Utilization of the rapamycin assay to visualize cytohesin association with Rab-positive compartments in epithelial cells
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- Open Access.
- Epithelial cell movement is a key process in development, tissue reparation, and tumor metastasis. Epithelial-mesenchymal transition is controlled by several factors, including Rab GTPases and the GEF cytohesins, which regulate ARF GTPases and the recycling of proteins to plasma membrane. To visualize association of cytohesins with various endosomal compartments, rapamycin has been used as a linker drug to induce migration of Rab-positive compartments to the cell-center. In this study, the rapamycin assay was optimized to quantify Rab-positive protein interactions and cytohesin recruitment to various endosomal populations within cells, and to establish a robust method of investigating protein associations with different endosomal compartments. HeLa cells were transfected with myc-FRB proteins, fluorescent compartment markers, and pBa-flag BicD2 FKBP before treatment with rapamycin. A 3-hour treatment time produced the most significant reduction in particle number (93%) and size (94%) as expected and transfection variations revealed myc-FRB proteins preferentially bind to their respective fluorescent compartment marker (p< 0.0001). Rab5a with Rab7, and Rab8a with Rab11 and EHD1 were also shown to associate with one another at increased levels. Transfection with cytohesin-2/ARNO did not show significant variable association with any myc-FRB proteins. Overall, a usable assay was developed that can continue to be used to analyze protein/endosomal interactions. Future studies will lead to a deeper understanding of protein interactions with endosomal compartments within the cell for application to drug discoveries regarding cancer and other related diseases.
- Dissertation Note:
- B.S. Pennsylvania State University 2019.
- Technical Details:
- The full text of the dissertation is available as an Adobe Acrobat .pdf file ; Adobe Acrobat Reader required to view the file.
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