Chromosome Scaffold is a Double-Stranded Assembly of Scaffold Proteins

Poonperm, R., Takata, H., Hamano, T. et al. Chromosome Scaffold is a Double-Stranded Assembly of Scaffold Proteins. Sci Rep 5, 11916 (2015). https://doi.org/10.1038/srep11916

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Kinesin family member 4 (KIF4) and condensins I and II are essential chromosomal proteins for chromosome organization by locating primarily to the chromosome scaffold. However, the mechanism of how KIF4 and condensins localize to the chromosome scaffold is poorly understood. Here, we demonstrate a close relationship between the chromosome localization of KIF4 and condensin I, but not condensin II, and show that KIF4 and condensin I assist each other for stable scaffold formation by forming a stable complex. Moreover, phosphorylation of KIF4 and condensin I by Aurora B and polo-like kinase 1 (Plk1) is important for KIF4 and condensin I localization to the chromosome. Aurora B activity facilitates the targeting of KIF4 and condensin I to the chromosome, whereas Plk1 activity promotes the dissociation of these proteins from the chromosome. Thus, the interdependency between KIF4 and condensin I, and their phosphorylation states play important roles in chromosome scaffold organization during mitosis

Localization of condensin and KIF4.

<p>(A) Mock, KIF4-depleted or SMC2-depleted HeLa cells were immunostained against SMC2 and CREST. DNA was counter stained with DAPI. Scale bar, 5 μm. Close-ups of the regions indicated by white boxes were shown on the right. (B) Mock, KIF4-depleted or SMC2-depleted HeLa cells were immunostained against KIF4 and CREST. DNA was counter stained with DAPI. Scale bar, 5 μm. Close-ups of the regions indicated by white boxes were shown on the right. (C) Quantification of relative mean fluorescence intensity of SMC2 or KIF4 staining in cells transfected with mock, SMC2- or KIF4-siRNA as indicated. Fluorescence intensities are normalized to the average pixel intensity of mock-transfected cells from the same experiment. Error bars represent the standard deviation. Two independent experiments were performed and data shown are from one representative experiment. (D) Western blot analysis of whole cell extract, chromatin fraction and cytoplasmic fraction prepared from mitotic mock, SMC2-depleted or KIF4-depleted HeLa cells. Tubulin and histone H3 were used as loading controls. (E) The band intensity of (D) was quantified using ImageJ software and normalized against protein amount in whole cell extract of mock. n = 3.</p

Effects of Aurora B and Plk1 inhibition on KIF4 and condensin I localization on chromosomes.

<p>(A) Immunofluorescence analysis of mitotic HeLa cells treated with DMSO as a control (DMSO) or 4 μM ZM447439 (ZM). Scale bar, 5 μm. Close-ups of the regions indicated by white boxes were shown in the bottom. (B) Immunofluorescence analysis of mitotic HeLa cells treated with DMSO as a control (DMSO) or 0.1 μM BI2536 (BI). Scale bar, 5 μm. Close-ups of the regions indicated by white boxes were shown in the bottom. (C) Quantification of relative mean fluorescence intensity of hCAP-H or KIF4 antibody in cells treated with DMSO, 4 μM ZM447439 (ZM) or 0.1 μM BI2536 (BI) as indicated. Fluorescence intensities are normalized to the average pixel intensity of DMSO-treated cells from the same experiment. Error bars represent the standard deviation. At least, three independent experiments were performed and data shown are from one representative experiment. (D) Physical interaction between KIF4, condensin I and condensin II. KIF4 was immunoprecipitated from chromatin-bound protein fractions extracted from mitotic HeLa cells treated without or with 4 μM ZM447439 (-ZM and +ZM, respectively). Co-immunoprecipitation of hCAP-H with KIF4 was analyzed by western blot. (E), (F) and (G) Western blot analysis of whole cell extract, chromatin fraction and cytoplasmic fraction prepared from mitotic mock, KIF4-depleted or hCAP-D2-depleted HeLa cells, respectively. The cells were treated with DMSO or 4 μM ZM447439 (ZM) or 0.1 μM BI2536 (BI). Tubulin and histone H3 were used as loading controls. (H), (I) and (J) The band intensity of (E), (F) and (G), respectively, was quantified using ImageJ software and normalized against protein amount in whole cell extract of mock. n = 3.</p

Interdependency between KIF4, condensin I and II.

<p>(A) Localization of hCAP-H (condensin I) and GFP-hCAP-D3 (condensin II) in mock- or KIF4-depleted HeLa cells were confirmed by immunostaining. DNA was counter stained with DAPI. Scale bar, 5 μm. Close-ups of the regions indicated by white boxes were shown on the right. (B) Localization of KIF4 in mock, hCAP-D2-depleted or hCAP-D3-depleted HeLa cells were confirmed by immunostaining. DNA was counter stained with DAPI. Scale bar, 5 μm. Close-ups of the regions indicated by white boxes were shown on the right. (C) Quantification of relative mean fluorescence intensity of GFP-hCAP-D3 or hCAP-H staining in cells transfected with mock or KIF4-siRNA as indicated. Fluorescence intensities are normalized to the average pixel intensity of mock-transfected cells from the same experiment. Error bars represent the standard deviation. Two independent experiments were performed and data shown are from one representative experiment. (D) Quantification of relative mean fluorescence intensity of KIF4 staining in cells transfected with mock, hCAP-D2—or hCAP-D3-siRNA as indicated. Fluorescence intensities are normalized to the average pixel intensity of mock-transfected cells from the same experiment. Error bars represent the standard deviation. Two independent experiments were performed and data shown are from one representative experiment. (E) Western blot analysis of whole cell extract, chromatin fraction and cytoplasmic fraction prepared from mitotic mock, hCAP-D2-depleted, hCAP-D3-depleted or KIF4-depleted HeLa cells. Tubulin and histone H3 were used as loading controls. (F) The band intensity of (E) was quantified using ImageJ software and normalized against protein amount in whole cell extract of mock. At least three independent experiments were performed. (G) Physical interaction between KIF4, condensin I and condensin II. KIF4 was immunoprecipitated from chromatin-bound or chromatin-unbound protein fractions. Co-immunoprecipitation of hCAP-H (condensin I) and hCAP-D3 (condensin II) with KIF4 was analyzed by western blot. n = 3.</p