The Drosophila mitotic spindle orientation machinery requires activation, not just localization

Abstract

The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins – called Pins, Gαi, Mud, and Dynein in flies – that exerts a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. In this study we revisit the canonical model and find that it is incomplete. Spindle orientation in the Drosophila follicular epithelium and embryonic ectoderm requires not only Pins localization but also direct interaction between Pins and the multifunctional protein Discs large. This requirement can be over‐ridden by interaction with another Pins interacting protein, Inscuteable.

AGS3 antagonizes LGN to balance oriented cell divisions and cell fate choices in mammalian epidermis

“…Studies of HeLa and keratinocyte cells in culture have shown that LGN is required for NuMA’s cortical recruitment through metaphase, but dispensable at anaphase, where instead, 4.1 G/R serves to anchor NuMA to the cortex ( Kiyomitsu and Cheeseman, 2013 ; Seldin et al, 2013 ). Similarly, a recent study in Drosophila follicular epithelium showed that Pins is recruited apically and laterally during mitosis, while Mud is lateral ( Neville et al, 2023 ).…”

“…This same study also showed that it is not the localization of Pins, but its upstream binding partner Insc, which is the best predictor of spindle orientation in Drosophila follicular epithelium, and raised the intriguing idea that there is a threshold level of Insc expression, above which it localizes apically—causing spindle misorientation—and below which it localizes laterally ( Neville et al, 2023 ). Since AGS3 can also bind Insc ( Izaki et al, 2006 ; Yuzawa et al, 2011 ), we also consider the possibility that cytoplasmic AGS3 may sequester Insc from the apical cortex in epidermal basal cells.…”

“…While it is not known whether aPKC phosphorylation of LGN impacts its spindle orienting capacity, loss of Prkci in the epidermis does result in a spindle orientation phenotype ( Niessen et al, 2013 ). On the other hand, the Dlg–LGN interaction is highly conserved throughout evolution ( Schiller and Bergstralh, 2021 ), and a non-phosphorylatable Drosophila pins S436A mutant is non-functional in an in vitro S2-induced polarity assay and in vivo in follicular epithelial cells ( Hao et al, 2010 ; Neville et al, 2023 ). Moreover, the equivalent mutation in vertebrate LGN (S401A) induces spindle misorientation in MDCK cells and the chick neural tube ( Saadaoui et al, 2014 ).…”

Fas2EB112: A Tale of Two Chromosomes

“…The popping out phenotype is enhanced by ectopic expression of Inscuteable, a protein that is normally expressed in neural progenitor cells (neuroblasts) in the developing nervous system (Bergstralh, Lovegrove, and St Johnston 2015; Kraut et al 1996). When it is expressed in the follicular epithelium, Inscuteable can reorient spindles so that they are parallel to the apical-basal axis, causing some daughter cells to be born outside the tissue plane (Bergstralh, Lovegrove, and St Johnston 2015; Neville et al 2023). This manipulation does not cause tissue disorganization by itself because the misplaced cells reintegrate.…”

Fas2EB112: a tale of two chromosomes

“…The popping out phenotype is enhanced by ectopic expression of Inscuteable, a protein that is normally expressed in neural progenitor cells (neuroblasts) in the developing nervous system ( Kraut et al 1996 ; Bergstralh et al 2015 ). When it is expressed in the follicular epithelium, Inscuteable can reorient spindles so that they are parallel to the apical–basal axis, causing some daughter cells to be born outside the tissue plane ( Bergstralh et al 2015 ; Neville et al 2023 ). This manipulation does not cause tissue disorganization by itself because the misplaced cells reintegrate.…”