We established experimental and analytical methodologies to follow individual molecule behavior in Drosophila embryos and then proceeded to collect data for thousand of molecules. We built a catalogue of single molecule behaviors for Spd-2, Cnn and dTACC. For each molecule the following were measured: time of incorporation and exit from the PCM, total binding duration, timing in regards to the phases of the cell cycle, distance from the centriole when bound and when exiting, movement throughout the PCM. The main conclusion coming from these studies was that both Spd-2 and Cnn bind to the PCM at the centre, close to Asl and then move outwards to the edge of the PCM, after which they exit. We call this movement FLUXING.
The major conclusions from this study are 1) molecules of Spd-2 are incorporated to the PCM mostly during early s-phase, with few incorporating during mitosis. Molecules of Cnn, in reverse, are mostly incorporated during mitosis and late S-phase, with few incorporated during early S-phase; 2) Spd-2 molecules spend on average l.7 minutes in the PCM whereas Cnn molecules spend 2 minutes in the PCM; 3) Fluxing Spd-2 and Cnn molecules move at speeds between 3 and 30 nm/s through the PCM, with Spd-2 molecules on average moving faster than Cnn; 4) Flux speed is determined by several factors: molecules binding initially closer to the centriole tend to move faster through the PCM than those that bind further from it and flux speed is faster (for Spd-2) during earlier S-phase than during later S-phase. We found that dTACC bound and unbound from the centrosome at a fast rate and seemingly at random distance from the centre, in clear contrast to the directional outwards movement of Spd2 and Cnn.
In order to find the determinants behind the observed fluxing behavior of Spd2 and Cnn, we repeated the single molecule analysis with either a) colchicine to depolymerise microtubules, where we found that Cnn stopped fluxing and would accumulate at the centrosome, whereas Spd2 was seemingly unaffected; constructed and used mutants of Spd-2 that lack the canonical phosphorylation sites – Spd2ALL and Spd2CONS, or mutants of Polo kinase and found that these were still able to flux, particularly at a faster rate indicating that phosphorylation of Spd2/Cnn contributes to keeping these proteins at the centrosome during expansion. We’re currently developing models to describe how fluxing of these proteins contributes to centrosome growth.
In conclusion, the implementation of this action provided a detailed model of how centrosomal key components flux through the centrosome during the cell cycle, the determinants of said flux and how this flux contributes to centrosome size growth and maintenance of size, which are crucial aspects that ensure mitotic fidelity. These findings are being used to prepare a manuscript suitable for publication.
Over the course of this action these findings were regularly disseminated to the community at conferences and symposiums, both internally in the department and at international conferences.
