Regulation of Abscission Timing in Animal Cells – AbsCyStem

Abscission is the last step of cytokinesis that physically separates the cytoplasm of sister cells. This aspect of cell division is the least understood, and has just begun to be characterized molecularly. It is challenging to explore, as it takes place at the very end of cell division and manipulations of molecular components involved earlier in the cell cycle can mask their late involvement during abscission. Abscission also involves structures that are at the limit of optical resolution of most microscopes. Here, we propose to investigate the timing of abscission with unique temporal and spatial precision using live imaging and super-resolution microscopy, and to uncover novel pathways regulating abscission by performing complementary biochemical and genetic screens. We will take advantage of two powerful model systems, the Drosophila germline stem cell lineage and cultured human cells to combine exceptional genetic tools existing in flies and great biochemistry available with cultured cells. Furthermore, the development of germ cells is an interesting case study, as germ cell switch from complete to incomplete cytokinesis to form germline cysts, a feature conserved from Drosophila to humans. Our first objective aims at developing new tools for describing abscission with unprecedented precision in both intact germline cells and human cells using microfluidic devises and super-resolution microscopy. We will design original FRET biosensors to monitor in real time the activities of both Aurora B and Cdk1 kinases during abscission. This unprecedented and quantitative description of abscission will be extremely useful for the molecular and mechanistic objective 2. In the second objective, our goal is indeed to define at the molecular level the regulatory landscape upstream and downstream of Aurora B and Cdk1, which we have recently identified as two key regulators of abscission timing in vivo. Despite being a master regulator of the cell cycle, Cdk1 function during abscission is completely novel and unexpected for the field. We will in particular use an analog-sensitive Cdk1 (“Shokat Cdk1”) which can specifically tag its substrates with a thiophosphate group and we will combine it with an original method for purifying midbodies to identify the marked targets of Cdk1 relevant for abscission. Objective 3 is exploratory as we want to uncover completely novel pathways regulating the duration of abscission. We will carry out a genetic screen for mutants affecting the number of germ cells per egg chamber as a simple read-out of abscission duration. Previous screens scoring binucleated cells were biased for cytokinesis failure occurring before abscission. In contrast, our original screen is specifically designed to uncover novel regulators of abscission.

All three objectives are based on solid preliminary data. Using PDMS microchip, we were able to film germarium for 17hr instead of 2hr previously. Also in support of Objective 1, we detected strong FRET signal at the midbody with a Cdk1 activity sensor. In support of Objective 2, we know that upstream regulators of Cdk1 affect the number of germ cells and thus may also regulate abscission timing. We are also currently testing Lgd as a novel target of Cdk1. We have also started a pilot screen for novel regulators of abscission as in Objective 3, and successfully identified the AMPK and ubiquitin pathways as novel and promising hits. Our project benefits from the very complementary skills of all three partners, which have already collaborated successfully: Jean-René Huynh (germ cell, Drosophila); Arnaud Echard (abscission, human cells); Olivier Gavet (FRET-based biosensors, human cells). This complementary between the three labs is both technical and also conceptual, as we aimed at uncovering the similarities and differences between Drosophila and human cells, and between complete and incomplete cytokinesis. Our proposal is relevant to the fields of reproduction, stem cell and cancer research.