School of Science Division of Life Science 40 How Cells Safeguard Mitochondria, the Powerhouse of the Cell? Supervisor: WANG Lan / LIFS Student: ZHANG Fangting / BCB Course: UROP 1100, Spring UROP 2100, Summer ATAD1 is a crucial protein involved in mitochondrial protein quality control. However, its interactors remain poorly characterized. In prior experiments, several candidate proteins, including AKAP1 and FKBP8, were identified as potential interactors. This study aimed to validate these interactions by constructing four plasmids (pFT01-pFT04) using Gibson assembly. The vectors were linearized via double digestion, and the inserts were PCR-amplified with homologous ends and subsequently gel-purified. The resulting plasmids were transformed into E. coli and purified using Miniprep for sequencing. The sequence-verified plasmids will be transfected into C2C12 cells, facilitating live cell imaging to investigate protein localization. Future work will utilize FLAG immunoprecipitation to confirm the interactions between AKAP1/FKBP8 and ATAD1, thereby enhancing our understanding of their roles in mitochondrial function. Mechanistic Investigation of Mitochondrial Quality Control Supervisor: WANG Lan / LIFS Student: LIU Shanqi / BCB-IRE Course: UROP 1000, Summer SidI is a tRNA-like protein from Legionella pneumophila, an intracellular bacterial pathogen and the causative organism of Legionnaires’ disease. It can combine with the ribosome and stop protein synthesis and even cause ribosome collision. To detect the structure of the SidI-ribosome complex the lab members designed some constructs. Previously, we tried replacing the GST tag to MBP- and Flag-tag to exclude the possibility that the SidI-ribosome crosslinked band is generated from a dissociated GST tag crosslinked with SidI, but the expression of neither MBP- or Flag- SidI had a satisfactory yielding, we, therefore, decided to cleave the GST tag off from SidI and use SidI crosslinked with ribosome directly for the subsequent Cryo-EM analysis. We optimized the cleavage strategy through two trials to minimize contamination, but further refinement is needed to efficiently remove the GST tag and purify SidI via FPLC for Cryo-EM screening. Mechanistic Investigation of Mitochondrial Quality Control Supervisor: WANG Lan / LIFS Student: YU Chun Ip William / BIBU Course: UROP 2100, Fall ATPase-associated diverse cellular activity domain-containing protein 3 (ATAD3) is a protein located at the inner membrane of the mitochondria and has an indispensable role in its quality control. Cells with disruption in ATAD3 showed reduced mitochondria number and abnormal cristae structure. Yet, the major role of ATAD3 remains an enigma and its interactors are still to be found. Subsequent to the previous report, this progress report aimed to study the potential interactors of ATAD3 with proximity labeling and the impact of long-term gene silencing on the mtDNA, and mRNA level through the introduction of short hairpin sequences into the C2C12 cell line, here we summarize and discuss the results.
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