@misc{oai:ir.soken.ac.jp:00001383,
 author = {小林, 聡子 and コバヤシ, サトコ and KOBAYASHI, Satoko},
 month = {2016-02-17, 2016-02-17},
 note = {Lichens are symbionts of fungi and algae (or cyanobacteria). Lichen forms a unique complex called thallus and produces large amount of lipids of various kinds. These lipids were secreted from the fungal cells and deposited in the thallus to form tremendous amount of crystalline materials. The crystalline materials are considered to have roles to protect the lichen from excess light, bacterial infection, freezing, and etc. This should be the reason that lichens can exhibit a wide ability of adaptations to extreme habitats even as those of alpine, desert, and polar region. From this aspect, we considered the significance of symbiosis of lichen must depend on the unusual lipid production and secretion.<br />　　As mentioned, the fungi produce lipids and secrete them out of cells. On the other hand, the algae were considered to provide precursor sugars for the fungi to produce lipids (Mosbach, 1969, Yamazaki et al., 1965.). Although fungi grown in the wild secrete large amount of lipids to form crystals, those grown in the culture both alone or with algae produce only small amounts and are found unable to form crystals in the culturing medium (Honegger, 1996).<br />　　This study is focused on the mode and the mechanism of the lipid secretion as we considered that the existence of large amount of crystals is signifying the symbiosis of lichens.<br />　　To begin the study we first respectively isolated the fungi and the algae from the thallus of Myelochroa leucotyliza. The isolated samples were cultured both alone or with each other. The cultured samples and the lichen in the wild were examined by means of quick freezing and replicated or substituted electron microscopy. Next, it seemed essential to identify the nature of crystals. X-ray and electron microscope diffraction methods and TLC analysis on the re-crystallized lipid extracted from the wild lichen did the identification. The major component of the re-crystallized material was atranorin.<br />　　According to the observations on the morphology of wild and cultured samples with electron microscopy the following results were obtained; 1) atranorin is the predominant component of the crystalline materials in the thallus, 2) the dynamic morphological change of the plasmamembrane of the fungi occurred in parallel to the activities of lipid secretion, 3) the zymogen granules contained in the fungal cytoplasm exocytose during the lipid secretion, 4) the amount of lipid bodies contained in the fungi inversely changed with those of the crystal deposition outside of the cell.<br />　　It was considered that if we could promote the cultured fungi into actively lipid-secreting cells, and if we could find crystals around the cells, we would have an opportunity to understand the entire scheme of the way the cell secretes lipid and the significance of symbiosis of lichens. Along with this consideration, we cultured fungi in the medium with higher concentration of sugar than usual. According to Hamada's report (1993) that in these condition fungi synthesize large amount of lipid. We detected neither crystal by EM or existence of atranorin by TLC. It was observed, however, that in the fungal cytoplasm cultured in this sugar fortified medium existence of multiple lipid bodies together with numbers of zymogen granules. The appearance of these accumulations of multiple organelles suggested the cellular inhibition of secretions of both lipids and proteins. In order to release the inhibition, we added into the culturing medium various materials that had been reported to promote lipid secretion. Finally, we soaked the fungi growing in this sugar fortified culture with a few drops of the medium in which the algae had been cultured alone. The soaking looked the fungi to release the inhibition and made them actively exocytose zymogen granules. The plasmamembrane changed the shape with many invaginations of omega appearances. The crystals were found outside of the cells.<br />　　The amount and the size of lipid bodies decrease during the active secretion of zymogen granules. However, the lipid-bodies may consist of reservoirs of sterol-derivatives and triacylglycerols. It is considered that they should represent the indirect source of atranorin. Atranorin belongs to polyketides. It has to be produced via acetate-malonate pathway by the interaction of polyketide synthase, in which two phenolic units derived from acetate join to become atranorin. The cytosolic location of polyketide synthase has been reported by showing fluorescent of GFP of the GFP-tagged enzyme. Further, we observed using a fluorescent microscope the possible cytosolic location of atranorin under UV excitation. As atranorin is extremely hydrophobic, the lipid must have being bound with some associating protein in the cytoplasm. However, it is not known how atranorin reaches the inner side of the plasmamembrane.<br />　　The modes of lipid secretion so far survived in the long course of histology are as follows. They are 1) the simple diffusion, 2) the exocytic secretion, and 3) the endoplasmocrine secretion. The simple diffusion is the hypothesis in which lipid in the cytoplasm diffuses freely through the lipidic plasmamembrane. The secretion by exocytosis represents the hypothesis that lipids bound by the biological membrane secrete themselves as the same mechanism to that of protein secretion. Rhodin (1971)has interpreted the endoplasmocrine secretion of lipids. The lipid-body encircled by the tubules of smooth ER makes contact with plasmamembrane by some confusing manner and in the consequence intact lipid-body is exocytosed outside.<br />　　We chose two out of above mentioned three in regard to the mechanisms of lipid secretion of our fungi for the following reasons. 1) Upon incubation with ethanol, Kabakibi et al. (1998) demonstrated the release of fatty acid ethyl ester of Hep-G2 cell into the culturing medium. The secretion stopped when BFA was added to the medium. However, the secretion resumed when lipoprotein or albumin was introduced into the medium that contained BFA. They concluded that the release of the lipid had occurred via an independent pathway of vesicular transport. That is, regardless of whether or not the protein secretion was inhibited by BFA, the release of lipid actually occurred due to the added protein operating outside of the cell. 2) We also did the experiment along the line shown by these authors. To determine whether albumin can pull atranorin out of lipid membrane we prepared atranorin-containing phosphatidyl choline (PC) liposome. The obtained atranorin-PC-liposome was added with BSA-albumin and after extensive shaking the mixture was centrifuged. The pellet consisted of lipid-bileaflet membrane. The supernatant was examined using a spectrofluorometer to detect whether it contains atranorin. Added albumin actually pulled atranorin out of the lipid membrane. In order to try to isolate “effective” protein/or proteins that pull atranorin out of fungal cells we employed anionic columns. The columns separated plenty of proteins from the fungal colony. A few of them showed the extraction ability of atranorin from the atranorin-PC-liposome. However, many of them had no ability to pull atranorin out. The elution of the homogenate of the fungal colony by the “albumin column” showed the most effective pulling ability of atranorin out of the atranorin containing lipid artificial membrane.<br />　　Regarding the mode of atranorin secretion, we concluded as follows. 1) When humoral signals come from the symbiotic algae, the zymogen granules in the fungal cytoplasm exocytose the contents into the extracellular space. 2) The contents of the zymogen granules once secreted elicit atranorin locating on the other side of the membrane. Atranorin goes through the plasmamembrane in the mode of simple diffusion providing if there exists the force of proteins outside., application/pdf, 総研大甲第776号},
 title = {A Morphological Study of Lichen-Symbionts, Ascomycetous Fungus Myelochroa leucotyliza and Green Alga Trebouxia sp., with Special Reference to the Mechanism of Lipid (Atranorin) Secretions},
 year = {}
}