HP1a dynamics in the early drosophila embryo

The spatial segregation of heterochromatin into distinct, membrane-less nuclear compartments involves the binding of the heterochromatin protein 1 (HP1) to H3K9me2/3-rich genomic regions. While HP1 exhibits liquid-liquid phase separation properties in vitro, its mechanistic role in vivo on the structure and dynamics of heterochromatin remains largely unresolved. Here, using biophysical modeling, we systematically investigate the mutual coupling between self-interacting HP1-like molecules and the chromatin polymer. We reveal that the specific affinity of HP1 for H3K9me2/3 loci facilitates coacervation in nucleo, and promotes the formation of stable heterochromatin condensates at HP1 levels far below the concentration required in vitro to observe phase separation in purified protein assays. These heterotypic HP1-chromatin interactions give rise to a strong dependence of the nucleoplasmic HP1 density on the HP1-H3K9me2/3 stoichiometry, consistent with the thermodynamics of multicomponent p..., Flies homozygous for the expression of ectopic GFP-HP1a on the second chromosome (+, GFP-HP1a/CyO; +) were allowed to lay embryos on apple juice plates supplemented with yeast paste at 25°C. Embryos were collected by hand and dechorionated in 50% bleach before being mounted for live imaging. Embryos were imaged using a Zeiss 880 Airyscan microscope with a 63x/1.4 oil immersion objective. Time-lapse image stacks were collected every 30 seconds with a Z-spacing of 0.36μm. Imaging at least part of nuclear cycle 13 (NC 13) allowed the start of NC14 (time=0 sec) to be clearly defined as the first time point where circular nuclei appear following mitosis. All movies were Airyscan-processed using Zen 2.3 software and the first 72 time points of NC 14 (max36 minutes) were analyzed using Arivis software. Briefly, HP1a foci were segmented from the overall nuclear signal using an intensity threshold > 75% of the fluorescence intensity and a lower size threshold of 0.03 μm3 corresponding to appr..., These files can be opened in ImageJ/ FIJI and Arivis. Please check the file names for each file to make sure that the time and z axis of the data have been properly read by whichever software you would like to use. The time and z index is appended to each file name, so a cursory read of these will orient the user.Â