Presentation Title

Identification of Trypanosoma brucei Microtubule Inner Proteins (MIPs) using APEX2 Proximity Labeling

Faculty Mentor

Michelle Shimogawa, Kent Hill

Start Date

23-11-2019 10:45 AM

End Date

23-11-2019 11:30 AM

Location

60

Session

poster 4

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Trypanosoma brucei is a unicellular, eukaryotic parasite that causes African sleeping sickness, a neglected tropical disease endemic to sub Saharan Africa, where 65 million people live at risk. The flagellum of T. brucei has multiple key functions, including motility, sensing, and signaling, and is thus essential for the survival of the parasite. The axoneme of the flagellum is highly stable, yet sources of this stability are not completely known. There has been some attribution to the impact of microtubule inner proteins (MIPs); however, there are very few MIPs identified and characterized in any organism. A recent paper (Owa et al., 2019) has identified two proteins, FAP45 and FAP52, as MIPs within the flagella of Chlamydomonas spp; additionally, the Hill lab has cryo-electron tomography data demonstrating that T. brucei FAP45 and FAP52 homologs exist nearby other unknown MIPs. A possible method of identifying these unknown MIPs is to use APEX2 proximity labeling to biotinylate neighboring proteins of FAP45 and 52, allowing for affinity purification and identification by mass spectrometry. In this project, we successfully fused the APEX2-HA enzyme to the T. brucei homolog of FAP45 and demonstrated presence in flagellar axoneme-specific cell fractions by Western blotting. We then performed APEX2 proximity labeling and observed FAP45-specific biotinylation in cytoskeleton-specific fractions by Western blotting and along the flagellum through immunofluorescence microscopy. Future experiments will explore the resulting mass spectrometry data to identify additional candidate MIPs and attempt knockdown of FAP45 and FAP52 to identify possible mutant phenotypes.

M. Owa et al., Inner lumen proteins stabilize doublet microtubules in cilia and flagella. Nat Commun 10, 1143 (2019).

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Nov 23rd, 10:45 AM Nov 23rd, 11:30 AM

Identification of Trypanosoma brucei Microtubule Inner Proteins (MIPs) using APEX2 Proximity Labeling

60

Trypanosoma brucei is a unicellular, eukaryotic parasite that causes African sleeping sickness, a neglected tropical disease endemic to sub Saharan Africa, where 65 million people live at risk. The flagellum of T. brucei has multiple key functions, including motility, sensing, and signaling, and is thus essential for the survival of the parasite. The axoneme of the flagellum is highly stable, yet sources of this stability are not completely known. There has been some attribution to the impact of microtubule inner proteins (MIPs); however, there are very few MIPs identified and characterized in any organism. A recent paper (Owa et al., 2019) has identified two proteins, FAP45 and FAP52, as MIPs within the flagella of Chlamydomonas spp; additionally, the Hill lab has cryo-electron tomography data demonstrating that T. brucei FAP45 and FAP52 homologs exist nearby other unknown MIPs. A possible method of identifying these unknown MIPs is to use APEX2 proximity labeling to biotinylate neighboring proteins of FAP45 and 52, allowing for affinity purification and identification by mass spectrometry. In this project, we successfully fused the APEX2-HA enzyme to the T. brucei homolog of FAP45 and demonstrated presence in flagellar axoneme-specific cell fractions by Western blotting. We then performed APEX2 proximity labeling and observed FAP45-specific biotinylation in cytoskeleton-specific fractions by Western blotting and along the flagellum through immunofluorescence microscopy. Future experiments will explore the resulting mass spectrometry data to identify additional candidate MIPs and attempt knockdown of FAP45 and FAP52 to identify possible mutant phenotypes.

M. Owa et al., Inner lumen proteins stabilize doublet microtubules in cilia and flagella. Nat Commun 10, 1143 (2019).