Augmin complex activity finetunes dendrite morphology through non-centrosomal microtubule nucleation in vivo.

During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo.

Proximity mapping of human separase by the BioID approach.

Separase is a caspase-like cysteine protease that is best known for its essential role during the metaphase-to-anaphase transition when it cleaves the cohesin ring complex that keeps the sister chromatids together. Another important function of separase is to regulate the process of centriole separation, known as centriole disengagement, at the end of mitosis. We used proximity-dependent biotin identification (BioID) to expand our knowledge on the identity of separase's proximity interactors. We show that separase BioID labeled two domains at the mother centriole: an area underneath the centriolar appendages and another at the proximal end of the mother centriole. BioID analysis identified more than 200 proximity interactors of separase, one being the Alström Syndrome Protein 1 (ALMS1) at the base of centrioles. Other proximity interactors are the histone chaperons NAP1L1 and NAP1L4, which localize to the spindle poles during mitosis and the spindle assembly checkpoint proteins BUBR1, SKA1 and SKA3 that reside at kinetochores in early mitosis. Finally, we show that depletion of BUBR1 homolog from Caenorhabditis elegans delayed the recruitment of separase to mitotic chromosomes, and eventually anaphase onset.

Sensors at centrosomes reveal determinants of local separase activity.

Separase is best known for its function in sister chromatid separation at the metaphase-anaphase transition. It also has a role in centriole disengagement in late mitosis/G1. To gain insight into the activity of separase at centrosomes, we developed two separase activity sensors: mCherry-Scc1(142-467)-ΔNLS-eGFP-PACT and mCherry-kendrin(2059-2398)-eGFP-PACT. Both localize to the centrosomes and enabled us to monitor local separase activity at the centrosome in real time. Both centrosomal sensors were cleaved by separase before anaphase onset, earlier than the corresponding H2B-mCherry-Scc1(142-467)-eGFP sensor at chromosomes. This indicates that substrate cleavage by separase is not synchronous in the cells. Depletion of the proteins astrin or Aki1, which have been described as inhibitors of centrosomal separase, did not led to a significant activation of separase at centrosomes, emphasizing the importance of direct separase activity measurements at the centrosomes. Inhibition of polo-like kinase Plk1, on the other hand, decreased the separase activity towards the Scc1 but not the kendrin reporter. Together these findings indicate that Plk1 regulates separase activity at the level of substrate affinity at centrosomes and may explain in part the role of Plk1 in centriole disengagement.