A coiled-coil- and C2-domain-containing protein is required for FAZ assembly and cell morphology in Trypanosoma brucei

Abstract

SummaryTrypanosoma brucei, a flagellated protozoan parasite causing human sleeping sickness, relies on a subpellicular microtubule array for maintenance of cell morphology. The flagellum is attached to the cell body through a poorly understood flagellum attachment zone (FAZ), and regulates cell morphogenesis using an unknown mechanism. Here we identified a new FAZ component, CC2D, which contains coiled-coil motifs followed by a C-terminal C2 domain. T. brucei CC2D is present on the FAZ filament, FAZ-juxtaposed ER membrane and the basal bodies. Depletion of CC2D inhibits the assembly of a new FAZ filament, forming a FAZ stub with a relatively fixed size at the base of a detached, but otherwise normal, flagellum. Inhibition of new FAZ formation perturbs subpellicular microtubule organization and generates short daughter cells. The cell length shows a strong linear correlation with FAZ length, in both control cells and in cells with inhibited FAZ assembly. Together, our data support a direct function of FAZ assembly in determining new daughter cell length by regulating subpellicular microtubule synthesis.

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

“…Coverslips were blocked with 3% BSA in PBS at room temperature for 1 h, and then incubated with the primary antibody at room temperature for 1 h. The following primary antibodies were used: FITC-conjugated anti-HA mAb (1:400, Sigma-Aldrich), L8C4 (anti-PFR2 mAb, 1:50 dilution) (33), 1B41 (anti-␤-tubulin mAb, 1:400) (34), anti-TbSAS-4 pAb (1:400 dilution), anti-TbSAS-6 pAb (1:400 dilution) (10), anti-CC2D pAb (1:400 dilution) (12), and YL 1/2 (1:1,000 dilution, Millipore). After washing the coverslips three times with PBS, coverslips were incubated with FITC-or Alexa Fluor 594-conjugated secondary antibody at room temperature for 1 h. The coverslips were washed three times with PBS, and then mounted with DAPI-containing VECTASHIELD mounting medium (Vector Laboratories).…”

“…Knockdown of TbSAS-4 Causes Asymmetrical Cell DivisionThe length of the new FAZ filament is known to determine the cytokinesis cleavage plane in T. brucei (12). Therefore, a 2N2K cell with a short, new FAZ filament can undergo asymmetrical cytokinesis to produce a smaller-sized 1N1K daughter cell with a short FAZ filament (12).…”

“…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).…”

“…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).…”

Two distinct cytokinesis pathways drive trypanosome cell division initiation from opposite cell ends

“…The cell division plane in T. brucei is positioned by the newly assembled flagellum and its associated cytoskeletal structure termed the flagellum attachment zone (FAZ) filament (5,6). Thus, cytokinesis is initiated from the anterior tip of the new FAZ filament, and cleavage furrow ingression occurs uni-directionally along the longitudinal axis toward the posterior end of the cell (4, 7) without forming an actomyosin contractile ring at the cleavage furrow (8).…”