A spindle-independent cleavage furrow positioning pathway

The mitotic spindle determines the cleavage furrow site during metazoan cell division, but whether other mechanisms exist remains unknown. Here we identify a spindle-independent mechanism for cleavage furrow positioning in Drosophila neuroblasts. We show that early and late furrow proteins (Pavarotti, Anillin, and Myosin) are localized to the neuroblast basal cortex at anaphase onset by a Pins cortical polarity pathway, and can induce a basally displaced furrow even in the complete absence of a mitotic spindle. Rotation or displacement of the spindle results in two furrows: an early polarity-induced basal furrow and a later spindle-induced furrow. This spindle-independent cleavage furrow mechanism may be relevant to other highly polarized mitotic cells, such as mammalian neural progenitors. Elegant physical or genetic manipulations of the mitotic spindle have shown that the spindle determines the position of the cleavage furrow in a wide range of cells. Although this is a common mechanism for furrow formation, it may not be the only one, as cleavagefurrow position during the highly asymmetric mammalian meiotic divisions can be specified by a spindle-independent chromosomal cue. The spindle pathway for furrow positioning is initiated at the overlapping microtubules of the central spindle, where the ‘centralspindlin’ protein complex is assembled. Centralspindlin components include the kinesin Pavarotti (Zen-4 in Caenorhabditis elegans), the RACGAP50 Tumbleweed (Cyk-4 in C. elegans) and the RhoGEF Pebble (Ect-2 in C. elegans). After assembly, the centralspindlin complex moves to the cell cortex, possibly through a special population of stable microtubules, to form a cortical ring at the site of the central spindle. The centralspindlin ring subsequently recruits actomyosin and initiates cleavage furrow constriction. In contrast, astral microtubules typically inhibit furrow formation (Fig. 1a, left). Here we test whether the spindle-induced furrow model is sufficient to account for cleavage furrow positioning during asymmetric cell division of Drosophila neuroblasts. Neuroblasts establish molecular asymmetry during early prophase with the apical cortical localization of the Par complex (Bazooka; Par-6; atypical protein kinase C, aPKC) and the Pins complex (Partner of Inscuteable (Pins); Gai; Discs large (Dlg)). Subsequently, the scaffolding protein Miranda (Mira) and its cargo proteins Prospero (Pros), Brain tumour (Brat) and Staufen are localized to the basal cortex. The mitotic spindle aligns along the apical/basal axis at metaphase and becomes asymmetric during anaphase, with the apical half forming longer astral and central spindle microtubules. The cleavage furrow is displaced basally, generating a larger apical daughter cell and a smaller basal daughter cell. It has been assumed that the centralspindlin complex is the only mechanism for furrow positioning, because the furrow is always positioned adjacent to the central spindle, even in mutants that disrupt spindle asymmetry. One model is that the basal spindle pole is anchored at the basal cortex, resulting in a basal displacement of the central spindle and subsequent cleavage furrow (Fig. 1a, right). However, in neuroblasts, experiments such as spindle rotation, spindle displacement or spindle ablation have never been performed to test directly