Dynamic behaviors of liposomes caused by interactions between liposomal membranes and surfactant were studied by direct realtime observation by using high-intensity dark-field microscopy. Solubilization of liposomes by surfactants is thought to be a catastrophic event akin to the explosion of soap bubbles in the air; however, the actual process has not been clarified. We studied this process experimentally and found that liposomes exposed to various surfactants exhibited unusual behavior, namely continuous shrinkage accompanied by intermittent quakes, release of encapsulated liposomes, opening up, and inside-out topological inversion. L iposomes (which are closed membrane vesicles) have been well studied as simplified models of biological membranes (1-5) and are now used in a number of applications (5, 6), for example, as carriers of drug or DNA delivery or as artificial membranes for reconstructing membranous enzyme activities (7-9). Recently, many important phenomena affecting lipid bilayers, including their detergent solubilization, have been explored by using liposomes; such studies promote a better understanding of the biophysical properties of bilayer membranes and moreover will improve the handling of membrane proteins when they are isolated from or reconstructed into lipid bilayers (10-13). However, studies of intermediate stages in the detergent solubilization of liposomes are only now in progress (14-17), and the interaction mechanism between membranes and surfactants has remained unclear. Therefore, real-time approaches by using optical microscopy to study the dynamic behavior of liposomes are very important.High-intensity dark-field microscopy has enabled us to obtain real-time high-contrast images of giant unilamellar liposomes in aqueous solutions (18)(19)(20)(21)(22). In this study, we used such techniques to characterize the interactions between liposomal membranes and surfactants. Eight kinds of liposomes and various types of surfactants ( Fig. 1) were mixed in all possible combinations in a mixing chamber to generate a concentration gradient of each surfactant for microscope specimens, and morphological changes of liposomes exposed to those surfactants were monitored (18, 23). In the absence of surfactant, liposomal membranes were spherical, and thermal fluctuations of their spherical shape were largely suppressed by the surface tension of their membranes. Hereafter, this morphological state of liposomes will be called tense. In this study, we found several unusual behaviors of liposomes (which are published as supplemental data on the PNAS web site, www.pnas.org). Materials and MethodsPreparation and Observation of Liposomes. To prepare giant unilamellar liposome, liposome (total 1 mM lipid concentration) was made of phosphatidylcholine (PC) or of PC and one of seven other lipids (1:1, mol͞mol) in Hepes buffer (10 mM HepesNaOH, pH 7.0), as described previously (18,21,22). Lipids were dissolved in a chloroform͞methanol solution, 98:2 (vol͞vol), and mixed. The organic solvent was evaporated unde...

To study the mechanisms involved in membrane fusion, we visualized the fusion process of giant liposomes in real time by optical dark-field microscopy. To induce membrane fusion, we used (i) influenza hemagglutinin peptide (HA), a 20-aa peptide derived from the N-terminal fusion peptide region of the HA2 subunit, and (ii) two synthetic analogue peptides of HA, a negatively (E5) and positively (K5) charged analogue. We were able to visualize membrane fusion caused by E5 or by K5 alone, as well as by the mixture of these two peptides. The HA peptide however, did not induce membrane fusion, even at an acidic pH, which has been described as the optimal condition for the fusion of large unilamellar vesicles. Surprisingly, before membrane fusion, the shrinkage of liposomes was always observed. Our results suggest that a perturbation of lipid bilayers, which probably resulted from alterations in the bending folds of membranes, is a critical factor in fusion efficiency.giant liposome ͉ influenza hemagglutinin ͉ optical microscopy ͉ direct observation M embrane fusion plays an essential role in cellular activities such as exocytosis, endocytosis, and vesicle transport of various cellular organelles. Also, it is an essential step of infection by enveloped viruses. Because the fusion of biological membranes is so important, studies on the mechanism of membrane fusion have been performed. Many of those studies focused on membrane fusion driven by fusogenic peptides, for example, those derived from influenza hemagglutinin. Those studies demonstrated that there are several critical steps required for membrane fusion (1-4). To characterize membrane fusion, resonance-energy transfer and fluorescence-quenching methods have been commonly used (5, 6). The resonance-energy transfer method monitors the mixing of membrane lipids that occurs during fusion, whereas the f luorescence-quenching method monitors the mixing of vesicular contents. Light scattering to detect fusion kinetics, or electron microscopy to visualize the fusion process, has also been commonly used. Electron microscopy can provide snapshots of the changing membrane structures in detail (7), but it is not suitable for studying the time-dependent transition of the three-dimensional membrane morphology.For understanding the fusion mechanism, the real-time visualization of individual fusion processes is indispensable (6, 8). We used giant (1-20-m diameter) liposomes as a model for biological membrane vesicles, and we monitored their fusion process in real time by using optical high-intensity dark-field microscopy (8). Giant liposomes can be visualized by several optical microscopic methods (9-11). However, we have used dark-field microscopy because it gives the best high-contrast images of giant liposomes compared with other methods (8,(12)(13)(14)(15).To induce membrane fusion, we used three different peptides: HA, E5, and K5. HA is a 20-aa peptide derived from the N-terminal fusion peptide region of the influenza hemagglutinin HA2 subunit, whereas E5 and K5 are negativ...

One of the major difference between the in vivo and in vitro embryonic environments is the stiffness of the culture substrate. Xanthan gum (XG) and locust bean gum (LBG) are natural materials that are safe, inexpensive and easy to handle. In this study, we investigated the effects of using a polysaccharide culture substrate made from 1% XG and 1% LBG (XG‐LBG gel) on bovine embryonic development. Oocytes collected from bovine ovaries were subjected to maturation, and fertilization to generate embryos at an early developmental stage (>4 cell stage). Cleaved embryos were further cultured in a well of 96‐well cell culture plate coated with or without XG‐LBG gel for 5 days. While the developmental rate up to the blastocyst stage did not differ between the two culture systems (control, 38.0 vs. gel, 38.6%), blastocysts developed on the XG‐LBG gel produced significantly high cell numbers and ATP content. Embryos cultured on XG‐LBG gels for 24 hr had high expression levels of F‐actin and a highly even distribution of E‐cadherin. In addition, embryos developed on XG‐LBG gel demonstrated increased translocation of YAP to the nucleus and increased connective tissue growth factor (CTGF) protein levels (downstream of Hippo signalling). These findings suggest that soft culture substrates improve embryonic development by enhancing mechanotransduction, including YAP‐CTGF signalling.

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