Periodic Reporting for period 1 - ENDODEP (Molecular mechanisms of endosome departure from the spindle in asymmetric cell division)
Reporting period: 2017-11-01 to 2019-10-31
Asymmetric cell division (ACD) allows developing organisms to balance self-renewal and diversification of cells during development. ACD generates sibling cells that differ in terms of cell content and therefore adopt different fates. One means of achieving such biased segregation is by enrolling the endosomal machinery to package and ship elements of cell content to one or the other pole of the dividing cell. The first step in this asymmetric distribution: directed trafficking, has been described in good detail. The present project aimed at gaining understanding of the last step of the process: How are endosomes actually released from microtubules, into the target cell, after being trafficked?
We use the first division of the Drosophila sensory organ precursor (SOP) as a model of ACD. The SOP undergoes ACD and generates an anterior –pIIb- and a posterior –pIIa- cell. PIIb and PIIa have different Notch status and one way the asymmetry of Notch levels is achieved, is through the biased trafficking of Notch already present in the mother cell. A population of Notch-containing Rab5 endosomes marked with the protein Smad Anchor for Receptor Activation (SARA) is trafficked posteriorly, sliding along microtubules organized in two overlapping antiparallel bundles. Sara endosomes are preferentially released into PIIa upon cytokinesis. Collectively, Sara endosomes are first targeted to the overlapping, antiparallel area of the spindle, accumulate at this particular location for 500ms after the onset of anaphase B, when they start vacating this area, showing a preferential departure towards PIIa. Previously, levels of Sara at the endosome surface were found to affect targeting to the spindle and Klp98A has been identified as the motor in charge of Sara endosomes; thus both Sara and Klp98A are involved in the timely release of Sara endosomes in the posterior cell. Here, we aimed at gaining understanding of the mechanisms underlying departure by i) clarifying the interactions between Sara and the motor, and ii) measuring endosomal motility parameters: we hypothesized identifying changes in these parameters could hint at spatial or temporal changes in the microtubule-motor interactions. We found that the motor-endosome association persists after departure. We also found that although the endosome motility is impaired in Sara mutant and in Klp98A mutant backgrounds, their ‘partner’ (resp. Klp98A and Sara) localizes correctly at the endosome surface. We reconstructed 91 endosome tracks and are in the process of extracting and analyzing their motility parameters.
We use the first division of the Drosophila sensory organ precursor (SOP) as a model of ACD. The SOP undergoes ACD and generates an anterior –pIIb- and a posterior –pIIa- cell. PIIb and PIIa have different Notch status and one way the asymmetry of Notch levels is achieved, is through the biased trafficking of Notch already present in the mother cell. A population of Notch-containing Rab5 endosomes marked with the protein Smad Anchor for Receptor Activation (SARA) is trafficked posteriorly, sliding along microtubules organized in two overlapping antiparallel bundles. Sara endosomes are preferentially released into PIIa upon cytokinesis. Collectively, Sara endosomes are first targeted to the overlapping, antiparallel area of the spindle, accumulate at this particular location for 500ms after the onset of anaphase B, when they start vacating this area, showing a preferential departure towards PIIa. Previously, levels of Sara at the endosome surface were found to affect targeting to the spindle and Klp98A has been identified as the motor in charge of Sara endosomes; thus both Sara and Klp98A are involved in the timely release of Sara endosomes in the posterior cell. Here, we aimed at gaining understanding of the mechanisms underlying departure by i) clarifying the interactions between Sara and the motor, and ii) measuring endosomal motility parameters: we hypothesized identifying changes in these parameters could hint at spatial or temporal changes in the microtubule-motor interactions. We found that the motor-endosome association persists after departure. We also found that although the endosome motility is impaired in Sara mutant and in Klp98A mutant backgrounds, their ‘partner’ (resp. Klp98A and Sara) localizes correctly at the endosome surface. We reconstructed 91 endosome tracks and are in the process of extracting and analyzing their motility parameters.
Sara endosomes are canonically defined as SOP endosomes positive for anti-Delta uptakes during the 10-30 minute time window after internalisation, I started the project with producing labelled anti-Delta : I undertook hybridoma cell culture, purification and labelling. In order to understand the timeline of motor-endosome association/ dissociation, I combined these antibodies with an endogenous GFP-tagged Klp98A and imaged SOPs during cell division. I then sought to detect spatio-temporal changes in the dynamics of endosome behavior, which could hint at changes in the motor-microtubule interactions over time, or at different sites in the dividing cell. Using similar anti-Delta uptake conditions as in the previous step, I acquired 4D movies of dividing SOPs. I reconstructed the endosome tracks, adapting and writing Fiji and Matlab computational tools to register tracks in time and space. In order to examine the endogenous distribution of Sara, we generated two CRISPR-cas9 lines: Sara-GFP and Sara-Scarlet. I then used these lines to visualize Sara at endogenous levels in the tissues. I characterized the diverse Sara-positive vesicles using anti-Delta, Dextran and mBSA uptakes and assessed the changes in the distribution of Sara during cell division. Next, I used these tools to address the interactions between Klp98A and SARA by looking at: a) the distribution of Sara-GFP in a Klp98A null background, b) the distribution of Klp98A-GFP vesicles in a Sara mutant background.
We found that the departure of Sara endosomes from the spindle is likely caused by a change in the motor-microtubule interaction rather than in the motor-endosome links. We will test this by comparing motility parameters of 91 space- and time-registered endosome tracks we acquired.
Visualizing for the first time the endogenous distribution of Sara, we found it is present at the surface of vesicles that have a wide range of sizes, of which canonical 'Sara endosomes' only match the smaller group. We also found that this group (Sara+- iDelta+) is the only one remaining in the SOP throughout cytokinesis.
Regarding the interactions between Sara and Klp98A, we found that both Sara and Klp98A maintain their normal localization at the surface of anti-Delta-positive endosomes when Klp98A and Sara, respectively, are lacking. In both cases however, the endosome motility is impaired.
The ongoing work was presented at two international meetings, in the fields of organelle dynamics and Drosophila biology.
We found that the departure of Sara endosomes from the spindle is likely caused by a change in the motor-microtubule interaction rather than in the motor-endosome links. We will test this by comparing motility parameters of 91 space- and time-registered endosome tracks we acquired.
Visualizing for the first time the endogenous distribution of Sara, we found it is present at the surface of vesicles that have a wide range of sizes, of which canonical 'Sara endosomes' only match the smaller group. We also found that this group (Sara+- iDelta+) is the only one remaining in the SOP throughout cytokinesis.
Regarding the interactions between Sara and Klp98A, we found that both Sara and Klp98A maintain their normal localization at the surface of anti-Delta-positive endosomes when Klp98A and Sara, respectively, are lacking. In both cases however, the endosome motility is impaired.
The ongoing work was presented at two international meetings, in the fields of organelle dynamics and Drosophila biology.
The labor-intensive acquisition of a large set of movies capturing the behavior of Sara endosomes during the first division of SOPs, the tracking of 91 Sara endosomes therein and the time and space registration of the tracks lay the ground for further studies aiming at gaining understanding of endosome motility. The dataset can be further exploited to propose a mathematical model of endosome behavior in the departure phase and can be compared with vertebrate (Zebra fish) data when these become available.
By developing two new CRISPR lines, we were able to visualize the distribution of SARA at endogenous levels for the first time. Available in two wavelengths, our newly developed tools can be combined with other fluorescent tools, to further explore the role SARA plays in various pathways, gain understanding of how it circulates among endosomes and cells. It will also allow putting the generalisation of our findings to the test by investigating the role of SARA in other models of cell division in fly tissues. For the first time, this will also enable the simultaneous visualization of the motor and the SARA protein, both expressed at endogenous levels.
The systematic exploration of the Motor-Marker (SARA) relationship is also a novel step in trying to dissect out the role of individual molecular actors in the processes of spindle targeting, asymmetric trafficking and asymmetric release.
By developing two new CRISPR lines, we were able to visualize the distribution of SARA at endogenous levels for the first time. Available in two wavelengths, our newly developed tools can be combined with other fluorescent tools, to further explore the role SARA plays in various pathways, gain understanding of how it circulates among endosomes and cells. It will also allow putting the generalisation of our findings to the test by investigating the role of SARA in other models of cell division in fly tissues. For the first time, this will also enable the simultaneous visualization of the motor and the SARA protein, both expressed at endogenous levels.
The systematic exploration of the Motor-Marker (SARA) relationship is also a novel step in trying to dissect out the role of individual molecular actors in the processes of spindle targeting, asymmetric trafficking and asymmetric release.