Assembly of the Paraflagellar Rod and the Flagellum Attachment Zone Complex During the Trypanosoma brucei Cell Cycle

Abstract

Trypanosomes possess a single flagellum that is attached to their cell body via the flagellum attachment zone (FAZ). The FAZ is composed of two structures: a cytoplasmic filament complex and four microtubules situated next to it. There is a complex transmembrane crosslinking of this FAZ to the paraflagellar rod (PFR) and axoneme within the flagellum. We have partially purified the FAZ complex and have produced monoclonal antibodies both against the FAZ and the paraflagellar rod. The two antibodies against the FAZ (L3B2 and L6B3) recognise the cytoplasmic filament in immunofluorescence and in immunoelectron microscopy. On western blot, they detect a doublet of high molecular weight (M(r) 200,000). Two anti-PFR antibodies (L13D6 and L8C4) recognise the paraflagellar rod in immunofluorescence, but show a difference on Western blot: L13D6 recognises both major PFR proteins, whereas L8C4 is specific for only one of them. Using these new antibodies we have shown that although the growth of both cytoplasmic FAZ filament and external PFR are related, their growth initiates at different time points during the cell cycle and the two structures elongate at distinct rates.

The flagellum of Trypanosoma brucei: New tricks from an old dog

“…Components of the T. brucei FAZ are also mostly unknown, although the ultrastructure of the FAZ has been characterized in detail (Vickerman, 1962a;Sherwin and Gull, 1989;Gull, 1999;Kohl et al, 1999). The cytoplasmic side of the FAZ is defined by an electron-dense filament of unknown composition, together with four specialized microtubules that associate with a membranous compartment.…”

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

“…To test this possibility, we immunostained the control and TbSAS-4 RNAi cells with anti-CC2D antibody (12), the 1B41 antibody (34), and the L8C4 (anti-PFR2) antibody (33) to label the FAZ filament, the FAZ-associating quartet microtubules, and the flagellum, respectively (Fig. 3, A and B).…”

A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei

“…Fixed cells were then rehydrated with PBS and incubated in blocking buffer (1% BSA and 0.1% Triton X-100 in PBS) for 1 h at room temperature. Cells were then incubated with the primary antibody diluted in PBS containing 1% BSA at room temperature for 1 h. The following primary antibodies were used: FITC-conjugated anti-HA mAb (1:400 dilution, Sigma-Aldrich), anti-protein A pAb (1: 400 dilution, SigmaAldrich), anti-TbPLK pAb (1:1,000 dilution), L3B2 (1:20 dilution) (22), YL 1/2 mAb (1:1,000 dilution) (23), 20H5 (anti-Centrin mAb, 1:400 dilution) (24), PS54 (anti-phospho-Ser-54 of TbCentrin2, 1: 30,000 dilution) (16), and anti-TbSAS-6 pAb (1:1,000 dilution) (25). After three washes with wash buffer (0.1% Triton X-100 in PBS), cells were incubated with FITCconjugated or Cy3-conjugated secondary antibody at room temperature for another hour.…”

“…To examine whether flagellum detachment in SPBB1 RNAi cells was caused by defective assembly of the FAZ filament, cells were immunostained with the L3B2 antibody, which labels the FAZ1 protein in the FAZ filament (22,29), to monitor the integrity of the FAZ filament. In the non-induced control cells, all of the 2N2K cells contained two full-length FAZ filaments and two attached flagella.…”