Fig. 2.

Mutation of the Ca²⁺ binding sites in FCaBP does not affect Ca²⁺-dependent flagellar membrane localization, possibly due to dimerization with endogenous wildtype FCaBP. (A) The flagellar retention of FCaBP^E151Q/E188Q is Ca²⁺-dependent. Untransfected epimastigotes and transfectants expressing the N-terminal 24 amino acids of FCaBP fused to GFP (FCaBP^1–24-GFP) or the FCaBP Ca²⁺-binding mutant FCaBP^E151Q/E188Q-myc were permeabilized and washed with buffers containing or lacking (EDTA) Ca²⁺. Cells were then analyzed by direct fluorescence (GFP) or immunofluorescence microscopy with paraflagellar rod [PFR (T. cruzi PAR1)]- or FCaBP-specific antisera. DNA was stained with DAPI. Both endogenous FCaBP and FCaBP^E151Q/E188Q-myc were washed out of the flagellum upon Ca²⁺ chelation. (B) Recombinant wildtype FCaBP and the Ca²⁺-binding mutant FCaBP^E151Q/E188Q form dimers in a Ca²⁺-independent manner. Size exclusion chromatography of recombinant wildtype FCaBP and Ca²⁺ binding mutant FCaBP^E151Q/E188Q were conducted in the presence of 2 mM CaCl₂ (solid line) or 2 mM EDTA (dashed line). The absorbance at 280 nm is plotted vs elution volume. Both wildtype FCaBP and FCaBP^E151Q/E188Q elute as dimers in both Ca²⁺-bound and Ca²⁺-free states. Multi-angle light scattering confirmed that FCaBP is a dimer in both Ca²⁺-free and Ca²⁺-bound states (not shown).