SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament

Abstract

The centrosome in animals constitutes the cell's microtubule-organizing center that nucleates spindle assembly, and is characterized by a symmetrical array of nine microtubule triplets emanating from a cartwheel structure in the very proximal region of the centriole. Biogenesis of centrioles requires many regulatory proteins, including SAS-6, SAS-4/CPAP, and BLD10/CEP135, which are thought to constitute the core ancestral module involved in centriole assembly (1). The three proteins have distinct functions in centriole biogenesis. SAS-6 assembles to the cartwheel (2, 3) and BLD10/CEP135 forms the pinhead that connects the cartwheel spokes to the A-microtubules of the microtubule triplets (4, 5), whereas SAS-4/CPAP controls the elongation of centriolar microtubules (6 -9).Trypanosoma brucei, a flagellated protozoan and the causative agent of human sleeping sickness, does not have centrosomes, but it possesses the basal body as its microtubule-organizing center to nucleate the assembly of a motile flagellum. The basal body is characterized by an array of nine microtubule triplets, with the conserved SAS-6 protein forming the cartwheel structure (10), similar to that of the centrosome in metazoa. The flagellum is attached, along most of its length, to the cell body via a specialized cytoskeletal structure termed the flagellum attachment zone (FAZ), 2 which consists of three main domains: the structure connecting the paraflagellar rod (PFR) to the flagellar membrane, the structure connecting the flagellar membrane and the cell body membrane, and a cytoplasmic filament (often referred to as the FAZ filament) and its associated microtubule quartet (11)(12)(13)(14)(15)(16)(17). In addition to mediating flagellum-cell body attachment, the FAZ filament also defines the cytokinesis cleavage plane (11,12), with the distal tip of the new FAZ filament marking the site of cytokinesis initiation (18 -20). Moreover, during the cell cycle in the procyclic form, the elongation of the FAZ filament positions the newly assembled basal body toward the cell posterior (21), which is crucial for organelle segregation and cell division (10,(22)(23)(24)(25).Trypanosomatids, a group of kinetoplastid parasites consisting of T. brucei, Trypanosoma cruzi, and Leishmania spp., appear in a variety of different morphological forms during their life cycle and are distinguished by the relative position of the kinetoplast (mitochondrial DNA) and the nucleus and by the position, length, and cell body attachment of the flagellum (26). In the tsetse fly vector, T. brucei differentiates from the trypomastigote form to the epimastigote form, which undergoes asymmetrical cell division to produce a long epimastigote cell and a short epimastigote cell (26). How life cycle transitions in T. brucei are regulated is largely unknown, but recent work has begun to uncover the regulators involved in this process. ALBA3 and ALBA4, two closed related RNA-binding proteins, are the first factors found to be involved in life cycle transitions in T. brucei (27). Anothe...

An EF-hand-containing Protein in Trypanosoma brucei Regulates Cytokinesis Initiation by Maintaining the Stability of the Cytokinesis Initiation Factor CIF1

“…The new FAZ tip appears to comprise many proteins, which likely play distinct functions, including cytokinesis initiation (8,11) and life cycle transition (23). The cytokinesis regulators, including the three components of the chromosomal passenger complex (TbAUK1, TbCPC1, and TbCPC2), TbPLK, and CIF1 (aka TOEFAZ1), localize to the new FAZ tip only (8 -11), whereas TbSAS-4, which controls life cycle transition from the epimastigote form to the trypomastigote form, and its associated proteins, FAZ6, FAZ11, FAZ13, and FAZ14, are enriched at the anterior tip of both the new and old FAZs (23). Despite the functional distinction between CIF1 and TbSAS-4, BioID with CIF1 as the bait was able to detect TbSAS-4 and its associated FAZ tip proteins (Table 1), indicating that TbSAS-4 and its partner proteins are located at the new FAZ tip in close proximity to CIF1.…”

“…The affinity purification and mass spectrometry were repeated three times, and the proteins that were only detectable in CIF1-BirA*-HA overexpression cells from all three experimental repeats were considered CIF1-binding proteins and near neighbors (supplemental Table S1). Among these proteins are nine known FAZ filament proteins, FAZ1-FAZ4 (20 -22), FAZ9 (10,20), FAZ10 (19), FAZ15 (10), CC2D (3), and KMP-11 (22), and four known FAZ tip proteins, FAZ6 (20), FAZ11 (19), FAZ14 (23), and TbSAS-4 (23) ( Table 1).…”

“…Affinity purification of biotinylated proteins was performed according to our previous publication (23). CIF1-BirA*-HA was overexpressed by induction with 0.5 g/ml tetracycline for 24 h, and cells (ϳ2.5 ϫ 10 9 ) were incubated with 50 M biotin for an additional 24 h. Cells were washed 3 times with PBS and treated with PEME buffer (100 mM PIPES, pH 6.9, 2 mM EGTA, 0.1 mM EDTA, 1 mM MgSO 4 ) containing 0.5% Nonidet P-40.…”

Filling the Void: Proximity-Based Labeling of Proteins in Living Cells

“…More importantly, BioID identified a novel protein interacting partner with lamin A, SLAP75, which was found to associate with the nuclear envelope. BioID has been similarly used to identify uncharacterized PPIs and constituents within largely insoluble cellular structures such as the mammalian cell-cell junctions [13–18] and trypanosome bilobe and flagella [19–22], structures that are often refractory to traditional study with methods such as affinity complex purification. As the labeling radius of BioID is limited, it can be used to map the population and spatial distribution of proteins within large structures by application to proteins throughout the structure, as was demonstrated at the nuclear pore complex [12].…”

Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance

“…Subsequently, two new pBBs are assembled next to the two mBBS, and one mBB/pBB pair moves to the posterior region of the cell (9, 10). T. brucei contains the evolutionarily conserved SAS-4 and SAS-6 homologs (11, 12) and a highly divergent BLD10 homolog (4). While TbSAS-6 is functionally conserved (11), TbSAS-4 is not localized to the basal body and plays a distinct function in life cycle transitions (12), and TbBLD10 has not been experimentally confirmed as a bona fide component of the basal body.…”