Major Professor: Dr. Scott Stagg
Title: Investigating the Functional Role of TFG in-situ Using Cryo-electron Tomography.
Cryo-electron tomography (Cryo-ET) is a technique to determine the three-dimensional (3D) structure of biological specimens. It provides the opportunity to image the molecular and supramolecular compartments within an unperturbed environment of cells at nanometer resolution. In order to achieve high-resolution images in Cryo-ET, the specimen should be sufficiently thin (<5um) to allow diffusion of electrons without a major alteration to their initial direction and energy. Since the thickness of typical eukaryotic cells is more than 5um, Cryo-ET structural analysis has been conventionally restricted to small bacterial cells, isolated viruses or peripheral regions of the eukaryotic cells. Techniques, such as ultramicrotomy of chemically fixed cells and vitreous sectioning have enabled visualisation of thicker cells and polymorphic structures. However, the methods are technically challenging to be implemented and subjected to various physical defects resulting in poor quality images. An alterative approach to achieve an intact thin section of the cellular samples is utilizing focused ion beam (FIB). In this technique, a high current focused ion beam (usually gallium, Ga+) is used to precisely remove materials from the surface of the specimen at targeted sites. The thin slices (lamella) achieved through this technique are shown to result in high-resolution Cryo-ET images . Based on this novel development, one aspect of my research focuses on electron-tomography (ET) of the mammalian cells lamella, in order to investigate the function and morphological structure of Trk-fused gene protein (TFG). Studies have suggested that TFG forms polymeric network to tether the interface of Golgi apparatus and endoplasmic reticulum (ER). Other studies have also indicated that TFG plays a role in un-coating of COPII vesicles. In addition, the inhibition of TFG function has been reported to associate with certain genetic gait disorders such as hereditary spastic paraplegia (HSP). However, the exact function of TFG is yet to be entirely understood. For this purpose, the wild type retinal pigment epithelial (RPE1) cell line is grown on electron microscopy (EM) grid and has gone through a FIB-milling process. Several tomograms of the lamellas (~ 200 nm thick) have been collected at the near nucleus regions and reconstructed by tomography. Our tomograms reveal some information regarding the architecture of ER and certain transport vesicles; however, investigating the TFG component within the crowded interior of the cell remains challenging. As part of a future work, the mutant RPE1 cell line will be induced to overexpress its TFG contents and the procedures of FIB-milling and Cryo-ET will be implemented to visualise the cluster of TFG and reconstruct its 3D structure to obtain a better understanding of its functional role.
MOB Graduate Student
Major Professor: Dr. Huan-Xiang Zhou/Dr. Hong Li
Title: Competitive Interactions between Macromolecular Crowders and Ligand with Maltose Binding Protein.
Macromolecular crowding has been known to affect the thermodynamics of biomolecular interactions and protein folding. Within this perspective, the volume exclusion effects have gained precedence over the history of this field. Using a well studied protein-ligand pair, we show that the soft interaction effects of both protein and polymeric crowders are competitive in nature. We also show that said soft interaction effects are sensitive to subtle changes in the protein’s conformation, thereby elucidating that protein-crowder non-specific interactions are not necessarily random.