Wippich, F. et al. Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling. Cell 152, 791–805 (2013). Sawle, L. & Ghosh, K. A theoretical method to compute sequence dependent configurational properties in charged polymers and proteins. J. Chem. Phys. 143, 085101 (2015). Trivedi, P. et al. The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex. Nat. Cell Biol. 21, 1127–1137 (2019).

Larson, A. G. et al. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236–240 (2017). HeLa cells (ATCC, CCL-2.2) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% foetal bovine serum (FBS, Gibco) at 37 °C in the presence of 5% CO 2. The Ki-67 KO HCT116 cell line was described previously 46 and was cultured in high-glucose DMEM supplemented with 10% FBS and penicillin–streptomycin at 37 °C in the presence of 5% CO 2. Cells were transfected with the plasmids using PEI-MAX (Polysciences). To induce mitotic arrest, cells were treated with 0.2 µM nocodazole for 15 h. For microscopic observation, cells on a cover glass (Matsunami Glass) were fixed with 4% paraformaldehyde at room temperature for 15 min and mounted with Vectashield (Vector Laboratories) containing Hoechst 33342. Protein purification Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017). Replacement parts, including batteries, line cords, additional handsets, etc, are available from the product helpdesk. a, b, Ki-67 reversibly associates and dissociates from mitotic chromosomes upon ammonium acetate treatment. Ki-67-KO cells expressing EGFP-Ki-67 were treated with 100 mM ammonium acetate for 10 min and then returned to normal culture medium after washing with PBS. Time-lapse fluorescence images are shown in (a). Bar, 5 µm. The total EGFP signal intensity at the chromosomes was quantified and plotted along the time course (each line shows the data from an individual cell (n = 4)) (b). c, Ki-67 shows liquid-like behaviour on the mitotic chromosome periphery. FRAP analysis of EGFP-(WT) 8-LR expressed in HeLa cells. Time-lapse fluorescence images before and after bleaching are shown. The region surrounded by a circle was bleached using 488-nm laser light and the fluorescence intensity in the area was measured and plotted (mean ± SD, n = 38) (right panel). The fitting result from 38 cells is shown. Signal intensity was quantified using MetaMorph (Molecular Devices). Curve-fitting was performed using Python2 or 3 with accompanying libraries (Numpy, Scipy, Pandas, Matplotlib), using the equation described in Supplementary Note. Bar, 2 µm. d, Fluorescence images of mitotic HeLa cells expressing LR-free RDs. EGFP-fused WT R12 ((WT) 4, (WT) 8) and phosphomimetic mutants ((Pm9) 4, (Pm9) 8), were expressed in HeLa cells. Cells were fixed, stained with Hoechst33342, and observed by confocal fluorescence microscopy. Bar, 5 μm. e, Localization of R12 repeat (EGFP-(WT) 4-LR, EGFP-(WT) 8-LR, EGFP-(A9) 4-LR) and EGFP-(A9) 8-LR) in mitotic HeLa cells. DNA was stained with Hoechst33342. Magnified images (square (3.5×3.5 µm)) are shown in the insets. Bar, 5 µm. Source numerical data are available in Source Data.Lyophilized protein was dissolved into dissolving buffer (2 M guanidine hydrochloride, 100 mM Tris–HCl pH 8.0 and 10 mM HEPES) to a final concentration of 4 mM. For fluorescence microscopic observation, protein was incubated with 10 µM ATTO488-maleimide (ATTO-TEC) at room temperature for 1 h and then with 5 mM dithiothreitol at room temperature for 1 h or at 4 °C overnight. The labelled protein solution was diluted in droplet buffer (50 mM HEPES, 100 mM NaCl and 15% (w/v) PEG3350 (Sigma-Aldrich), pH 7.4) at a 1:100 ratio, incubated at room temperature for 30 min and transferred to a 96-well clear-bottom plate (Greiner Bio-One) for microscopic observation (FV3000, Olympus). The final concentration of protein was 40 μM unless otherwise indicated. For protein with multiple repeats, the final protein concentration is indicated in the figure legend. For the turbidity assay, protein in dissolving buffer was sequentially diluted with the same buffer, and then mixed with droplet buffer at 1:50. The mixture was incubated at room temperature for 10 min and transferred to a microcuvette. The optical density at 600 nm (OD 600) was measured using a V-630 spectrophotometer (JASCO). The C sat value was defined by the concentration at which the turbidity was at half-maximal value 10. The data obtained were fitted using the equation ( Supplementary Note) in OriginPro (v.9.8). In vitro phosphorylation by CDK1

Using a crawler, a cybercriminal could feasibly search through your device searching and important data. If this person is then able to transfer this data onto their own device, they may have harvested enough to impersonate you or even access your bank accounts and other financials. Seydoux, G. The P granules of C. elegans: a genetic model for the study of RNA–protein condensates. J. Mol. Biol. 430, 4702–4710 (2018). If you ever find your smart battery running low, then public charging ports can really be your saviour. But using these charging stations can really come with a darkside that you should be aware of. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012). Cuylen-Haering, S. et al. Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly. Nature 587, 285–290 (2020).Li, H. et al. SysPTM: a systematic resource for proteomic research on post-translational modifications. Mol. Cell. Proteom. 8, 1839–1849 (2009). E. coli cells (BL21‐CodonPlus(DE3)‐RIL, Agilent Technologies) harbouring the expression vector for hexahistidine (His 6)-tagged Ki-67 fragments were cultured in Luria–Bertani medium. Protein expression was induced by adding 0.1–1.0 mM IPTG, and the cultures were further incubated at 20 °C or 37 °C overnight. The cells were collected by centrifugation (5,000 g, 15 min, 20 °C) and stored at −80 °C until use. For purification under a denaturing condition, the cell pellet was subjected to two rounds of freeze–thaw cycles and was finally dissolved in urea-containing buffer (8 M urea, 10 mM Tris–HCl, 100 mM NaH 2PO 4, 5 mM 2-mercaptoethanol and 10 mM imidazole, pH 8.0) at 4 °C for overnight to 2 days. The lysate was centrifuged (10,000 g, 4 °C, 30 min) and the supernatant was collected and mixed with Ni-NTA agarose beads (Qiagen). The beads were gently agitated at 4 °C for 1 h, washed with wash buffer (8 M urea, 10 mM Tris–HCl, 100 mM NaH 2PO 4, 10 mM 2-mercaptoethanol and 20 mM imidazole, pH 8.0), and His 6-tagged proteins were eluted with elution buffer (8 M urea, 10 mM Tris–HCl, 100 mM NaH 2PO 4, 5 mM 2-mercaptoethanol and 100, 300 or 500 mM imidazole, pH 8.0). Eluted proteins were sequentially dialysed at 4 °C against dialysis buffer 1 (0.1% (v/v) trifluoroacetic acid (TFA) and 2 mM 2-mercaptoethanol) for 3 h, dialysis buffer 2 (0.05% (v/v) TFA and 2 mM 2-mercaptoethanol) for 3 h or overnight and, finally, dialysis buffer 3 (0.05% (v/v) TFA) for 3 h. The purified proteins were lyophilized (FDU‐2200, EYELA) and stored at 4 °C. Das, S., Eisen, A., Lin, Y.-H. & Chan, H. S. A lattice model of charge-pattern-dependent polyampholyte phase separation. J. Phys. Chem. B 122, 5418–5431 (2018). Simultaneous connections. Premium providers permit using the same account to protect multiple devices, while free services don’t have this luxury.