Generation of Clathrin(CLH) RNAi in T. brucei
Disciplines
Cell Biology
Abstract (300 words maximum)
Trypanosoma brucei is a unicellular eukaryote parasite found in sub-Saharan Africa. T. brucei causes human African trypanosomiasis (HAT) (sleeping sickness), a disease that is deadly untreated individuals. Fexinidazole is one of the drugs used to treat HAT.
Endocytosis is the engulfing of particles or fluids into cells via vesicles. Clathrin is an essential protein in the endocytosis pathway of trypanosomes. Clathrin is a scaffold protein comprised of three heavy and three light chains that coats intracellular vesicles during endocytosis. Other proteins such as AP2, dynamin, and actin are important for endocytosis. Trypanosome endocytosis differs from human endocytosis pathways. The process in T. brucei takes place at a specicalized region termed the flagellar pocket. Trypanosomes lacks adaptor protein 2 (AP2) compared to other eukaryotes. Also, dynamin is nonessential in T. brucei. This study contributes to the molecular and cellular biology of the endocytosis process in T. brucei.
The purpose for this study is to gain further knowledge about the mechanism of Clathrin mediated endocytosis process in T. brucei. We proceeded with this study by constructing an RNAi (Ribonucleic Acid interference) construct, we first performed cloning of the CLH RNAi construct. Next, we transfect CLH-RNAi construct into SM-mSCARLET cells. Following, we performed selection and screening for positive CLH-RNAi transfectants. Lastly, we performed western blot analysis. After induction with tetracycline (24hr), there was a depletion in cell growth rate and observation of “Big Eye” phenotype in the flagellar pocket caused by the depletion of CLH protein.
In conclusion, we observed that CLH is an essential protein in the T. Brucei endocytosis process. RNAi cell line was confirmed after CLH protein depletion was observed through western blot analysis (induction with tetracycline 24hr).
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Kojo Mensa-Wilmot
Generation of Clathrin(CLH) RNAi in T. brucei
Trypanosoma brucei is a unicellular eukaryote parasite found in sub-Saharan Africa. T. brucei causes human African trypanosomiasis (HAT) (sleeping sickness), a disease that is deadly untreated individuals. Fexinidazole is one of the drugs used to treat HAT.
Endocytosis is the engulfing of particles or fluids into cells via vesicles. Clathrin is an essential protein in the endocytosis pathway of trypanosomes. Clathrin is a scaffold protein comprised of three heavy and three light chains that coats intracellular vesicles during endocytosis. Other proteins such as AP2, dynamin, and actin are important for endocytosis. Trypanosome endocytosis differs from human endocytosis pathways. The process in T. brucei takes place at a specicalized region termed the flagellar pocket. Trypanosomes lacks adaptor protein 2 (AP2) compared to other eukaryotes. Also, dynamin is nonessential in T. brucei. This study contributes to the molecular and cellular biology of the endocytosis process in T. brucei.
The purpose for this study is to gain further knowledge about the mechanism of Clathrin mediated endocytosis process in T. brucei. We proceeded with this study by constructing an RNAi (Ribonucleic Acid interference) construct, we first performed cloning of the CLH RNAi construct. Next, we transfect CLH-RNAi construct into SM-mSCARLET cells. Following, we performed selection and screening for positive CLH-RNAi transfectants. Lastly, we performed western blot analysis. After induction with tetracycline (24hr), there was a depletion in cell growth rate and observation of “Big Eye” phenotype in the flagellar pocket caused by the depletion of CLH protein.
In conclusion, we observed that CLH is an essential protein in the T. Brucei endocytosis process. RNAi cell line was confirmed after CLH protein depletion was observed through western blot analysis (induction with tetracycline 24hr).