Ayako Yamada

Time-Resolved Tracking of a Minimum Gene Expression System Reconstituted in Giant Liposomes

Individual expression: We describe a method that allows the observation of real‐time gene expression in a large number of individual giant liposomes encapsulating identical genetic material. We followed the gene expression profiles from DNA and mRNA templates coding for different proteins. Although the average profiles of individual liposomes were similar to those measured in bulk solution, strong variability between individual liposomes was observed at both transcription and translation.

Entrapping desired amounts of actin filaments and molecular motor proteins in giant liposomes.

We have successfully prepared cell-sized giant liposomes encapsulating desired amounts of actoHMM, a mixture of actin filament (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), in the presence of 5 mM MgCl 2 and 50 mM KCl. We employed a spontaneous transfer method to prepare those liposomes. In the absence of HMM, F-actin was distributed homogeneously inside the liposomes. In contrast, when F-actin was encapsulated in liposomes together with HMM, network structures were generated. Such network structures are attributable to the cross-linking of F-actin by HMM.

Electroformation of Giant Phospholipid Vesicles on a Silicon Substrate: Advantages of Controllable Surface Properties

We introduce the use of silicon (Si) as a substrate for the electroformation of giant phospholipid vesicles. By taking advantage of the tunability of silicon surface properties, we varied the organization of the phospholipid film on the electrode and studied the consequences on vesicle formation. In particular, we investigated the effects of Si surface chemistry and microtopology on the organization of the phospholipid film and the properties of the final vesicles. We established correlations between chemical homogeneity, film defects, and resulting vesicle size distribution. By considering phospholipid films that are artificially fragmented by electrode microstructures, we showed that the characteristic size of vesicles decreases with a decrease in microstructure dimensions. We finally proposed a way to control the vesicle size distribution by using a micropatterned silicon dioxide layer on a Si substrate.

Photocontrol of single-chain DNA conformation in cell-mimicking microcompartments.

It has been well established that the regulation of gene activity is strongly dependent on the higher-order structure of genomic DNA molecules. Several strategies have thus been developed to control the higher-order structure of long DNA molecules. Most of them have been based on the use of chemical compounds that bind to DNA to neutralize its charge, such as polyamines, multivalent metal cations, cationic surfactants, cationic polymers, nanoparticles, or crowding agents such as hydrophilic polymers. Depending on the concentration of these additives, DNA exhibits a folded or unfolded conformation. Nevertheless, with all these strategies, it is impossible to act in a reversible way on the DNA higher-order structure under a constant chemical composition. Moreover, for transfection applications, compacting DNA is an essential step to allow the entry of DNA into the cell. In most cases, however, DNA remains in a compact conformation inside the cell, which can significantly alter the DNA gene expression. Using an external stimulus to control DNA higherorder structure within a cell-sized compartment has thus became an important challenge. On the other hand, motivated by the perspective of DNA vectorization, preparation of artificial cells or biochemical microreactors, many scientists have attempted to encapsulate DNA into cell-like microcompartments, for example, cellsized liposomes or phospholipid-coated microdroplets. Consequently, various successful strategies have been proposed to prepare DNA–liposome complexes or encapsulate DNA inside liposomes. In most cases encapsulated DNA molecules were typically smaller than a few thousands base pairs. However, in nature, genomic DNA molecules can be much larger, up to hundreds of kbp (kilo base pairs). To the best of our knowledge, no method has been proposed to encapsulate efficiently, in a controlled way, and without degradation, DNA molecules that are larger than 1 kbp into cell-sized liposomes. One paper reported the encapsulation of T4 DNA molecules, but the data were not sufficient to draw conclusions about the integrity of encapsulated DNA chains. Another strategy was to encapsulate DNA in a compact state, but DNA molecules remained in their compact state once they were encapsulated. Very recently, Le Ny and Lee made a breakthrough by proposing a system where DNA higher-structure can be controlled by light in a reversible manner. This was achieved by adding to a DNA solution a photosensitive cationic surfactant, azobenzene trimethylammonium bromide surfactant (AzoTAB). The apolar tail of the surfactant contains an azo group, which is mainly in the trans (more hydrophobic) conformation under visible conditions. Under UV illumination (365 nm), the azo group photoisomerizes into the cis (more hydrophilic) conformation. They demonstrated that there exists an AzoTAB concentration range for which DNA is in the compact state under dark/visible conditions but in the unfolded state under UV illumination, that is, DNA higher-order structure can be controlled by light. In this study, the authors mainly characterized the average property of many DNA chains in solution. Here, we characterized the single-chain conformational behavior of long genomic DNA as a function of AzoTAB concentration and time of UV illumination. We established that the transition has a first-order character at the single-chain level. We studied the single-chain unfolding upon UV illumination and evidenced two mechanisms of single-chain DNA unfolding. Then we applied this strategy to unfold genomic DNA molecules that are encapsulated in cell-mimicking microcompartments. To this end, DNA that was compacted by AzoTAB under visible conditions was encapsulated into cell-sized microdroplets that were coated with various phospholipids prior to UV light exposition. We studied the unfolding process of individual DNA molecules inside the microdroplets. We could successfully unfold most of the DNA molecules when the phospholipid was anionic (DOPS phospholipid). We thus demonstrated how an external stimulus, here light, can be used to control the higher-order structure of individual genomic DNA molecules within cell-sized phospholipid-coated microcompartments. By using fluorescence microscopy (FM), we characterized the conformation of individual T4 DNA molecules at a very low DNA concentration (0.1 mm) in Tris–HCl buffer (10 mm, pH 7.4). To the DNA solution, we added azobenzene trimethylammonium bromide (AzoTAB, Figure 1A) at various concentrations under dark conditions (most of AzoTAB molecules are in the trans conformation, that is, more hydrophobic). Depending on AzoTAB concentration, we distinguished three regimes with re[a] M. Geoffroy, Dr. D. Baigl Department of Chemistry, Ecole Normale Sup rieure 24 rue Lhomond, 75005 Paris (France) Fax: (+33)1-4432-2402 E-mail : damien.baigl@ens.fr [b] Prof. M. Sollogoub, S. Guieu Institut de Chimie Mol culaire (FR2769) Laboratoire de Chimie Organique (UMR CNRS 7611) UPMC Universit Paris 06 4, Place Jussieu, C. 181, 75005 Paris (France) [c] Dr. A. Yamada, Dr. A. Est vez-Torres, Prof. K. Yoshikawa Department of Physics, Kyoto University Kitashirakawa oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan) [d] Dr. A. Est vez-Torres, Prof. K. Yoshikawa, Dr. D. Baigl Spatio-Temporal Order Project, ICORP JST (Japan Science and Technology Agency) Kyoto University, Kyoto 606-8502 (Japan) A video clip is available as Supporting information on the WWW under http://www.chembiochem.org or from the author.

β-Lactotensin derived from bovine β-lactoglobulin exhibits anxiolytic-like activity as an agonist for neurotensin NTS2 receptor via activation of dopamine D1 receptor in mice

J. Neurochem. (2011) 119, 785–790.

Characterization of Tyr-Leu-Gly, a novel anxiolytic-like peptide released from bovine αS-casein

We found previously that dipeptide YL exhibits orally active anxiolytic activity comparable to diazepam. The YL sequence is often observed in the primary structure of natural food proteins. In the present study, we investigated whether YL and YL analogues are released from bovine αS‐casein by gastrointestinal proteases. YLG, corresponding to αS1‐casein (aa 91–93), was more effectively released from αS‐casein than YL by pepsin‐pancreatin digestion, mimicking gastrointestinal enzymatic conditions. Using the synthetic model peptide, we determined that trypsin cleaved the N terminus of YLG, and elastase and carboxypeptidase contributed to cleave the C‐terminus. YLG exhibited orally active anxiolytic‐like activity in the elevated plus maze and open‐field tests in mice. The anxiolytic‐like activity of YLG was inhibited by WAY100135, SCH23390 or bicuculline, antagonists of serotonin 5‐HT1A, dopamine D1, and GABAA receptors, respectively; however, YLG had no affinity for these receptors. The pepsin‐pancreatin digest of αS‐Casein also exhibited anxiolytic‐like activity. Meanwhile, anxiolytic‐like activity of α‐casozepine, an αS1‐casein‐derived decapeptide with YL sequence in the N terminus, was blocked by WAY100135, SCH23390, or bicuculline, equally to YLG and YL; however, it was not detected in the pepsin‐pancreatic digest. Taken together, we found that YLG is released after pepsin‐pancreatic digestion of αS‐casein and exhibits potent anxiolytic‐like activity via activation of serotonin, dopamine, and the GABA receptor system.—Mizushige, T., Sawashi, Y., Yamada, A., Kanamoto, R., Ohinata, K. Characterization of Tyr‐Leu‐Gly, a novel anxiolytic‐like peptide released from bovine αS‐casein. FASEB J. 27, 2911‐2917 (2013). www.fasebj.org

Construction of Cell-Sized Liposomes Encapsulating Actin and Actin-Cross-linking Proteins

To shed light on the mechanism underlying the active morphogenesis of living cells in relation to the organization of internal cytoskeletal networks, the development of new methodologies to construct artificial cell models is crucial. Here, we describe the successful construction of cell-sized liposomes entrapping cytoskeletal proteins. We discuss experimental protocols to prepare giant liposomes encapsulating desired amounts of actin and cross-linking proteins including molecular motor proteins, such as fascin, alpha-actinin, filamin, myosin-I isolated from brush border (BBMI), and heavy meromyosin (HMM). Subfragment 1 (S-1) is also studied in comparison to HMM, where S-1 and HMM are single-headed and double-headed derivatives of conventional myosin (myosin-II), respectively. In the absence of cross-linking proteins, actin filaments (F-actin) are distributed homogeneously without any order within the liposomes. In contrast, when actin is encapsulated together with an actin-cross-linking protein, mesh structures emerge that are similar to those in living motile cells. Optical microscopic observations on the active morphological changes of the liposomes are reported.

All-or-none switching of transcriptional activity on single DNA molecules caused by a discrete conformational transition

Recently, it has been confirmed that long duplex DNA molecules with a size larger than several tens of kilo-base pairs (kbp), exhibit a discrete conformational transition from an unfolded coil state to a folded compact state upon the addition of various kinds of chemical species that usually induce DNA condensation. In this study, we performed a single-molecule observation on a large DNA, Lambda ZAP II DNA (∼41kbp), in a solution containing RNA polymerase and substrates along with spermine, a tetravalent cation, at different concentrations, by use of fluorescence staining of both DNA and RNA. We found that transcription, or RNA production, is completely inhibited in the compact globule state, but is actively performed in the unfolded coil state. Such an all-or-none effect on transcriptional activity induced by the discrete conformational transition of single DNA molecules is discussed in relation to the mechanism of the regulation of large-scale genetic activity.

Folding transition into a loosely collapsed state in plasmid DNA as revealed by single-molecule observation

The conformational transition of a plasmid DNA, pGEG.GL3 (12.5 kbp, circular), induced by spermine(4+) was studied through the observation of individual DNA by fluorescence microscopy. We deduced the change in the hydrodynamic radius R H from an analysis of the Brownian motion of single DNA molecules. R H decreases in a continuous manner with an increase in spermine(4+), in contrast to the large discrete on/off change for long linear DNA. Just after the transition to the collapsed state, a small number of DNA molecules tend to form an assembly, which disperses in the bulk solution without precipitation.

Spontaneous Generation of Giant Liposomes from an Oil/Water Interface

Giant liposomes, phospholipid vesicles with a bilayered membrane and a size greater than 1 mm, have attracted a strong scientific interest mainly motivated by trying to synthesize artificial cells. Indeed, giant liposomes have been shown to be the best primary model of cells in terms of size, membrane composition, or the ability to encapsulate biologically relevant molecules. Hence, various methodologies for the preparation of cell-sized liposomes have been proposed, such as natural swelling, freezing and thawing, electroformation, extraction from a cell membrane, transfer of microdroplets, and microfluidic methods. Among them, the widely used natural swelling method, in which liposomes generate from a dry lipid film, is usually described as a spontaneous phenomenon, and its mechanism has been investigated. Here, we report a new spontaneous phenomenon in a biphasic (oil/water) environment that leads to the formation of giant liposomes with a diameter ranging from 1 to 100 mm. We observed that giant liposomes spontaneously formed when a horizontal liquid interface was made between an upper oil phase containing phospholipid and a lower water phase. Our method mainly differs from natural swelling in that liposomes are obtained from a liquid oil/water interface at which the lipid organization and dynamics differ from that on a usual solid substrate. In this study, we used DOPC phospholipid dissolved in mineral oil at various concentrations, and characterized the peculiar phospholipid organization at the interface as well as the obtained giant liposomes by confocal microscopy. An oil/water interface was prepared in a cylindrical observation chamber made of poly(dimethyloxane) (PDMS) walls bound to a glass microscope cover slide (Figure 1). A thin layer of water was placed at the bottom of the observation chamber, which was topped up with a thin layer of mineral oil containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid and a small fraction of 1-oleoyl-2-[12-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC) fluorescent phospholipid. First, the experiment was performed with a DOPC concentration of 1 mm in oil and a temperature of 25 8C. One minute after interface preparation, we observed the formation of small phospholipid aggregates randomly distributed at the oil/water interface, which presented some irregularities. (Figure 2A top) The presence of liposomes was not detected in either phase. With time, the interface became more indented as phospholipid aggregates slowly reorganized. 18 h after interface preparation, phospholipid molecules were arranged into a dotted pattern at the interface, as shown by the fluorescence of NBD-PC, Figure 1. Experimental set-up.