2012年
VISUALIZATION OF TGN-ENDOSOME TRAFFICKING IN MAMMALIAN AND DROSOPHILA CELLS
IMAGING AND SPECTROSCOPIC ANALYSIS OF LIVING CELLS: OPTICAL AND SPECTROSCOPIC TECHNIQUES
- ,
- 巻
- 504
- 号
- 開始ページ
- 255
- 終了ページ
- 271
- 記述言語
- 英語
- 掲載種別
- 論文集(書籍)内論文
- DOI
- 10.1016/B978-0-12-391857-4.00013-6
- 出版者・発行元
- ELSEVIER ACADEMIC PRESS INC
Mannose 6-phosphate receptors (MPRs) are known to be shuttled between the trans-Golgi network (TGN) and endosomes, thereby several lysosomal hydrolases are delivered through the endocytic pathway into lysosomes. This interorganellar transport is mediated by transport intermediates, now called transport carriers. Previous studies employing green fluorescent protein (GFP)-based live-cell imaging demonstrated that these transport carriers are pleiomorphic structures composed of tubular and vesicular elements. Introducing a time-axis into light microscopic observations enabled us to identify transport carriers that are derived from or targeted at a distinct organelle. In this study, we describe several methods for the observation of GFP-tagged MPRs. Photobleaching the peripheral region of a cell before a time-lapse observation allows us to monitor TGN-derived transport carriers for longer periods (more than 4 min). Events of their targeting into endosomes can be visualized by dual-color imaging of both GFP-MPRs and fluorescently tagged transferrin that is internalized by cells. By using a technique of fluorescence recovery after photobleaching (FRAP), we can analyze overall cycling kinetics of MPRs in a single cell. Transport of MPRs is regulated by several cytosolic factors like clathrin adaptors, AP1, and GGAs. The adaptors on the TGN membranes are exchanging with their cytosolic pool, which can also be analyzed by FRAP. In addition, the relationships of the MPR-containing transport carriers that left the TGN and the adaptors can be visualized by dual-color imaging. A similar system of membrane transport and its regulation is well documented in drosophila cells. As Drosophila melonogaster has only a single MPR (LERP), API, or GGA, it is an ideal model system for the understanding of specific functions of each cytosolic factor. To visualize these molecules in drosophila cells, however, we need to consider that multiple Golgi dots exist scattered in the cytoplasm. Thus, the Golgi dots or endosomes should be identified before live-cell imaging.
- リンク情報
- ID情報
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- DOI : 10.1016/B978-0-12-391857-4.00013-6
- ISSN : 0076-6879
- PubMed ID : 22264539
- Web of Science ID : WOS:000303896400013