Huntingtin S421 phosphorylation increases kinesin and dynein engagement on early endosomes and lysosomes

Huntingtin (htt) acts as a scaffolding protein that recruits motor proteins to vesicular cargoes, enabling it to regulate kinesin- and dynein-dependent transport. To maintain the native stoichiometry of huntingtin with its interacting partners, we used CRISPR/Cas9 to induce a phosphomimetic mutation of the endogenous htt at S421 (S421Dhtt). Using single-particle tracking, optical tweezers, and immunofluorescence, we examined the effects of this mutation on the motility of early endosomes and lysosomes. In S421Dhtt cells, early endosomes and lysosomes exhibited more outward motility towards the cell periphery compared to wild-type (WT) cells. In contrast, overexpression of htt had variable effects on the processivity, run length, and directional bias of both early endosomes and lysosomes. Kinesins and dyneins exerted greater forces on early endosomes and lysosomes in cells expressing S421Dhtt. Additionally, endosomes had higher binding rates, increased resistance to detachment under load, and enhanced recruitment of kinesins and dyneins in S421Dhtt cells. These data indicate that phosphorylation of the endogenous huntingtin causes early endosomes and lysosomes to move towards the cell periphery by activating both kinesins and dyneins. Summary Statement Huntingtin phosphorylation at S421 increases the outward motility of early endosomes and lysosomes by enhancing the microtubule binding and force generation of kinesin and dynein.