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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.

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.

During development, morphogen gradients encode positional information to pattern morphological structures during organogenesis

The transforming growth factor-β (TGF-β)-type morphogens are conserved throughout the animal kingdom. TGF-β-type molecules form spatial concentration gradients whose length scales with the size of growing, developing organs. Scaling of these morphogens can also be mediated by death, adjusting the size of the tissue to the range of the gradient. Death-mediated scaling might provide a molecular toolbox exploited by cancer cells.

Morphogen gradients are fundamental to establish morphological patterns in developing tissues

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.

Capillary morphogenesis gene 2 (CMG2/ANTXR2) is a cell surface receptor for both collagen VI and anthrax toxin. Biallelic loss-of-function mutations in CMG2 lead to a severe condition, hyaline fibromatosis syndrome (HFS). We have here dissected a network of dynamic interactions between CMG2 and various actin interactors and regulators, describing a different behavior from other extracellular matrix receptors. CMG2 binds talin, and thereby the actin cytoskeleton, only in its ligand-free state. Extracellular ligand binding leads to src-dependent talin release and recruitment of the actin cytoskeleton regulator RhoA and its effectors. These sequential interactions of CMG2 are necessary for the control of oriented cell division during fish development. Finally, we demonstrate that effective switching between talin and RhoA binding is required for the intracellular degradation of collagen VI in human fibroblasts, which explains why HFS mutations in the cytoskeleton-binding domain lead to dysregulation of extracellular matrix homeostasis.

Summary Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. Here, we describe a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. We show that trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, we demonstrate that recycling endosomes are essential for Delta activity. Our results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments.

To measure the chemical-mechanic states of lipid membranes, once needs various tools, many of which being incompatible with cell biology protocols. Applying lessons from nature, we developed a mechanosensitive fluorescent probe, the twisted dithienothiophene. This push-pull probe, change planarization state in function of his environment, and we have taken full advantage of this mechano-probe potential and we calibrated based on membrane tension, fluidity and different lipid composition by measuring the push-pull fluorescence lifetime. Likewise, we are able to use this fluorescent probe on life cells, for visualize differences between organelles, as well as to distinguish lipids properties among cells cultured on classic plates or in extracellular matrix.

Recent studies have uncovered a key role of endocytosis during Notch signalling after the asymmetric division of the fly sensory organ precursor cells (SOP): directional signalling is mediated by differential endocytosis of the ligand Delta and the Notch effector Sanpodo in one of the SOP daughters, the pIIb. Here we show a novel mechanism of directional signalling based on the trafficking of Delta/Notch molecules already internalized in the SOP and targeted subsequently to the pIIa daughter. We show that, in the SOP, internalized Delta and Notch traffic to an endosome characterized by the localization of the endosomal protein Sara. During SOP division, Sara endosomes and Notch/Delta therein move first to the central spindle and then to the pIIa cell in a process mediated by the Par-complex. Subsequently, in pIIa (but not pIIb), Notch appears cleaved in Sara endosomes in a process that requires gamma-secretase and Delta internalization implying that the intracellular Notch tail has been released to mediate transcription of Notch target genes. We thus uncovered two phases during biased Notch signalling: (i) in the mother, receptor molecules internalized into Sara endosomes are targeted to the pIIa, thereby increasing the level of Notch signaling in pIIa and decreasing it in pIIb and, (ii) subsequently, biased signaling is also mediated by endocytic trafficking leading to Sanpodo downregulation and Delta recycling/activation in the pIIb daughter cell.