Masatoshi Ichikawa

Optical transport of a single cell-sized liposome

A closed phospholipid membrane vesicle, or giant liposome, with a diameter of ∼10 μm, constitutes a model of a living cell. Since the phospholipid membrane is about 5 nm thick, it is extremely difficult to trap an individual giant liposome with a laser. We report here the intact transport of an individual cell-sized liposome using an infrared laser. It is shown that the optical attractive potential on a liposome with an inner solution of higher refractivity is about one order of magnitude greater than that of a liposome with the same solution both inside and outside. Since this cell-sized liposome is rather stable, the optical manipulation of liposomes could be useful for the realization of a μm-scaled laboratory for use in biochemistry and molecular biology.

Physicochemical analysis from real-time imaging of liposome tubulation reveals the characteristics of individual F-BAR domain proteins.

The Fer-CIP4 homology-BAR (F-BAR) domain, which was identified as a biological membrane-deforming module, has been reported to transform lipid bilayer membranes into tubules. However, details of the tubulation process, the mechanism, and the properties of the generated tubules remain unknown. Here, we successfully monitored the entire process of tubulation and the behavior of elongated tubules caused by four different F-BAR domain family proteins (FBP17, CIP4, PSTPIP1, and Pacsin2) using direct real-time imaging of giant unilamellar liposomes with dark-field optical microscopy. FBP17 and CIP4 develop many protrusions simultaneously over the entire surface of individual liposomes, whereas PSTPIP1 and Pacsin2 develop only a few protrusions from a narrow restricted part of the surface of individual liposomes. Tubules formed by FBP17 or CIP4 have higher bending rigidities than those formed by PSTPIP1 or Pacsin2. The results provide striking evidence that these four F-BAR domain family proteins should be classified into two groups: one group of FBP17 and CIP4 and another group of PSTPIP1 and Pacsin2. This classification is consistent with the phylogenetic proximity among these proteins and suggests that the nature of the respective tubulation is associated with biological function. These findings aid in the quantitative assessment with respect to manipulating the morphology of lipid bilayers using membrane-deforming proteins.

Spontaneous mode-selection in the self-propelled motion of a solid/liquid composite driven by interfacial instability.

Spontaneous motion of a solid/liquid composite induced by a chemical Marangoni effect, where an oil droplet attached to a solid soap is placed on a water phase, was investigated. The composite exhibits various characteristic motions, such as revolution (orbital motion) and translational motion. The results showed that the mode of this spontaneous motion switches with a change in the size of the solid scrap. The essential features of this mode-switching were reproduced by ordinary differential equations by considering nonlinear friction with proper symmetry.

Structural Change of DNA Induced by Nucleoid Proteins: Growth Phase-Specific Fis and Stationary Phase-Specific Dps

The effects of nucleoid proteins Fis and Dps of Escherichia coli on the higher order structure of a giant DNA were studied, in which Fis and Dps are known to be expressed mainly in the exponential growth phase and stationary phase, respectively. Fis causes loose shrinking of the higher order structure of a genome-sized DNA, T4 DNA (166 kbp), in a cooperative manner, that is, the DNA conformational transition proceeds through the appearance of a bimodal size distribution or the coexistence of elongated coil and shrunken globular states. The effective volume of the loosely shrunken state induced by Fis is 30-60 times larger than that of the compact state induced by spermidine, suggesting that cellular enzymes can access for DNA with the shrunken state but cannot for the compact state. Interestingly, Dps tends to inhibit the Fis-induced shrinkage of DNA, but promotes DNA compaction in the presence of spermidine. These characteristic effects of nucleotide proteins on a giant DNA are discussed by adopting a simple theoretical model with a mean-field approximation.

How environmental solution conditions determine the compaction velocity of single DNA molecules

Understanding the mechanisms of DNA compaction is becoming increasingly important for gene therapy and nanotechnology DNA applications. The kinetics of the compaction velocity of single DNA molecules was studied using two non-protein condensation systems, poly(ethylene glycol) (PEG) with Mg2+ for the polymer-salt-induced condensation system and spermine for the polyamine condensation system. The compaction velocities of single tandem λ-DNA molecules were measured at various PEG and spermine concentrations by video fluorescent microscopy. Single DNA molecules were observed using a molecular stretching technique in the microfluidic flow. The results show that the compaction velocity of a single DNA molecule was proportional to the PEG or spermine concentration to the power of a half. Theoretical considerations indicate that the compaction velocity is related to differences in the free energy of a single DNA molecule between the random coil and compacted states. In the compaction kinetics with PEG, acceleration of the compaction velocity occurred above the overlap concentration while considerable deceleration occurred during the coexistence state of the random coil and the compacted conformation. This study demonstrates the control factors of DNA compaction kinetics and contributes toward the understanding of the compaction mechanisms of non-protein DNA interactions as well as DNA–protein interactions in vivo.

Optically driven transport into a living cell

We report a method for transporting foreign substances into a desired living cell through the use of laser trapping. A single giant DNA molecule can be delivered into a cytoplasmic space if the DNA is folded into a compact state. We also find that microparticles of zeolite are very effective as a vehicle for transferring foreign compounds into a living cell by optical manipulation.

Reconstruction of Active Regular Motion in Amoeba Extract: Dynamic Cooperation between Sol and Gel States.

Amoeboid locomotion is one of the typical modes of biological cell migration. Cytoplasmic sol–gel conversion of an actomyosin system is thought to play an important role in locomotion. However, the mechanisms underlying sol–gel conversion, including trigger, signal, and regulating factors, remain unclear. We developed a novel model system in which an actomyosin fraction moves like an amoeba in a cytoplasmic extract. Rheological study of this model system revealed that the actomyosin fraction exhibits shear banding: the sol–gel state of actomyosin can be regulated by shear rate or mechanical force. Furthermore, study of the living cell indicated that the shear-banding property also causes sol–gel conversion with the same order of magnitude as that of shear rate. Our results suggest that the inherent sol–gel transition property plays an essential role in the self-regulation of autonomous translational motion in amoeba.

Dynamical formation of lipid bilayer vesicles from lipid-coated droplets across a planar monolayer at an oil/water interface

Recently, the transfer method has been shown to be useful for preparing cell-sized phospholipid bilayer vesicles, within which desired substances at desired concentrations can be encapsulated, with a desired asymmetric lipid composition. Here, we investigated the transfer process of water-in-oil (W/O) droplets coated by phospholipid monolayers across an oil/water interface by both experimental observation and theoretical modeling. Real-time experimental observation of the transfer revealed that the transfer process is characterized by three kinetic regimes: a precontact process (approaching regime), an early fast process (entering regime), and a late slow process (relaxation regime). In addition, bigger droplets require much more time to transfer than smaller droplets. We propose a theoretical model to interpret this kinetic process. Our theoretical model reproduces the essential aspects of the transfer kinetics, including its size-dependence.

Tilt control in optical tweezers

Laser trapping of micrometer-sized objects floating in water is investigated through the use of a tilted laser beam. With a change in the tilt direction, the orientation of the trapped object can be easily controlled when the object has an asymmetric body or nonuniform refractive index, such as nanowires, living cells, and so on. The method enables efficient orientation control under laser trapping through a simple setup. This method for tilt control may be useful for high-performance laser trapping in bioengineering and microsurgery in single living cells.

Micro-segregation induced by bulky-head lipids: formation of characteristic patterns in a giant vesicle

Bulky-head lipids, such as glycolipids, play indispensable roles in cellular membranes. However, little is known about the effects that bulky-head lipids have on the physicochemical properties of the membrane. In this study, we examined the effects of a giant vesicle containing poly(ethylene glycol)-conjugated cholesterol (PEG-Chol), as a model of natural bulky-head lipids, on phase separation in the membrane. We used a lipid combination that included the saturated phospholipid DPPC, the unsaturated phospholipid DOPC and Chol, which is known to cause phase separation into two liquid phases. This phase separation is classified as a first-order phase transition under the criterion of Landau, and thus micro-domains tend to show coarse-graining up to a global pair of domains, i.e., mono-domains, so as to minimize interfacial instability. In contrast to such coarsening in the ternary system, we show here the generation of stable micro-domains in the presence of PEG-Chol above a critical composition. The transition from global- to micro-segregation is interpreted theoretically in terms of the competition between two physical effects, i.e., steric repulsive interaction between the bulky-head groups of PEG-Chol and the cost in line energy along the domain boundaries. Interestingly, among the micro-domain structures, a network pattern of domains appears as an intermediate state in which small domains are connected to each other. We examined the stability of the network pattern under local heating using a focused laser, and confirmed self-recovery of the pattern. Based on these observations, natural bulky-head lipids in cells may also stabilize the domain structure like a lipid raft.