| Publication note | Here, we demonstrate that the inner-centromere targeting region of the CPC can phase-separate in vitro and provide strong evidence that the CPC at the inner-centromere exist in a phase-separated state. We also demonstrate that the CPC’s ability to undergo phase-separation is important for its inner-centromere localization and function. We tested whether subunits of the CPC could phase-separate in vitro. We expressed Survivin, Borealin, and the N-terminal 58 amino acids of INCENP (ISB) in E. coli. This set of proteins has all the activities required to localize the CPC to the inner-centromere. We observed spontaneous ISB phase-separation under conditions of high protein concentration, low salt concentration or the presence of the molecular crowding agent PEG-3350. Coacervates formed with GFP-ISB were fluorescent, demonstrating they are composed of CPC protein. (Organism: Homo sapiens; Cell line: U2OS cells,HeLa cells) |
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| Material state | liquid,hydrogel : The ISB coacervates display liquid-like properties that are similar to those observed in phase-separating proteins. They are highly circular in shape, the larger coacervates undergo shear upon placement of a coverslip, and the coacervates undergo fusion at early time points after induction of phase-separation. ISB coacervates quickly (~1 min) mature to have gel-like properties. | ||
| LLPS region | 1-280 | Key domains | _ : _ |
| Protein partner | Survivin,Borealin,H3 ( O15392,O13024,P68431 ) |
We observed that α-satellite DNA, histone H3.3 polynucleosomes, Histone H3.1 mononucleosomes, H3T3ph mononucleosomes and microtubule bundles all induce phase-separation under conditions where ISB exists in a homogeneous phase on its own. |
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| RNA partner | _ |
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| Others | _ |
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| PTM | _ |
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| Mutation | p.R132A/K133A/R139A/K141A/R142A/K147A/K148A/R149A/K157A/K159A/K161A/R162A/R165A/R175A/E177A/K183A,p.R139A/K141A/R142A/K147A/K148A/R149A/K157A/K159A,p.A139_K160del |
We mutated 8 positively charged residues in the 139-160 region of Borealin to alanine (p.R139A/K141A/R142A/K147A/K148A/R149A/K157A/K159A). We also mutated 5 additional positively charged residues surrounding 139-160aa region to alanine (p.R132A/K133A/R139A/K141A/R142A/K147A/K148A/R149A/K157A/K159A/K161A/R162A/R165A/R175A/E177A/K183A). Both of these mutants were defective in phase-separation. BorealinΔ139-160(p.A139_K160del) was also deficient in the Optodroplet assay. ISB lacking 139-160 amino acids of Borealin was deficient in both spontaneous and DNA induced phase-separation. |
| Alternative splicing | _ | |
| Repeat | _ | |
| Oligomerization | _ | |