Final Report Summary - MGSVCT (Mitochondrial genome segregation via the cytoskeleton in trypanosomes)
We took three different strategies to identify new components. Firstly, we purified cytoskeleton materials, which contain our target proteins, to immune mice to harvest antibodies against the targets. By this way, we obtained more than 300 positive cells samples recognised basal body or tripartite attachment complex (TAC). These antibodies could be used for the identification of their target proteins later, as well as markers for future researches.
Secondly, those cytoskeleton materials were also used for mass spectrometry, a sensitive way to directly identify proteins. We found 845 proteins and selectively tested 24 of them by fluorescence labelling. Two novel basal body proteins stood out from the test: one localised in both basal body and pro-basal body, which is named CCDC77 after its human homolog, and another one localised between basal body and pro-basal body termed basal body connector protein-1 (BBCP-1). BBCP-1 is the first protein known to be in such a special position, which attracts our great interests.
Thirdly, we also enriched the proteins strongly binding to BBCP-1, and identified 88 proteins by mass spectrometry. Based on bioinformatics analysis, 17 of them are likely candidates of novel basal body proteins, which still required follow-on works to confirm.
We select these two new found proteins BBCP-1 and CCDC77 to study their role in Trypanosoma brucei basal body. Firstly, we used ribonucleic acid interference (RNAi) technique to abolish the synthesis of protein BBCP-1 and CCDC77. The abolishment of CCDC77 caused significant growth defeats in bloodsteam and insect forms, for BBCP-1 it eventually is even lethal. Thus, we put all our effort to try to understand the function of BBCP-1. The depletion of BBCP-1 resulted in asymmetric division of mitochondrial deoxyribonucleic acid (DNA) within 24 hours (bloodstream form) / 72 hours (insect form). In basal body labelled with YFP, RNAi of BBCP-1 led to splitting of new basal body and new pro-basal body within 8 hours (bloodstream forms) / 24 hours (insect form). RNAi of BBCP-1 resulted in mobility defeat by inflation of flagellar pocket in a later time point, 12 hours (bloodstream forms) / 72 hours (insect form). Electron microscopy of RNAi cells confirmed all sorts of defeats above in BBCP-1 RNAi cells.
Our works we have added great value in understanding the biology of basal body / centrosome. The works we planned to do will create a state-of-art platform for biological researches of TAC / basal body / centrosome.
We have shown BBCP-1 is required to retain the normal position of pro-basal body, which is essential for cell cycle progress, mitochondrial DNA maintenance and cell motility. This is up-to-date the first protein known to have such function. This finding advances our knowledge of the basal body/centrosome composition, assembly and division cycle. The identification of BBCP-1 opens a field to study its interacting proteins, as well as fascinates the mining of its orthologues in other eukaryotes. Its special RNAi phenotype provides a useful tool to study the biology of basal body / centrosome, and eventually an important tool to other eukaryotic researches.
Secondly, those cytoskeleton materials were also used for mass spectrometry, a sensitive way to directly identify proteins. We found 845 proteins and selectively tested 24 of them by fluorescence labelling. Two novel basal body proteins stood out from the test: one localised in both basal body and pro-basal body, which is named CCDC77 after its human homolog, and another one localised between basal body and pro-basal body termed basal body connector protein-1 (BBCP-1). BBCP-1 is the first protein known to be in such a special position, which attracts our great interests.
Thirdly, we also enriched the proteins strongly binding to BBCP-1, and identified 88 proteins by mass spectrometry. Based on bioinformatics analysis, 17 of them are likely candidates of novel basal body proteins, which still required follow-on works to confirm.
We select these two new found proteins BBCP-1 and CCDC77 to study their role in Trypanosoma brucei basal body. Firstly, we used ribonucleic acid interference (RNAi) technique to abolish the synthesis of protein BBCP-1 and CCDC77. The abolishment of CCDC77 caused significant growth defeats in bloodsteam and insect forms, for BBCP-1 it eventually is even lethal. Thus, we put all our effort to try to understand the function of BBCP-1. The depletion of BBCP-1 resulted in asymmetric division of mitochondrial deoxyribonucleic acid (DNA) within 24 hours (bloodstream form) / 72 hours (insect form). In basal body labelled with YFP, RNAi of BBCP-1 led to splitting of new basal body and new pro-basal body within 8 hours (bloodstream forms) / 24 hours (insect form). RNAi of BBCP-1 resulted in mobility defeat by inflation of flagellar pocket in a later time point, 12 hours (bloodstream forms) / 72 hours (insect form). Electron microscopy of RNAi cells confirmed all sorts of defeats above in BBCP-1 RNAi cells.
Our works we have added great value in understanding the biology of basal body / centrosome. The works we planned to do will create a state-of-art platform for biological researches of TAC / basal body / centrosome.
We have shown BBCP-1 is required to retain the normal position of pro-basal body, which is essential for cell cycle progress, mitochondrial DNA maintenance and cell motility. This is up-to-date the first protein known to have such function. This finding advances our knowledge of the basal body/centrosome composition, assembly and division cycle. The identification of BBCP-1 opens a field to study its interacting proteins, as well as fascinates the mining of its orthologues in other eukaryotes. Its special RNAi phenotype provides a useful tool to study the biology of basal body / centrosome, and eventually an important tool to other eukaryotic researches.