Final Report Summary - ENDASYM (Asymmetric endosomes in asymmetric cell division and tumorigenesis)
Asymmetric endosomal trafficking regulates directional signalling during asymmetric cell division. To study this phenomenon, we used as a model system the Sensory Organ Precursor cells (SOPs) of the Drosophila notum. Downstream SOP division, biased Notch signalling mediates cell fate decisions of the sibling pIIa and pIIb daughter cells. It has been shown that the Notch receptor and its ligand Delta internalised in the mother cell traffic through specialised early endosomes characterised by the presence of the protein Sara. In the course of asymmetric cell division, Sara endosomes target Notch and Delta to the pIIa daughter cell, contributing this way to its definition as the signal-receiving cell.
As very little is known about the mechanisms governing the asymmetric dynamics of signalling endosomes, this project addressed the question: how is the motility of Sara endosomes regulated during asymmetric cell division?
The main findings of this project can be subdivided into two parts: 1/ the role of Klp98a in the asymmetric motility of Sara endosomes; 2/ Uif, a direct link between Sara endosomes asymmetry and the control of Notch signalling.
1/ We performed a small scale RNAi screen and identified a molecular motor specifically present on Sara endosomes: the kinesin-3 Klp98a. We showed that Klp98a is present on Sara endosomes and is asymmetrically segregated to the pIIa cell during SOP division. In collaboration with the Schmidt lab in Leiden (The Netherlands), we have designed a Matlab software that tracks endosomes during SOP mitosis (fig. 1). This tool permitted to show that Klp98a controls the asymmetric motility of Sara endosomes (fig. 2).
We also addressed when and where specific forces are necessary for the asymmetric motility of Sara endosomes. In collaboration with the Bacri lab in Paris (France), we have developed magnetic micro-manipulation as a novel tool to probe Sara endosomes dynamics: upon application of a magnetic force, we were able to specifically manipulate the dynamics of Sara endosomes (fig. 3). We have also developed a correlative live imaging and lineage analysis assay that revealed that mis-segregation of Sara endosomes in the pIIb cell was not sufficient to disturb cell fate decisions downstream SOP division (fig. 4).
Using magnetic micro-manipulation experiments we also showed that the forces crucial for the asymmetry of Sara endosomes are exerted at late anaphase/cytokinesis on the central spindle. We addressed the existence of a putative asymmetric organisation of the mitotic spindle, and discovered that there are unequal quantities of acetylated tubulin on the two halves of the central spindle (fig. 5). This asymmetric cytoskeletal organisation could provide a biochemical basis for the asymmetric motility of Sara endosomes.
2/ We also discovered a new molecular partner of the Sara protein: the protein Uninflatable (Uif). We have shown that Uif is present on Sara endosomes and that it controls the asymmetric movements of Sara endosomes (fig. 6).
We found that Uninflatable plays a role in the control of Notch signalling also in tissues in which asymmetric cell division is not involved (notum and wing) (fig. 7). Uif is present at the apical membrane and we found that this apical pool is asymmetrically segregated to the pIIa cell during SOP division. Our working model is that the endosomal pool of Uif plays a role in the control of Sara endosomes motility, while the apical pool plays a different function in core Notch signalling.
This project has thus enabled to elucidate novel mechanisms involved in the endosomal control of signalling during asymmetric cell division. Unravelling how a defined cytoskeleton machinery enables directed motility of organelles in a short time window is a breakthrough for the cell biology community. Besides, Uninflatable appears as a new Notch machinery component, and elucidating its mode of action will reveal a new Notch regulation mechanism.
As very little is known about the mechanisms governing the asymmetric dynamics of signalling endosomes, this project addressed the question: how is the motility of Sara endosomes regulated during asymmetric cell division?
The main findings of this project can be subdivided into two parts: 1/ the role of Klp98a in the asymmetric motility of Sara endosomes; 2/ Uif, a direct link between Sara endosomes asymmetry and the control of Notch signalling.
1/ We performed a small scale RNAi screen and identified a molecular motor specifically present on Sara endosomes: the kinesin-3 Klp98a. We showed that Klp98a is present on Sara endosomes and is asymmetrically segregated to the pIIa cell during SOP division. In collaboration with the Schmidt lab in Leiden (The Netherlands), we have designed a Matlab software that tracks endosomes during SOP mitosis (fig. 1). This tool permitted to show that Klp98a controls the asymmetric motility of Sara endosomes (fig. 2).
We also addressed when and where specific forces are necessary for the asymmetric motility of Sara endosomes. In collaboration with the Bacri lab in Paris (France), we have developed magnetic micro-manipulation as a novel tool to probe Sara endosomes dynamics: upon application of a magnetic force, we were able to specifically manipulate the dynamics of Sara endosomes (fig. 3). We have also developed a correlative live imaging and lineage analysis assay that revealed that mis-segregation of Sara endosomes in the pIIb cell was not sufficient to disturb cell fate decisions downstream SOP division (fig. 4).
Using magnetic micro-manipulation experiments we also showed that the forces crucial for the asymmetry of Sara endosomes are exerted at late anaphase/cytokinesis on the central spindle. We addressed the existence of a putative asymmetric organisation of the mitotic spindle, and discovered that there are unequal quantities of acetylated tubulin on the two halves of the central spindle (fig. 5). This asymmetric cytoskeletal organisation could provide a biochemical basis for the asymmetric motility of Sara endosomes.
2/ We also discovered a new molecular partner of the Sara protein: the protein Uninflatable (Uif). We have shown that Uif is present on Sara endosomes and that it controls the asymmetric movements of Sara endosomes (fig. 6).
We found that Uninflatable plays a role in the control of Notch signalling also in tissues in which asymmetric cell division is not involved (notum and wing) (fig. 7). Uif is present at the apical membrane and we found that this apical pool is asymmetrically segregated to the pIIa cell during SOP division. Our working model is that the endosomal pool of Uif plays a role in the control of Sara endosomes motility, while the apical pool plays a different function in core Notch signalling.
This project has thus enabled to elucidate novel mechanisms involved in the endosomal control of signalling during asymmetric cell division. Unravelling how a defined cytoskeleton machinery enables directed motility of organelles in a short time window is a breakthrough for the cell biology community. Besides, Uninflatable appears as a new Notch machinery component, and elucidating its mode of action will reveal a new Notch regulation mechanism.
