Fig. 2: Plasmodium kinesin-8B motor domains are MT depolymerases.
From: Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission
a Representative TIRF-M kymographs of Pbkinesin-8B-MD depolymerising paclitaxel-stabilised MTs in the presence of ATP (middle) and AMPPNP (right). Depolymerisation occurs at both MT ends in both conditions; b Paclitaxel-stabilised-MT depolymerisation rate (nm/s) for Pbkinesin-8B-MD and Pfkinesin-8B-MD in the presence of ATP or AMPPNP compared to the no-kinesin control. Error bars represent the mean ± SD and individual measurements are also plotted. Ordinary one-way ANOVA was performed in Prism. Significance values are displayed as asterisks, ****p-values < 0.0001; ***p-value = 0.0002. nno-kinesin-8B = 112 ends, nPbkinesin-8B-MD,ATP = 56 ends, nPbkinesin-8B-MD,AMP = 68 ends, nPfkinesin-8B-MD,ATP = 103 ends, nPfkinesin-8B-MD,AMP = 92 ends. c Cryo-EM image showing protofilaments peeling from MT wall and forming ring-like structures (observed in two independent experiments). d Cryo-EM image showing ring structures formed by incubating tubulin and Pbkinesin-8B-MD in the presence of AMPPNP. A representative ring structure is highlighted with green circle. Scale bar in c, d = 50 nm. The rings were observed in three independent experiments. e Representative 2D class average (7906 particles) of AMPPNP-dependent Pbkinesin-8B-MD-induced tubulin ring structure; the outer ring is formed by curved αβ-tubulin dimers (white arrowhead), while and inner ring is formed by individual Pbkinesin-8B-MD density (white arrow). Scale bar = 10 nm. f Pbkinesin-8B-MDE1023A (olive green) does not exhibit MT-stimulated ATPase activity in the enzyme-coupled assay used for measuring ATPase activity (n = 3 for each point, mean ± SD), while WT Pbkinesin-8B-MD (dark green) induces a decrease in NADH absorbance (340 nm), both in the presence of 1 μM GMPCPP MTs. g Paclitaxel-stabilised-MT depolymerisation rate (nm/s) for Pbkinesin-8B-MD and Pbkinesin-8B-MDE1023A in the presence of ATP compared to the no-kinesin control. Error bars represent the mean ± SD and individual measurements are also plotted. Ordinary one-way ANOVA was performed in Prism. Significance values are displayed as asterisks, ****p-values < 0.0001; nno kinesin-8B = 54 ends, nPbkinesin-8B-MD, ATP = 60 ends, nPbkinesin-8B-MDE1023A,,ATP = 56 ends.
