Trypanin, a Component of the Flagellar Dynein Regulatory Complex, Is Essential in Bloodstream Form African Trypanosomes
Figure 2
TPN Localizes to the Flagellum in Procyclic and Bloodstream Form Trypanosomes
(A–C) Immunofluorescence of procyclic (A) and bloodstream form (B) cells. Cytoskeletons were prepared by detergent extraction and used for immunofluorescence with anti-TPN monoclonal antibodies. Phase-contrast images are shown in the left panels and merged images are shown in the right panels with antibody staining in red and DAPI staining in blue. (C) Western blot showing that TPN is quantitatively associated with the flagellar cytoskeleton (P2). Whole cell lysates (L), or the indicated subcelluar fractions were prepared as described [21] from bloodstream form cells. Intact cytoskeletons (P1) were separated from detergent-soluble proteins (S1) by centrifugation at 1,000 × g, then further extracted with 0.5 M NaCl to depolymerize the subpellicular cytoskeleton, leaving the flagellar cytoskeleton (P2) intact. The intact flagellar cytoskeleton (P2) was separated from solubilized proteins (S2) by centrifugation at 16,000 × g.
(D–F) Cytoskeletons from procyclic (D) and bloodstream form (E and F) cells, prepared by extraction with 1% NP-40 to remove cellular membranes as described [21,45]. (D and E) Cells were extracted in solution then spotted onto slides and viewed directly. (F) Cells were adhered to glass coverslips prior to detergent-extraction. Scalebars are 10 μm.