Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde trafficking

Abstract

Mutations in the microtubule (MT)-binding protein doublecortin (DCX) or in the MT-based molecular motor dynein result in lissencephaly. However, a functional link between DCX and dynein has not been defined. Here, we demonstrate that DCX negatively regulates dynein-mediated retrograde transport by reducing dynein's association with MTs and by disrupting the composition of the dynein motor complex. Previous work showed an increased binding of the adaptor protein C-Jun-amino-terminal kinase-interacting protein 3 (JIP3) to dynein in the absence of DCX. Using purified components, we demonstrate that JIP3 forms an active motor complex with dynein and its cofactor dynactin with two dyneins per complex. DCX competes with the binding of the second dynein, resulting in a velocity reduction of the complex. We conclude that DCX negatively regulates dynein-mediated retrograde transport through two critical interactions by regulating dynein binding to MTs and by regulating the association of JIP3 to the dynein motor complex.

Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases

“…One possible explanation is that previous work did not use polarity-marked microtubules (Sun et al, 2011; Watt et al, 2015); thus, it is possible that the motility they detected was in fact minus-end-directed and dynein-driven. Based upon our data, and consistent with the recent reports on the activation of dynein by JIP3 (Rao et al, 2022; Singh et al, 2022) we conclude that JIP3 and JIP4 robustly activate dynein motility, with only marginal activation of kinesin under the conditions tested. Consistent with these observations, labeled JIP3 and JIP4 both move almost exclusively retrograde in neuronal axons (Fig.…”

“…Concurrent with our study, two other groups have shown that purified recombinant truncated JIP3 is sufficient to activate dynein-mediated motility in vitro (Rao et al, 2022; Singh et al, 2022). Our study adds to this growing body of work, as the approaches used here (1) include endogenous binding partners, which negates the need to use truncated constructs; (2) is performed at physiological temperature on dynamically growing microtubules; and (3) includes competing kinesin complexes.…”

“…Our study adds to this growing body of work, as the approaches used here (1) include endogenous binding partners, which negates the need to use truncated constructs; (2) is performed at physiological temperature on dynamically growing microtubules; and (3) includes competing kinesin complexes. Our average JIP3 velocity (∼1.8 µms -1 ) is higher than that observed by the other groups [0.7 µms -1 (Rao et al, 2022); 1 µms -1 (Singh et al, 2022)], most likely due to the more physiological assay temperature (37°C vs. room temperature). It is, however, consistent with previous observations of dynein activation using lysate assays performed at 37° (Fenton et al, 2021).…”

“…Previous work has reported that the Drosophila JIP3 ortholog Sunday driver (syd) activates kinesin-1 (Sun et al, 2011). However, these assays were performed using mammalian cell lysate at room temperature on stabilized microtubules without polarity labelling, and the average velocities (0.6-1.0 µms -1 ) and run lengths (3-5.5 µm) observed suggest that minus end-directed motility may have dominated in their assays, as these values are more consistent with dynein-mediated transport (Olenick et al, 2016; Urnavicius et al, 2018; Fenton et al, 2021; Canty et al, 2021; Fu et al, 2014; Fu and Holzbaur, 2013; Rao et al, 2022; Singh et al, 2022; Sun et al, 2011). Similar assays performed with mammalian JIP3 (Watt et al, 2015) resulted in velocities (∼0.25 µms -1 ) more consistent with kinesin-1 activation; however, the relatively short run lengths (∼0.75 µm) suggest that JIP3 may require additional effectors to fully activate kinesin-1 motility.…”

“…Further, JIP3/4 can bind to the dynactin subunit p150 Glued (Fig. 1 A) and truncated JIP3 can activate dynein motility in a purified system (Montagnac et al, 2009;Rao et al, 2022). However, JIP3 and JIP4 can also interact with the kinesin-1 complex via interactions (Fig.…”