Neurons dispose of hyperactive kinesin into glial cells for clearance.

Microtubule-based kinesin motor proteins are crucial for intracellular transport, but their hyperactivation can be detrimental for cellular functions. This study investigated the impact of a constitutively active ciliary kinesin mutant, OSM-3CA, on sensory cilia in C. elegans. Surprisingly, we found that OSM-3CA was absent from cilia but underwent disposal through membrane abscission at the tips of aberrant neurites. Neighboring glial cells engulf and eliminate the released OSM-3CA, a process that depends on the engulfment receptor CED-1. Through genetic suppressor screens, we identified intragenic mutations in the OSM-3CA motor domain and mutations inhibiting the ciliary kinase DYF-5, both of which restored normal cilia in OSM-3CA-expressing animals. We showed that conformational changes in OSM-3CA prevent its entry into cilia, and OSM-3CA disposal requires its hyperactivity. Finally, we provide evidence that neurons also dispose of hyperactive kinesin-1 resulting from a clinic variant associated with amyotrophic lateral sclerosis, suggesting a widespread mechanism for regulating hyperactive kinesins.

The physiological cargo adaptor of kinesin-2 functions as an evolutionary conserved lockpick.

Specific recognition of cellular cargo and efficient transport to its correct intracellular destination is an infrastructural challenge faced by most eukaryotic cells. This remarkable deed is accomplished by processive motor proteins that are subject to robust regulatory mechanisms. The first level of regulation entails the ability of the motor to suppress its own activity. This autoinhibition is eventually relieved by specific cargo binding. To better understand the role of the cargo during motor activation, we dissected the activation mechanism of the ciliary homodimeric kinesin-2 from Caenorhabditis elegans by its physiological cargo. In functional reconstitution assays, we identified two cargo adaptor proteins that together are necessary and sufficient to allosterically activate the autoinhibited motor. Surprisingly, the orthologous adaptor proteins from the unicellular green algae Chlamydomonas reinhardtii also fully activated the kinesin-2 from worm, even though C. reinhardtii itself lacks a homodimeric kinesin-2 motor. The latter suggested that a motor activation mechanism similar to the C. elegans model existed already well before metazoans evolved, and prompted us to scrutinize predicted homodimeric kinesin-2 orthologs in other evolutionarily distant eukaryotes. We show that the ciliate Tetrahymena thermophila not only possesses a homodimeric kinesin-2 but that it also shares the same allosteric activation mechanism that we delineated in the C. elegans model. Our results point to a much more fundamental role of homodimeric kinesin-2 in intraflagellar transport (IFT) than previously thought and warrant further scrutiny of distantly related organisms toward a comprehensive picture of the IFT process and its evolution.

Kinesin-2 from C. reinhardtii Is an Atypically Fast and Auto-inhibited Motor that Is Activated by Heterotrimerization for Intraflagellar Transport.

Construction and function of virtually all cilia require the universally conserved process of intraflagellar transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, on average, 10 kinesin-2 motors "line up" in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each other's way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4] but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit fully activating FLA8/10 for IFT in vivo. Finally, we linked fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and both complexes spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors work mostly alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.

Myosin Va's adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro.

Pigment organelles, or melanosomes, are transported by kinesin, dynein, and myosin motors. As such, melanosome transport is an excellent model system to study the functional relationship between the microtubule- and actin-based transport systems. In mammalian melanocytes, it is well known that the Rab27a/melanophilin/myosin Va complex mediates actin-based transport in vivo. However, pathways that regulate the overall directionality of melanosomes on the actin/microtubule networks have not yet been delineated. Here, we investigated the role of PKA-dependent phosphorylation on the activity of the actin-based Rab27a/melanophilin/myosin Va transport complex in vitro. We found that melanophilin, specifically its C-terminal actin-binding domain (ABD), is a target of PKA. Notably, in vitro phosphorylation of the ABD closely recapitulated the previously described in vivo phosphorylation pattern. Unexpectedly, we found that phosphorylation of the ABD affected neither the interaction of the complex with actin nor its movement along actin tracks. Surprisingly, the phosphorylation state of melanophilin was instead important for reversible association with microtubules in vitro. Dephosphorylated melanophilin preferred binding to microtubules even in the presence of actin, whereas phosphorylated melanophilin associated with actin. Indeed, when actin and microtubules were present simultaneously, melanophilin's phosphorylation state enforced track selection of the Rab27a/melanophilin/myosin Va transport complex. Collectively, our results unmasked the regulatory dominance of the melanophilin adaptor protein over its associated motor and offer an unexpected mechanism by which filaments of the cytoskeletal network compete for the moving organelles to accomplish directional transport on the cytoskeleton in vivo.