Files
Abstract
Trypanosoma brucei is a protozoan responsible for the disease Human African Trypanosomiasis. Fluorescence microscopy has proven to be an invaluable asset for revealing cellular morphology of the trypanosome. Recent advances in super-resolution microscopy have allowed the observation of details previously only seen in electron microscopy techniques. But the dearth of fluorescent membrane probes compatible with super-resolution microscopy has limited its effectiveness in studies of membrane biology. In Chapter 2 we present optimized protocols for the use of mCLING and report its utility in both fixed and live trypanosomes as a super resolution-compatible fluorescent marker of the plasma membrane, flagellar membrane, and endocytic vesicles. In Chapter 3, we report an effort to characterize signaling pathways of trypanosome casein kinase TbCK1.2. TbCK1.2 regulates kinetoplast division and cytokinesis and is essential for cell viability. Delineation of protein kinase signaling pathways in the trypanosome is hampered by several factors. First, 60% of proteins in T. brucei lack homologs in model eukaryotes. Second, substrates for T. brucei kinases have not been defined experimentally. Combined, these facts preclude bioinformatic approaches for kinasepathway prediction. Here, we present a bottom-up approach to characterize the signaling pathways of TbCK1.2 and identify its endogenous kinase substrates. The strategies presented in this study may find general use for identifying proteins involved in pathways regulated by protein kinases in other obscure eukaryotes. In Chapter 4, we identify evidence of proteome adaptation to loss of the TbCK1.2 pathway protein BBP59. Most genes in T. brucei are not required for proliferation of BSF trypanosomes, but few studies have directly asked “why?”. Here we begin to address this question by studying trypanosomes lacking a BBP59 gene and identifying alterations in the proteome that may promote survival. Our results suggest that the concepts of “essential” and “non-essential” genes may need to be re-evaluated. Modern proteomic techniques have enabled and simplified highly sensitive, quantitative, global proteomic studies that can easily identify “silent” changes in the proteome associated with gene knockout. We propose a molecular definition of “essential gene” as one whose loss necessitates proteome remodeling for survival.