Plasmodium kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission
Fig 2
Kinesin-8X shows ATPase, gliding motor and depolymerization activities.
(A) Schematic protein organisation of PBANKA_0805900 (Pbkinesin-8X) and PF3D7_0319400 (Pfkinesin-8X) showing their full-length sequence and central location of the motor domain, Pbkinesin-8X (green) and Pfkinesin-8X (blue). (B-D) Activities of Pb (top—green) and Pf (bottom–blue) kinesin-8X motor domains in three kinesin assays. (B) MT stimulated ATPase activity; data fitted to an adapted Michaelis-Menten equation with calculated Vmax, Km and V0 parameters. V0 was included as a term to aid the curve fitting and account for the non-zero basal ATPase activity of the kinesins in absence of MTs. Error bars represent the mean +/- SD for each MT concentration, n = 3. (C) MT gliding activity measured by TIRF microscopy; left, the average motility (nm/s) and individual data points are plotted. The difference between Pbkinesin-8X and Pfkinesin-8X velocity is statistically significant (t-test P <0.0001). Error bars represent the mean +/- SD; right, an exemplar kymograph demonstrates plus-end directed MT gliding using polarity-marked MTs (schematic above). (D) MT depolymerization measured using TIRF microscopy; depolymerization rate (nm/s) in the presence of ATP and AMPPNP is compared to a control in the absence of nucleotide. Error bars present the mean +/- SD, for Pbkinesin-8X control n = 77, AMPPNP n = 74, ATP n = 93, and for Pfkinesin-8X control n = 112, AMPPNP n = 116, ATP n = 117. An ordinary one-way ANOVA was performed on the depolymerisation data for Pbkinesin-8X and Pfkinesin-8X separately in Prism to establish the significance of the nucleotide-dependent differences. Significance values are displayed as asterisks, all p-values were <0.0001 (****) comparing control with the presence of AMPPNP or ATP and comparing activity in the presence of AMPPNP or ATP.