Polarized branched Actin modulates cortical mechanics to produce unequal-size daughters during asymmetric division.

The control of cell shape during cytokinesis requires a precise regulation of mechanical properties of the cell cortex. Only few studies have addressed the mechanisms underlying the robust production of unequal-sized daughters during asymmetric cell division. Here we report that unequal daughter-cell sizes resulting from asymmetric sensory organ precursor divisions in Drosophila are controlled by the relative amount of cortical branched Actin between the two cell poles. We demonstrate this by mistargeting the machinery for branched Actin dynamics using nanobodies and optogenetics. We can thereby engineer the cell shape with temporal precision and thus the daughter-cell size at different stages of cytokinesis. Most strikingly, inverting cortical Actin asymmetry causes an inversion of daughter-cell sizes. Our findings uncover the physical mechanism by which the sensory organ precursor mother cell controls relative daughter-cell size: polarized cortical Actin modulates the cortical bending rigidity to set the cell surface curvature, stabilize the division and ultimately lead to unequal daughter-cell size.

Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants.

Asymmetric cell division gives rise to two daughter cells that inherit different determinants, thereby acquiring different fates. Polarized trafficking of endosomes containing fate determinants recently emerged as an evolutionarily conserved feature of asymmetric cell division to enhance the robustness of asymmetric cell fate determination in flies, fish and mammals. In particular, polarized sorting of signalling endosomes by an asymmetric central spindle contributes to asymmetric cell division in Drosophila melanogaster. However, how central spindle asymmetry arises remains elusive. Here we identify a moonlighting function of the Elongator complex-an established protein acetylase and tRNA methylase involved in the fidelity of protein translation-as a key factor for central spindle asymmetry. Elongator controls spindle asymmetry by stabilizing microtubules differentially on the anterior side of the central spindle. Accordingly, lowering the activity of Elongator on the anterior side using nanobodies mistargets endosomes to the wrong cell. Molecularly, Elongator regulates microtubule dynamics independently of its acetylation and methylation enzymatic activities. Instead, Elongator directly binds to microtubules and increases their polymerization speed while decreasing their catastrophe frequency. Our data establish a non-canonical role of Elongator at the core of cytoskeleton polarity and asymmetric signalling.

Endosomal Trafficking During Mitosis and Notch-Dependent Asymmetric Division.

Endocytosis is key in a number of cell events. In particular, its role during cell division has been a challenging question: while early studies examined whether endocytosis occurs during cell division, recent works show that, during division, cells do perform endocytosis actively. More importantly, during asymmetric cell division, endocytic pathways also control Notch signaling: endocytic vesicles regulate the presence, at the plasma membrane, of receptors and ligands at different levels between the two-daughter cells. Both early and late endocytic compartments have been shown to exert key regulatory controls by up-regulating or down-regulating Notch signaling in those cells. This biased Notch signaling enable finally cell fate assignation and specification which play a central role in development and physiology. In this chapter, we cover a number of significant works on endosomal trafficking evincing the importance of endocytosis in Notch-mediated cell fate specification during development.

Sara phosphorylation state controls the dispatch of endosomes from the central spindle during asymmetric division.

During asymmetric division, fate assignation in daughter cells is mediated by the partition of determinants from the mother. In the fly sensory organ precursor cell, Notch signalling partitions into the pIIa daughter. Notch and its ligand Delta are endocytosed into Sara endosomes in the mother cell and they are first targeted to the central spindle, where they get distributed asymmetrically to finally be dispatched to pIIa. While the processes of endosomal targeting and asymmetry are starting to be understood, the machineries implicated in the final dispatch to pIIa are unknown. We show that Sara binds the PP1c phosphatase and its regulator Sds22. Sara phosphorylation on three specific sites functions as a switch for the dispatch: if not phosphorylated, endosomes are targeted to the spindle and upon phosphorylation of Sara, endosomes detach from the spindle during pIIa targeting.

Polarized endosome dynamics by spindle asymmetry during asymmetric cell division.

During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division. Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila. In flies, this has been generalized to stem cells in the gut and the central nervous system, and, in zebrafish, to neural precursors of the spinal cord. However, the mechanism of asymmetric endosome segregation is not understood. Here we show that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. We demonstrate this mechanism by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. Our data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division.

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division.

Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis [1-6]. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate [1-6]. We have previously shown that Notch/Delta molecules internalized in the mother cell traffic through Sara endosomes and are directed to the pIIa daughter [6]. Here we show that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. Our data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis.

Formation of adherens junctions leads to the emergence of a tissue-level tension in epithelial monolayers.

Adherens junctions and desmosomes integrate the cytoskeletons of adjacent cells into a mechanical syncitium. In doing so, intercellular junctions endow tissues with the strength needed to withstand the mechanical stresses encountered in normal physiology and to coordinate tension during morphogenesis. Though much is known about the biological mechanisms underlying junction formation, little is known about how tissue-scale mechanical properties are established. Here, we use deep atomic force microscopy (AFM) indentation to measure the apparent stiffness of epithelial monolayers reforming from dissociated cells and examine which cellular processes give rise to tissue-scale mechanics. We show that the formation of intercellular junctions coincided with an increase in the apparent stiffness of reforming monolayers that reflected the generation of a tissue-level tension. Tension rapidly increased, reaching a maximum after 150 min, before settling to a lower level over the next 3 h as monolayers established homeostasis. The emergence of tissue tension correlated with the formation of adherens junctions but not desmosomes. As a consequence, inhibition of any of the molecular mechanisms participating in adherens junction initiation, remodelling and maturation significantly impeded the emergence of tissue-level tension in monolayers.