| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所-臨床醫學組 === 102 === Wnt proteins are important signaling morphogenes that are implicated in a number of signal transduction pathways in the cell and aberrant Wnt signaling has been linked to both neurodegenerative diseases and cancers in humans. The mechanism of Wnt secretion from their cells of origin is not yet clear. Wntless is a conserved transmembrane protein that carries Wnts from the endoplasmic reticulum to the golgi and plasma membrane for secretion out of the cell. Interference with Wntless function results in the sequestration of Wnts in endoplasmic reticulum and golgi thus inhibited secretion to target cells. In the present study, the orientation of Wntless inside the cytoplasm and plasma membrane of Wnt-secreting cells was investigated. Monoclonal antibodies against the Wntless C-terminus and N-terminus from Balb/c mice were generated and used in immunofluorescence and confocal microscopy to observe the localization of Wntless inside the cytoplasm and plasma membrane and its localization with the retromer complex subunit Vps35. Immunofluorescence results indicated inisignificant interaction of retromer subunit Vps35 with Wntless in unstimulated HEK293 cells and Wntless was mainly localized in the membrane. Its orientation inside the plasma membrane was found to occur with the N-terminus projecting outside, relative to Vps35. The C-terminus of Wntless projected inside towards the cytoplasm relative to Vps35. Wntless C-terminus colocalized with Vps35 in Wnt-3a stimulated HEK 293 cells, indicating an interaction while the N-terminus did not colocalize with Vps35. Wntless was also found to break apart into two parts, in a time dependant manner, after stimulation of cells with Wnt3a. This disassembly of Wntless occurred through the C-terminus.
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