Mitochondrial Dynamics in Crithidia fasciculata and Trypanosoma brucei

Open Access
Author:
Malfara, Madeline Frances
Area of Honors:
Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Megan Lindsay Povelones, Thesis Supervisor
  • Hans Schmidt, Honors Advisor
  • Elizabeth Anne Dudkin, Faculty Reader
Keywords:
  • Crithidia fasciculata
  • DLP
  • kinetoplastids
  • Trypanosoma brucei
  • CRISPR/Cas9
Abstract:
Trypanosoma brucei and Crithidia fasciculata are eukaryotic parasites that serve as model organisms for a variety of biological processes. Since these organisms diverged early in eukaryotic history, they offer insights into important pathways that have been conserved throughout the eukaryotic lineage. For T. brucei, there are well-developed techniques for studying the function of genes, such as RNA interference (RNAi). C. fasciculata have traditionally been a useful model for biochemistry; however, there are not yet well-established genetic techniques to investigate the role of different genes. We are particularly interested in the mitochondrion of these parasites, since it has many unusual features. To study pathways for mitochondrial dynamics in kinetoplastids, we have created and validated mitochondrial-GFP (mitoGFP)-expressing T. brucei and C. fasciculata cell lines. In this background, we have performed RNAi in T. brucei to confirm some of the published phenotypes of dynamin-like protein (DLP). We have also created a CRISPR/Cas9 C. fasciculata cell line, which can be used to knockdown many different proteins, including DLP. After confirming the expression of the CRISPR/Cas9 plasmid, we plan to knockdown pf16, a central axoneme protein of the flagellum. This proof-of-principle experiment will show that we can use this system to rapidly study gene function in C. fasciculata. We also plan to fuse a destabilizing domain construct to DLP in C. fasciculata, in order to study the role of what is likely an essential protein.