|Budget Amount *help
¥32,300,000 (Direct Cost: ¥32,300,000)
Fiscal Year 1999: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1998: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 1997: ¥10,100,000 (Direct Cost: ¥10,100,000)
Fiscal Year 1996: ¥14,600,000 (Direct Cost: ¥14,600,000)
1. Morphological changes of liposomes.
(1). Shape change caused by microtubule assembly
In order to elucidate the role of the cytoskeleton in cellular morphogenesis, model systems using lipsomes encapsulating cytoskeletal proteins have been developed using tubulin or actin. Upon polymerization of the cytoskeleton, liposomes are transformed into characteristic shapes, which depend on the types of encapsulated cytoskeletal proteins. Two tubular projections developed from antipodal positions on spherical liposomes when microtubules polymerized. Both projections were straight, rigid, uniform in diameter and elongated in opposite directions. These observations indicate that the cytoskeleton can generate mechanical forces capable of transforming the phospholipid membrane.
(2). Stabilization of bipolar liposomes by MAPs
(3). Binding between microtubules and liposomes in the presence of MAPs
2. Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking
the transformation of liposomes encapsulating actin and its crosslinking proteins, fascin, α-actinin, or filamin, using real-time high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-actin polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. The differences in morphology among transformed liposomes indicate that actin-crosslinking proteins determine liposome shape by organizing their specific actin networks. Morphological analysis reveals that the crosslinking manner, i.e. distance and angular flexibility between adjacent crosslinked actin filaments, is essential for the morphogenesis rather than their binding affinity and stoichiometry to actin filaments.
3. Opening-up of liposomes membranes by talin
Morphological changes liposomes caused by interactions between liposomal membranes and talin, a cytoskeletal submembranous protein, were studied by direct, real-time observation using high-intensity dark-field microscopy. Surprisingly, when talin was added to a liposome solution, liposomes opened stable holes and were transformed into cup-shaped liposomes. The holes became larger with increasing talin concentration, and finally the cup-shaped liposomes were transformed into lipid bilayer sheets. These morphological changes were reversed by protein dilution, i.e., the sheets could be transformed back into closed spherical liposomes. We demonstrated that talin was localized mainly along the membrane verges, presumably avoiding exposure of its hydrophobic portion at the edge of the lipid bilayer. This is the first demonstration that a lipid bilayer can stably maintain a free verge in aqueous solution. This finding refutes the established dogma that all lipid bilayer membranes inevitably form closed vesicles, and suggests that talin is a useful tool for manipulating liposomes. Less