Journal: bioRxiv

Article Title: CDK-regulated phase separation seeded by histone genes ensures precise growth and function of Histone Locus Bodies

doi: 10.1101/789933

Figure Lengend Snippet: (A) Schematic of the Histone Locus Body and the Drosophila histone locus located on the left arm of the second chromosome (2L). (B) Representative images of HLBs (GFP-Mxc) during S phase of syncytial cycles 11-14. HLBs show homogeneous size distribution dependent on cycle stage. Scale bars, 10µm. c, cycle. (C) Schematic of HLB size quantification. i) HLBs were visualized by expressing a GFP-Mxc fusion protein in the presence of endogenous Mxc. ii) Multiple z stack images were acquired with confocal microscopy. iii) HLBs were segmented using threshold-based computational segmentation. iv) HLB size was approximated by measuring the trapezoidal cone shaped space occupied by multiple HLB masks. v) HLB ‘Total Fluorescence’ was quantified by multiplying the approximated size by the average fluorescence intensity level of the HLB masks. (D) HLB total fluorescence as a function of time for cycles 11-14. Shaded green area, SEM. (E) Fraction of nuclei with paired chromosomes over time. (F) Comparison between the average Mxc total fluorescence of fused HLBs (‘paired (fused) HLB’) and that of two HLBs that have not fused (‘unpaired HLBs multiplied by 2’). c, cycle. M, mitosis. A.U., arbitrary units.

Article Snippet: GFP-Mxc AP22 fragment was generated by mutagenizing serines and threonines of 2 nd -23 rd putative phosphorylation sites (Ser/Pro or Thr/Pro) from the N-terminus to alanines (Ala) on the mxc gene (GenScript).

Techniques: Expressing, Confocal Microscopy, Fluorescence

Journal: bioRxiv

Article Title: CDK-regulated phase separation seeded by histone genes ensures precise growth and function of Histone Locus Bodies

doi: 10.1101/789933

Figure Lengend Snippet: (A) Top: Example of HLB fusion upon chromosome pairing during S phase in Drosophila embryos. Bottom: Simulation of HLB fusion event and quantification of droplet circularity during the fusion. (B) Quantification of HLB circularity index during fusion events. Multiple quantifications were performed, aligned by the fusion timing (t=0), and averaged. Shaded area, SEM. (C) Schematic of HLB FRAP experiment. (D) Example of unbleached (black) versus bleached (red) HLB fluorescence level quantification. Dim red line indicates original data and the solid red line indicates the same data after noise correction, which is used for analysis in panel (E). (E) Logarithm of the difference between bleached (red in (D)) and unbleached (black in (D)) HLB fluorescence from (D) over time. The initial fit (dashed green line) was generated from all data points (blue and light blue points). From this initial fit, points whose residuals were greater than 2 standard deviations were removed from the dataset (light blue points). The best fit line was then computed on the dataset lacking the outliers (solid green line). The export rate k off was measured from the slope of the solid green line. (F) HLB recovery time (on logscale) as a function of the HLB total fluorescence. (G) Phase diagram for a liquid-liquid phase separation. At a given temperature and concentration, a mixture of macromolecules with the potential to undergo liquid-liquid phase separation becomes thermodynamically unstable and separates into two different phases with concentrations of ρ - and ρ + . Red area indicates the portion of the phase diagram in which phase separation occurs. Thus, ρ - and ρ + can be determined for any given temperature (horizontal dotted line). Red triangles and yellow circles represent other soluble proteins that do not participate in the phase separation; while green circles are the phase-separated component (e.g. GFP-Mxc). Adapted from . (H) Quantification of average Mxc concentration inside HLB versus HLB total fluorescence. (I) Numerical solution of Cahn-Hilliard (CH) equation in 2D lattice reveals that HLB growth proceeds in three stages: 1) seeding level-dependent fast growth, 2) slower growth independent of the seeding level, and 3) equilibrium phase where HLB size is only dependent on nuclear Mxc concentration. (J) A particle-based simulation of HLB growth in the 3D lattice shows that HLB size (y-axis) depends on the seeding level (shades of gray) and Mxc concentration (x-axis) in the first regime indicated as “1” in (I). (K) Experimental data of HLB total fluorescence inside each nucleus and nuclear Mxc concentration, for WT (∼200 histone repeats) and histone deficiency heterozygote mutant (∼100 histone repeats). A.U., arbitrary units.

Article Snippet: GFP-Mxc AP22 fragment was generated by mutagenizing serines and threonines of 2 nd -23 rd putative phosphorylation sites (Ser/Pro or Thr/Pro) from the N-terminus to alanines (Ala) on the mxc gene (GenScript).

Techniques: Fluorescence, Generated, Concentration Assay, Mutagenesis

Journal: bioRxiv

Article Title: CDK-regulated phase separation seeded by histone genes ensures precise growth and function of Histone Locus Bodies

doi: 10.1101/789933

Figure Lengend Snippet: (A) Schematic of the non-phosphorylateable mutant Mxc (Mxc AP22 ). Out of the 36 consensus SP or TP Cdk phosphorylation sites found in the Mxc protein, numbers 2-23 throughout the N-terminus were mutated to alanines. (B) HLB total fluorescence for embryos laid by WT (green), Cyclin E/Cdk2 double heterozygotes (blue), Mxc AP22 (black) mothers, and Cdk2 inhibitor injected (orange) embryos. (C) Confocal images of HLBs for WT (top left), GFP-Mxc AP22 expressing embryos (top right), DMSO control injected embryos (bottom left), and Cdk2 inhibitor injected embryos (bottom right). (D) Nuclear Mxc concentration for WT (green), Cyclin E/Cdk2 double heterozygotes (blue), Cdk2 inhibitor injected (orange), and Mxc AP22 embryos (black). (E) Mxc concentration inside HLBs for various genetic conditions altering cell cycle regulators. (F) The simple dependency of HLB size vs Mxc concentration (From ) still holds with different genetic conditions. A.U., arbitrary units. c, cycle. M, mitosis. Scale bars, 5µm.

Article Snippet: GFP-Mxc AP22 fragment was generated by mutagenizing serines and threonines of 2 nd -23 rd putative phosphorylation sites (Ser/Pro or Thr/Pro) from the N-terminus to alanines (Ala) on the mxc gene (GenScript).

Techniques: Mutagenesis, Fluorescence, Injection, Expressing, Concentration Assay