smectic liquid crystalline structure. We here report the muscles auto-oscillatory property named spontaneous oscillatory contraction (SPOC) [1], and a mathematical model to explain its mechanism [2, 3]. Also, sarcomere dynamics observed during heartbeat are described. The second topic is the micromechanics of the meiotic spindle, a bipolar assembly of microtubules with chromosomes [4]. The third topic is the demonstration of a contractile actin ring spontaneously formed inside a water-in-oil droplet, which can be considered as an artificial cell model [5].

Processive Nanostepping of Formin mDia1 Loosely Coupled with Actin Polymerization

Formins are actin-binding proteins that construct nanoscale machinery with the growing barbed end of actin filaments and serve as key regulators of actin polymerization and depolymerization. To maintain the regulation of actin dynamics, formins have been proposed to processively move at every association or dissociation of a single actin molecule toward newly formed barbed ends. However, the current models for the motile mechanisms were established without direct observation of the elementary processes of this movement. Here, using optical tweezers, we demonstrate that formin mDia1 moves stepwise, observed at a nanometer spatial resolution. The movement was composed of forward and backward steps with unitary step sizes of 2.8 and -2.4 nm, respectively, which nearly equaled the actin subunit length (∼2.7 nm), consistent with the generally accepted models. However, in addition to steps equivalent to the length of a single actin subunit, those equivalent to the length of two or three subunits were frequently observed. Our findings suggest that the coupling between mDia1 stepping and actin polymerization is not tight but loose, which may be achieved by the multiple binding states of mDia1, providing insights into the synergistic functions of biomolecules for the efficient construction and regulation of nanofilaments.

1P212 Measuring the lamellarity of giant liposomes prepared by inverted emulsion method(13A.Biological & Artifical membrane: Structure & Property,Poster,The 51st Annual Meeting of the Biophysical Society of Japan)

The inverted emulsion method of liposome formation is pushing water-inoil droplets through the water/oil interface. This method is efficient for encapsulating proteins and is thus widely used for modeling cells. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively (e.g., dependency on lipid composition). Here we prepared fluorescently labeled liposomes and analyzed the fluorescence intensity of the membrane of individual liposomes under the microscope. By comparing the intensities of the membrane before and after quenching of the fluorescent dye on the outermost monolayer and monitoring the permeability of the inner and the outer buffers in the presence of hemolysin, we concluded that >90% of liposomes were unilamellar.

Accurate polarity control and parallel alignment of actin filaments for myosin-powered transport systems

An actin filament is a micrometer-long biological filament, which serves as a track of transport systems in cells. The filament exhibits polarity while myosin transports a cargo along it unidirectionally. Here, we established a method to align many actin filaments parallel with each other on a substrate with uniform (>99%) polarity as assessed by myosin movements. This actin array is an ideal candidate for the construction of a nano-scale unidirectional transport system powered by myosin.