FIGURE 8:
NDK5H121A expression from a genomic transgene in WT cells impaired flagellar motility, length, RS assembly, and RS phosphorylation. (A) The percentages of clones with swimmers (S), paralyzed cells (P), or a mixture (P/S) expressing NDK5 (first bar) or NDK5H121A (second bar) from one experiment. Average flagellar length (B) and flagellar-length (Fla. length) distribution (C) of representative strains cultured in minimal media showed that flagellar lengths of P strains (P1) were between the control and ndk5, whereas P/S strains (P/S1) had flagella of normal length. Asterisks indicate statistically significant differences (p < 0.01, n = 50). (D) Western blot analysis of axonemes from transformants with different motility levels. RSPs in the flagella of P/S strains (P/S1 and P/S2) appeared normal but reduced in P strains (P1 and P2) (top). In addition, NDK5 and the scaffold protein RSP3 were hypophosphorylated (arrow), especially NDK5H121A (triangle). IC140, a subunit of the inner dynein arm, was the loading control. *Con., a WT strain transformed with pNDK5. Dashed line, the arbitrary line to distinguish exogenous proteins expressed from the transgenes. (E) The ratio of rapidly migrating NDK5 from the transgenes relative to total NDK5 in D. Ratio of exogenous protein in Con. strain represents the background. Exo., Exogenous.