Directional trafficking of cargo, a hallmark of living cells, relies on a polarised landscape of microtubule tracks. Here, I studied how the regulation of an endosomal motor affects polarised trafficking, using the asymmetric division of Drosophila Sensory Organ Precursor (SOP) cells as a model system. In this pathway, the PI(3)P-binding kinesin-3, Klp98A biases the motility of signalling endosomes towards the posterior daughter cell. Since these endosomes contain the cell fate determinants Notch and Delta, this contributes to an asymmetric cell fate. Mechanistically, Klp98A-mediated transport is achieved along the microtubule-based anaphase-specific spindle midzone, termed the central spindle. During SOP division, the central spindle becomes asymmetric, with a higher density of microtubules on the anterior side. Since Klp98A is a plus-end directed motor, this biases the motility of early endosomes into the posterior daughter cell, towards which more microtubule plus-ends point.

In this work, I focused on how the modulation of Klp98A activity can control polarised trafficking on three levels: i) via post-translational modifications, ii) through changes in microtubule binding, and iii) by polarising the microtubules Klp98A walks on.

By first using an in vitro reconstitution with purified motors on micropatterned supported lipid bilayers containing PI(3)P, I investigated how Klp98A is activated. Lipid-binding was sufficient to induce activation of Klp98A, so I proceeded to use both enzymatic-induced proximity labelling and a candidate-based approach to identify possible regulators of Klp98A activity. In vivo, modulation of two enzymes, p-21 activated kinase 1 (Pak1) and Sirtuin 2 (Sirt2), resulted in symmetric endosome segregation despite an asymmetric landscape of microtubule tracks. In the case of Pak1, this endosomal phenotype appeared to arise due to a delay in the targeting of endosomes to the central spindle, either directly due to modulation of post-translational modifications on the motor itself or through an intermediary.

Next, I focused on characterising how the loop 8 (L8) region of the Klp98A motor domain modulates its activity. This well-conserved loop within the kinesin-3 family is thought to be involved in microtubule binding and could thus be a central way by which the activity of this motor is regulated in vivo. To understand how L8 controls motor activity, I studied a previously isolated Klp98A mutant, Klp98A Δ6, which consists of a two amino-acid deletion and one amino-acid substitution within the L8 site. Strikingly, in vivo, Klp98A Δ6 localises to endosomes like the wild-type but leads to symmetric segregation of cell fate determinants in dividing SOP cells, phenocopying a complete loss of function of Klp98A. A combination of in vitro reconstitution, single-molecule assays and crystal-structure determination showed that, unexpectedly, Klp98A Δ6 retains wild-type microtubule binding affinity but is unable to move processively. The cause of this transport defect by Klp98A Δ6 is likely a consequence of a long-range structural rearrangement from the L8 site to the catalytic domain, preventing ATP hydrolysis independently of microtubule binding. Thus, it seems in wild-type Klp98A, L8 functions as a sensor, coupling microtubule binding to ATP hydrolysis.

Lastly, I investigated how the asymmetry of the central spindle, which polarises Klp98A motility, is established and maintained during SOP cell division. I demonstrated that at the cell cortex, the Par complex promotes central spindle symmetry breaking. Downstream of that, I found that Elongator, originally identified as a regulator of translation, promotes the formation of an asymmetric central spindle. Here, Elongator is enriched on the anterior side of the spindle, where it stabilises microtubules.

Altogether, this data sheds light on how polarised trafficking emerges from the synergistic effects of both the polarity within the network of microtubules and the local control of the activity of motors at the surface of endosomes.