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. Author manuscript; available in PMC: 2011 Jun 8.
Published in final edited form as: Curr Biol. 2010 May 13;20(11):1048–1052. doi: 10.1016/j.cub.2010.04.025

Figure 3. Somatic H1 Chromosome Binding is Enhanced by Phosphorylation.

Figure 3

(A) H1 kinase assay from metaphase CSF-arrested (MET) or interphase-arrested (INT) egg extracts for H1M, H10, and alanine-substituted H10 (-AA).

(B) Anti-6XHistidine immunofluorescence of reactions with H10 wild-type versus alanine (AA) or glutamic acid (EE) phosphorylation site mutants. Interphase or mitotic reactions were fixed separately, then mixed and processed together. Average H1:DNA (6XHistidine:Hoechst) fluorescence ratio is shown for mitotic chromosomes alone (center column) or mitotic chromosomes over interphase nuclei (right of figure).

(C) Sperm nuclei were remodeled directly into mitotic chromosomes in CSF egg extracts supplemented with 1 μM H1A-GFP or phosphorylation site mutants and the average H1:DNA (GFP:DAPI) fluorescence ratio was determined (center column).

(D) Time-lapse images of dividing cells in stage 13 embryos expressing H1-GFP (green) and H2B-RFP (red). The Cdk1 phosphorylation site alanine mutant (AA) binds to mitotic chromosomes with reduced affinity compared to wild-type H1A, H1A-EE, or H1M, causing chromatin to shift from yellow to red. H1-GFP signal was normalized to H2B-RFP levels for single cells at interphase and anaphase, and the anaphase:interphase fluorescence ratio was calculated for each individual cell and averaged (right of figure). Scale bars, 10 μm; error, standard error. 10–50 structures or cells were examined per condition. See also Figure S2 and Movies S3–5.