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

Huntingtin (HTT) is a scaffolding protein that recruits motor proteins to vesicular cargoes, enabling it to regulate kinesin-1, dynein, and myosin-VI-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 (HTT-S421D). Using single particle tracking, optical tweezers, and immunofluorescence, we examined the effects of this mutation on the motility of early endosomes and lysosomes. In HTT-S421D cells, lysosomes exhibit longer displacements and higher processive fractions compared to wild-type (HTT-WT) cells. Kinesins and dyneins exert greater forces on early endosomes and lysosomes in cells expressing HTT-S421D. Additionally, endosomes bind to microtubules faster and are more resistant to detachment under load. The recruitment of kinesins and dyneins to microtubules is enhanced in HTT-S421D cells. In contrast, overexpression of HTT had variable effects on the processivity, displacement, and directional bias of both early endosomes and lysosomes. These data indicate that phosphorylation of the endogenous huntingtin causes early endosomes and lysosomes to move longer distances and more processively by recruiting and activating both kinesin-1 and dynein.