APEX2 Proximity Proteomics Resolves Flagellum Subdomains and Identifies Flagellum Tip-Specific Proteins in Trypanosoma brucei

Abstract

Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum (cilium) that plays critical roles in transmission and pathogenesis. An emerging concept is that the flagellum is organized into subdomains, each having specialized composition and function. The overall flagellum proteome has been well studied, but a critical knowledge gap is the protein composition of individual subdomains. We have tested whether APEX-based proximity proteomics could be used to examine the protein composition of T. brucei flagellum subdomains. As APEX-based labeling has not previously been described in T. brucei, we first fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, a well-characterized axonemal complex. We found that DRC1-APEX2 directs flagellum-specific biotinylation, and purification of biotinylated proteins yields a DRC1 “proximity proteome” having good overlap with published proteomes obtained from purified axonemes. Having validated the use of APEX2 in T. brucei, we next attempted to distinguish flagellar subdomains by fusing APEX2 to a flagellar membrane protein that is restricted to the flagellum tip, AC1, and another one that is excluded from the tip, FS179. Fluorescence microscopy demonstrated subdomain-specific biotinylation, and principal-component analysis showed distinct profiles between AC1-APEX2 and FS179-APEX2. Comparing these two profiles allowed us to identify an AC1 proximity proteome that is enriched for tip proteins, including proteins involved in signaling. Our results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve the proteome composition of flagellum subdomains that cannot themselves be readily purified. IMPORTANCE Sleeping sickness is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei. The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need for approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei and has allowed identification of proximity proteomes for different flagellar subdomains that cannot be purified. This capacity opens the possibility to study the composition and function of other compartments. We expect this approach may be extended to other eukaryotic pathogens and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilium function is impaired.

A multi-adenylate cyclase regulator at the flagellar tip controls African trypanosome transmission

“…2c and Extended Data Fig. 2b) as expected from the presence of a transmembrane domain (TMD) and consistent with previous reports 17,18 . The probability of colocalization between two fluorescently tagged protein populations was determined by the coordinatebased colocalization (CBC) analysis method of Malkusch, et al 1) showed significant colocalization for CARP3-ACP1 (p < 0.05) that was in the same range as the positive control (CARP3-PAmCherry-CARP3mNG;…”

“…1e, Extended Data Fig. 1h), consistent with subcellular proteome studies 16,17,40 . CARP3 is concentrated at the tip of the parental and daughter flagella (Fig.…”

“…The trypanosome flagellar tip is a site with particular importance for interaction with host tissue surfaces 13 and forms a distinct microdomain. This is defined by exclusive localization of a subset of flagellar proteins [14][15][16][17] , including some members of the AC multigene family 18 . The cAMP microdomain concept was first described by Buxton and Brunton 19 and is now well established in different systems 20 including primary cilia 21,22 .…”

FAP106 is an interaction hub required for assembly of conserved and lineage-specific microtubule inner proteins at the cilium inner junction

“…mNeonGreen and APEX2-tagged cell lines were generated in the 29-13 background by C-terminal tagging (40) with pPOTv6-puro-puro-mNG or pPOTv7-blast-blast-APEX2-3xHA, respectively. pPOTv7-blast-blast-APEX2-3xHA contains the same APEX2-3xHA tag as in (48). For MC3-NG, a transient CRISPR-Cas9 system (49) was used to tag both alleles.…”

Paving the Way: Contributions of Big Data to Apicomplexan and Kinetoplastid Research

“…Proximity-dependent biotinylation approaches have been extremely useful for studying the protein interactome of T. brucei in various contexts. These include the identification of bilobe components ( Morriswood et al., 2013 ); changes resulting from the ectopic expression of developmentally-regulated RNA-binding proteins ( De Pablos et al., 2017 ); mapping the interactome of the T. brucei cytokinetic machinery ( Hilton et al., 2018 ); identification of key proteins required for microtubule quartet anchorage to basal bodies ( Dong et al., 2020 ); novel cytoskeleton-associated proteins essential for morphogenesis and cytokinesis ( Schock et al., 2021 ); and flagellum tip-specific proteins ( Vélez-Ramírez et al., 2021 ). Still within the umbrella of fluorescence, TrypTag has been an invaluable tool for the parasitology community.…”