Sequences in the stalk domain regulate auto-inhibition and ciliary tip localization of the immotile kinesin-4 KIF7

Abstract

The kinesin-4 member KIF7 plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by autoinhibition as the full-length protein is inactive for microtubule binding in cells. We identify a segment, the inhibitory coiled coil (inhCC), that is required for autoinhibition of KIF7 whereas the adjacent regulatory coiled coil (rCC) that contributes to autoinhibition of the motile kinesin-4's KIF21A and KIF21B, is not sufficient for KIF7 autoinhibition. Disease-associated mutations in the inhCC relieve autoinhibition and result in strong microtubule binding. Surprisingly, uninhibited KIF7 proteins did not bind preferentially to or track the plus ends of growing microtubules in cells, as suggested by previous in vitro work, but rather bound along cytosolic and axonemal microtubules. Localization to the tip of the primary cilium also required the inhCC and could be increased by disease-associated mutations regardless of the protein's autoinhibition state. These findings suggest that loss of KIF7 autoinhibition and/or altered cilium tip localization can contribute to pathogenesis of human disease.

Hedgehog-induced ciliary trafficking of kinesin-4 motor KIF7 requires intraflagellar transport but not KIF7’s microtubule binding

“…Because the KIF7 rigor variant localized along cytosolic microtubules rather than at their plus ends ( Figure 2 and Supplemental Figure S2), we were surprised that this variant localized at the cilium tip rather than along the shaft of the cilium ( Figure 3A ). It appears that this variant’s loss of autoinhibition ( Figure 2B ) alters its ability to enter and/or exit the cilium compartment, similar to the behavior of a pathogenic mutant that results in loss of autoinhibition ( Blasius et al. , 2021 ).…”

“…, 2014 ). We note that the expression level of KIF7 does not influence its localization to cytosolic microtubules or to the cilium tip ( Blasius et al. , 2021 ).…”

“…These findings do not support model 3 ( Figure 1 ) as the primary mechanism by which Hh stimulation causes an increase in KIF7 localization to the cilium tip. Further evidence against model 3 comes from our recent work demonstrating that Hh stimulation does not induce KIF7 to bind to the plus ends of microtubules in cells ( Blasius et al. , 2021 ).…”

“…These results suggest that a Hh-induced change in the ability of KIF7 to access the ciliary compartment (model 1; Figure 1 ) is unlikely to be the primary mechanism by which Hh stimulation causes an increase in KIF7 localization to the cilium tip. Further evidence against model 1 comes from our recent work demonstrating that the truncated version KIF7(1-558) localizes along the shaft of the primary cilium ( Blasius et al. , 2021 ), suggesting that the motor domain of KIF7 can access the cilium in the unstimulated state.…”

“…Image analysis was performed using ImageJ (National Institutes of Health [NIH]). Although the level of KIF7 expression does not influence its ability to localize to the tip of the primary cilium ( Blasius et al. , 2021 ), images were only acquired of ciliated cells expressing the indicated constructs at low levels.…”