Cell Structure and Function

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Recent indirect observations have suggested that various axonemal proteins in cilia and flagella of live cells undergo turnover independently of shortening or elongation of the axoneme. To gain direct evidence, here we examined using a FRAP (fluorescence recovery after photobleaching) technique whether actin, a subunit of inner arm dynein, is being turned over in Chlamydomonas flagella. Fluorescently labeled rabbit actin was introduced by electroporation into the cells of ida5oda1, a double mutant between oda1 lacking outer arm dynein and ida5 lacking several species of inner arm dyneins due to the absence of a conventional-type actin. In actin-loaded cells, flagella became motile and fluorescent due to incorporation of inner-arm dyneins containing the labeled actin. Cells were sandwiched between an agar layer and a coverslip so as to restrict flagellar movement. After a small portion of a flagellum was photobleached, the fluorescence intensity in the bleached area was monitored with a sensitive video camera. The fluorescence intensity in the photobleached region was found to recover 10–40% of the original level over several tens of minutes without changing its position. The time course and extent of the recovery varied greatly from one cell to another, suggesting that the turnover depends on cellular conditions. Western blot analysis indicated that 70–80% of flagellar actin was associated with the axoneme. Hence this experiment provides direct evidence that an axonemal component undergoes dynamic exchange in stationary flagella.

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Hepatocyte transplantation is expected to become a novel method for treatment of liver disease. However, many questions remain regarding this approach, especially concerning donor cells. To evaluate whether human amniotic epithelial cells can be used as a cell source for hepatocyte transplantation, hepatic gene expression and functions of human amniotic epithelial cells were analyzed. Reverse transcription-polymerase chain reaction analysis demonstrated that human amniotic epithelial cells expressed albumin, α₁-antitrypsin, and other hepatocyte-related genes. Cultivated human amniotic epithelial cells demonstrated albumin production, glycogen storage, and albumin secretion consistent with the hepatocyte gene expression profile. In organ culture, the amnion secreted 30-fold larger amounts of albumin than human amniotic epithelial cells in monolayer culture. Moreover, in organ culture the amnion also secreted α₁-antitrypsin. Following transplantation into mice, the amnion survived and secreted albumin. These observations suggest that transplantation of human amniotic epithelial cells and/or amnion could be novel therapeutic strategy for treatment of hepatic diseases, including α₁-antitrypsin deficiency.

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