UROP Proceedings 2020-21

School of Science Division of Life Science 15 Analysis of Surface Delivery of Frizzled6 Supervisor: GUO Yusong / LIFS Student: DANI Raissa Gavrila / BIOT Course: UROP1000, Summer This report investigated the function of SCAMP2 and Vti1b in vesicular trafficking from the Golgi Apparatus to the plasma membrane. SCAMP2 is a tetraspanning integral protein and Vti1b is a form of v-SNARE protein, both found in mammalian cells. We previously demonstrated siRNA knockdown procedures on both SCAMP2 and Vti1b separately. Post siRNA transfection, defects were observed for both SCAMP2 and Vti1b knockdown cells. Here, to further investigate whether or not the defects were the direct consequence of Vti1b knockdown and SCAMP2 knockdown, we generate rescue constructs for both genes that are resistant to siRNA knockdown. Both rescue constructs are generated using PCR mutagenesis by making primers with three base pairs mismatched on the siRNA annealing region. Since the experiment is currently in progress, we expected two possible outcomes after generating cell lines with the two rescue constructs. First, the defects will still be present indicating that the defects do not have a direct relationship to Vti1b or SCAMP2. On the other hand, if the defects disappear in the rescued Vti1b and SCAMP2 cell lines, this suggests that the defects indeed have a direct relationship to Vti1b and SCAMP2. Analysis of Surface Delivery of Epidermal Growth Factor Receptors Supervisor: GUO Yusong / LIFS Student: LI Chun Wa / BCB Course: UROP1000, Summer Epidermal growth factor receptor (EGFR) is a type 1 receptor tyrosine kinase involved in cell proliferation, differentiation, migration, and survival. As a vital receptor, the surface trafficking of this transmembrane protein is of great physiological significance. Rab12 is a potential mediator of EGFR transport with little attention. To study protein-proteininteraction of Rab12 with EGFR, immunoprecipitation can be applied for Rab12 purification, in which an affinity tag is required for RAb12 protein enrichment. Therefore, molecular cloning was done to generate FLAG tag at 3’ end of Rab12 gene based on Rab12-3xHA plasmid and pCMV14 (3xFLAG) plasmid, followed by immunofluorescence staining to verify the normal functioning of Rab123xFLAG protein by comparing with functionally-verified Rab12-3xHA. In this project, we successfully cloned Rab12-3xFLAG gene and undergone immunofluorescence imaging for verification. Analysis of Secretion of Insulin-like Growth Factor 2 Supervisor: GUO Yusong / LIFS Student: LEE Kai Ho / BICH Course: UROP1100, Summer TMED10 is one of the members of the P24 family proteins. In a previous study, it was found that TMED10 is important for Insulin-like growth factor 2 (IGF2) secretion, and hence may contributes to regulating skeletal myogenesis and myoblast differentiation. Meanwhile, TMED10 is composed of different domains, and the GOLD domain is suspected to play a role in specific cargo recognition. In this research, we want to study the importance of GOLD domain on IGF2 secretion by generating a TMED10 deletion mutant lacking the GOLD domain. Immunoprecipitation and RUSH-assay will then be conducted to see the effect of GOLD domain on the IGF2-TMED10 interaction and the secretion of IGF2.