| Language: | English |
|---|---|
| Type: | Article |
| Authors: | Kingo Takiguchi, Ayako Yamada, Makiko Negishi, Masato Honda, Yohko Tanaka-Takiguchi and Kenichi Yoshikawa |
| Journal: | Methods in Enzymology |
| ISSN: | 0076-6879 |
| Volume: | 464 |
| Chapter: | 3 |
| Year: | 2009 |
| Actual year: | 2009 |
| Pages: | 31-53 |
| doi: | 10.1016/s0076-6879(09)64003-9 |
| Abstract: | To shed light on the mechanism underlying the active morphogenesis of livingcells in relation to the organization of internal cytoskeletal networks, thedevelopment 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, a-actinin, filamin, myosin-I isolated frombrush border (BBMI), and heavymeromyosin (HMM). Subfragment 1 (S-1) is also studied in comparison to HMM, where S-1 and HMM are singleheaded 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. |
Kingo Takiguchi, Ayako Yamada, Makiko Negishi, Masato Honda, Yohko Tanaka-Takiguchi and Kenichi Yoshikawa, Methods in Enzymology, 464, 31-53 (2009)