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.
