MULTIDISCIPLINARY APPROACH TO STUDY THE SPATIOTEMPORAL ASPECTS OF DE NOVO PURINE BIOSYNTHESIS (PURINOSOME)
Open Access
- Author:
- Chan, Chung Yu
- Graduate Program:
- Engineering Science and Mechanics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 14, 2017
- Committee Members:
- Tony Jun Huang, Special Member
Bruce J. Gluckman, Committee Chair/Co-Chair
Corina S. Drapaca, Committee Member
Ibrahim Tarik Ozbolat, Committee Member
Tak Sing Wong, Outside Member
Stephen J. Benkovic, Special Member
Bruce J. Gluckman, Dissertation Advisor/Co-Advisor - Keywords:
- De novo purine biosynthesis
Purinosome
Metabolism
Super resolution imaging
Microfluidics
Surface acoustic wave - Abstract:
- The growth and proliferation of a single cell is tightly associated to its energy and metabolism. To maintain the homeostasis and the necessary growth of the cell, machinery related to energy and metabolism has to be regulated extensively. One of the metabolic pathway is de novo purine biosynthesis which involves the synthesis of purine from six enzymes in ten, highly conserved steps. De novo purine biosynthesis is also an important, validated target for cancer chemotherapeutics. With the discovery of metabolon – the purinosome in this metabolic pathway, a paradigm shift of understanding the regulation of metabolism emerges. While pioneer works on this non-membrane bound enzyme complex have been carried out, the complete understanding of function and mechanism of purinosome is not yet revealed. In this dissertation, we conduct a comprehensive study of the spatiotemporal aspects of the purinosome by multidisciplinary approaches across different fields. With our unique approaches, we believe that the study of its spatiotemporal aspects can provide significant and novel insights for cancer biology. Characterization of the purinosome is first performed with various biochemical assays. The average size and number distribution of purinosomes are measured in the cells by conventional fluorescence microscopy. With the morphological definition of purinosomes, we demonstrates that purinosome formation in cell is in accordance with the cell cycle phases. The results are further supported by the fact that purinosome formation is not a function of the enzyme protein expression. Using the biochemical approach, we establish that purinosome is a cellular biomarker in response to cellular purine requirements. Next, we seek to understand the spatial organization of purinosomes with other subcellular components in the cell. To obtain the highest spatial resolution possible, we employ Stochastic Optical Reconstruction Microscopy (STORM) to visualize and define their interaction at submicron level. Our results indicate that there is high probability of purinosomes dual-colocalized with both microtubules and mitochondria, suggestive the possible interplay among the three cellular components. The observation of purinosomes spatially organized with microtubules and mitochondria by STORM posts an intriguing question of how the interplay of the three cellular components would be. To parse the interaction among the three, the motion of the purinosome is temporally resolved by super-resolution confocal imaging. In general, purinosomes are found to have a high tendency to be actively transported by microtubules towards and away from mitochondria. Microtubules also provide stability for the spatial organization between purinosomes and mitochondria. Their high colocalization percentage could indicate a bioenergetics advantage between the two cellular components. Finally, the understanding of the purinosome in cell metabolism at the cell-cell interaction level is explored. To control the microenvironment and precisely position the cells of interest, we develop polystyrene-based microfluidic device and standing surface acoustic wave (SSAW) tweezers. We validate both systems for cell culture and purinosome study. SSAW powers are optimized and their effect in instantaneous cell viability and long term cell proliferation are investigated. Using the optimized SSAW power, we confirm that optimal SSAW is suitable for the study of purinosome formation during cell-cell interaction. Taken together, multidisciplinary approach is essential for the study of spatiotemporal aspects of purinosome. With the advantages and capabilities in different approaches, the regulation and function of purinosome is further elucidated. The notions presented in this dissertation offer significant foundation for the future study of similar enzyme complex system in the cell.