Functional Exploration of Heterotrimeric Kinesin-Ii on Intraflagellar Transport and Ciliary Length in Chlamydomonas reinhardtii

Heterotrimeric kinesin-2 (kinesin-II) serves as the anterograde motor for intraflagellar transport (IFT) that is essential for ciliary assembly. The functional significance of requiring two non-identical motor subunits in kinesin-II for IFT is not clear. In addition, the velocity of anterograde IFT varies significantly in different organisms, e.g. ~2.2 mm s-1 in Chlamydomonas and ~0.5 mm s-1 in mammalian cells. The mechanism underlying this difference and the impact of kinesin-II velocity on IFT and ciliary assembly remain elusive. To address these questions, we have studied human (HsKinesin-II), Chlamydomonas (CrKinesin-II) and chimeric kinesin-IIs in vitro and in vivo. We showed that the motor subunits (FLA10 and FLA8) of CrKinesin-II could self-interact respectively and the resulting homodimeric motors with two identical heads could function in vitro and in vivo. However, these motors could not interact with KAP (for FLA8) or with IFT complex (for FLA10), which necessitates heterodimer formation. Motility analyses of Crkinesin-II, HsKinesin-II and chimeric kinesin-IIs in vitro and in vivo suggest that the difference in the velocity of anterograde IFT is accounted for by the species-specificity of kinesin-II. Expressing the chimeric kinesin-II (KIF3B’/FLA10) reduced the velocity of anterograde IFT in Chlamydomonas by almost three-fold, which led to a significant reduction in IFT injection rate but only a mild decrease in ciliary length (~15%). Finally, theoretical modeling analysis reveals a non-linear scaling relationship between IFT velocity and ciliary length, implying a negative feedback in which the level of ciliary IFT proteins, in return, regulates the IFT injection rate.