Periodic Reporting for period 1 - PIADINE (Deciphering the role of PI(4,5)P2 and its modulators PPK-1 and CSNK-1 in nematode asymmetric division)
Período documentado: 2020-10-01 hasta 2022-09-30
Originally, the project was focused on spindle positioning during the first mitosis of one-cell stage C. elegans embryos. In agreement with all parts concerned, the project quickly steered towards a focus in cell polarity establishment, which happens a bit earlier in the cell cycle. We took this decision because we identified a role for CSNK-1 (one of the proteins at the center of the fellowship application) in this process. Since cell polarity is defining the following steps of the cell cycle, including mitotic spindle positioning, we decided to address this biological problem to start with.
C. elegans zygote is a great model system to investigate cell polarity, which results in the establishment of the anterior-posterior body axis. This system is attractive because of its large dimensions (50x30µm), optical accessibility, and the evolutionary conservation of most of the molecular players, which can be targeted using genetics and functional genomics.
Following fertilization of the oocyte by the sperm, the newly formed zygote exhibits homogenous actomyosin dependent cortical contractions, and the presence of anterior-PAR proteins throughout the cell cortex. The sperm derived centrosomes mature in the zygote, through the recruitment of dedicated proteins, including SPD-2 and AIR-1. Once mature, the centrosomes contact the embryonic cortex, thus initiating polarity establishment. The first visible event of polarity establishment is a cortical actomyosin flow that moves away from the point of contact made by the centrosomes. This flow displaces also the anterior-PAR proteins, leaving the cortex accessible to posterior-PAR proteins. Polarity establishment ends when the cortical flow has moved the anterior-PARs to one half of the embryo cortex (the anterior half) and the posterior-PARs are localized on the other half (the posterior one), defining in this manner the anterior-posterior axis of the animal.
SPD-2 and AIR-1 depletion or inactivation were previously shown to impair centrosome maturation. In this condition, the embryo still undergoes polarity establishment, but in an aberrant manner since it usually harbors two posterior poles rather than one, each marked with PAR-2. CSNK-1 depletion was reported previously not to affect polarity establishment.
C. elegans zygote is a great model system to investigate cell polarity, which results in the establishment of the anterior-posterior body axis. This system is attractive because of its large dimensions (50x30µm), optical accessibility, and the evolutionary conservation of most of the molecular players, which can be targeted using genetics and functional genomics.
Following fertilization of the oocyte by the sperm, the newly formed zygote exhibits homogenous actomyosin dependent cortical contractions, and the presence of anterior-PAR proteins throughout the cell cortex. The sperm derived centrosomes mature in the zygote, through the recruitment of dedicated proteins, including SPD-2 and AIR-1. Once mature, the centrosomes contact the embryonic cortex, thus initiating polarity establishment. The first visible event of polarity establishment is a cortical actomyosin flow that moves away from the point of contact made by the centrosomes. This flow displaces also the anterior-PAR proteins, leaving the cortex accessible to posterior-PAR proteins. Polarity establishment ends when the cortical flow has moved the anterior-PARs to one half of the embryo cortex (the anterior half) and the posterior-PARs are localized on the other half (the posterior one), defining in this manner the anterior-posterior axis of the animal.
SPD-2 and AIR-1 depletion or inactivation were previously shown to impair centrosome maturation. In this condition, the embryo still undergoes polarity establishment, but in an aberrant manner since it usually harbors two posterior poles rather than one, each marked with PAR-2. CSNK-1 depletion was reported previously not to affect polarity establishment.
When I used stringent SPD-2 inactivation conditions, including together with CSNK-1 depletion, I identified a novel polariy phenotype whereby PAR-2 is absent from the cell cortext of most embryos, an observation that prompted our study and which has just been submitted for publication. In this project I used primarily genetic and RNAi-mediated inactivation approaches, followed by time lapse microscopy microscopy and image analysis to quantify the resulting phenotypes. This led to an interesting separation of function between SPD-2 and AIR-1 in the process of polarity establishment.
Results coming from my project will push the cell polarity knowledge since they clarify part of the interplay between centrosomal proteins in this process.