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Chromosome Scaffold is a Double-Stranded Assembly of Scaffold Proteins.

Poonperm R, Takata H, Hamano T, Matsuda A, Uchiyama S, Hiraoka Y, Fukui K - Sci Rep (2015)

Bottom Line: This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes.We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity.Our model provides new insights into chromosome higher order structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, JAPAN.

ABSTRACT
Chromosome higher order structure has been an enigma for over a century. The most important structural finding has been the presence of a chromosome scaffold composed of non-histone proteins; so-called scaffold proteins. However, the organization and function of the scaffold are still controversial. Here, we use three dimensional-structured illumination microscopy (3D-SIM) and focused ion beam/scanning electron microscopy (FIB/SEM) to reveal the axial distributions of scaffold proteins in metaphase chromosomes comprising two strands. We also find that scaffold protein can adaptably recover its original localization after chromosome reversion in the presence of cations. This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes. We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity. Our model provides new insights into chromosome higher order structure.

No MeSH data available.


Related in: MedlinePlus

Depletion of hCAP-E or Topo IIα, but not KIF4, disrupts construction of a double-stranded chromosome scaffold (DCS) and proper chromosome organization.a,b,c, Maximum intensity projections of z-stack images obtained by 3D-SIM of KIF4-, hCAP-E- and Topo IIα-depleted HeLa-wt cells, respectively, immunostained for targeted scaffold proteins as indicated. Scale bars, 2 μm. DNA is shown in blue. Insets show magnified views of the white boxes in a,b and d as indicated. Scale bars, 250 nm.
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f3: Depletion of hCAP-E or Topo IIα, but not KIF4, disrupts construction of a double-stranded chromosome scaffold (DCS) and proper chromosome organization.a,b,c, Maximum intensity projections of z-stack images obtained by 3D-SIM of KIF4-, hCAP-E- and Topo IIα-depleted HeLa-wt cells, respectively, immunostained for targeted scaffold proteins as indicated. Scale bars, 2 μm. DNA is shown in blue. Insets show magnified views of the white boxes in a,b and d as indicated. Scale bars, 250 nm.

Mentions: To investigate which scaffold proteins are required for construction of the DCS, individual scaffold proteins were depleted from HeLa-wt cells using siRNA (Supplementary Fig. 4). DCS was often visualized by 3D-SIM observations in mock-treated chromosomes (Fig. 3a), similar to the results in non-transfected HeLa-wt mitotic cells (Fig. 1c, Supplementary Fig. 1c). After KIF4 depletion, hCAP-E and Topo IIα had slightly more dispersed distributions in broader chromosomes (Fig. 3b) compared to mock-treated chromosomes (Fig. 3a); the DCS was still observed (Fig. 3b, insets). This indicates that KIF4 may not be directly involved in formation of the DCS. In hCAP-E-depleted cells, chromosomes had a highly distorted structure and the chromosome scaffold was not constructed (Fig. 3c). Topo IIα was highly diffused throughout the chromosomes, while KIF4 was greatly reduced with depletion of hCAP-E (Fig. 3c, Supplementary Fig. 4b). Because KIF4 knockdown also reduced hCAP-E level on the chromosomes (Fig. 3b, Supplementary Fig. 4b). These results indicate the interdependency of KIF4 and condensin for localization to the chromosome scaffold5. In Topo IIα-depleted cells, chromosomes had an elongated shape. Although, a single dotted line liked distribution of hCAP-E and KIF4 was observed on the chromatid axes, no double strands were detected (Fig. 3d). These results signify that condensin is primarily important for construction of the chromosome scaffold10, and that Topo IIα should be responsible for construction the DCS, because, without Topo IIα, although the chromosome scaffold could be established, double strands could not be observed (Fig. 3d).


Chromosome Scaffold is a Double-Stranded Assembly of Scaffold Proteins.

Poonperm R, Takata H, Hamano T, Matsuda A, Uchiyama S, Hiraoka Y, Fukui K - Sci Rep (2015)

Depletion of hCAP-E or Topo IIα, but not KIF4, disrupts construction of a double-stranded chromosome scaffold (DCS) and proper chromosome organization.a,b,c, Maximum intensity projections of z-stack images obtained by 3D-SIM of KIF4-, hCAP-E- and Topo IIα-depleted HeLa-wt cells, respectively, immunostained for targeted scaffold proteins as indicated. Scale bars, 2 μm. DNA is shown in blue. Insets show magnified views of the white boxes in a,b and d as indicated. Scale bars, 250 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487240&req=5

f3: Depletion of hCAP-E or Topo IIα, but not KIF4, disrupts construction of a double-stranded chromosome scaffold (DCS) and proper chromosome organization.a,b,c, Maximum intensity projections of z-stack images obtained by 3D-SIM of KIF4-, hCAP-E- and Topo IIα-depleted HeLa-wt cells, respectively, immunostained for targeted scaffold proteins as indicated. Scale bars, 2 μm. DNA is shown in blue. Insets show magnified views of the white boxes in a,b and d as indicated. Scale bars, 250 nm.
Mentions: To investigate which scaffold proteins are required for construction of the DCS, individual scaffold proteins were depleted from HeLa-wt cells using siRNA (Supplementary Fig. 4). DCS was often visualized by 3D-SIM observations in mock-treated chromosomes (Fig. 3a), similar to the results in non-transfected HeLa-wt mitotic cells (Fig. 1c, Supplementary Fig. 1c). After KIF4 depletion, hCAP-E and Topo IIα had slightly more dispersed distributions in broader chromosomes (Fig. 3b) compared to mock-treated chromosomes (Fig. 3a); the DCS was still observed (Fig. 3b, insets). This indicates that KIF4 may not be directly involved in formation of the DCS. In hCAP-E-depleted cells, chromosomes had a highly distorted structure and the chromosome scaffold was not constructed (Fig. 3c). Topo IIα was highly diffused throughout the chromosomes, while KIF4 was greatly reduced with depletion of hCAP-E (Fig. 3c, Supplementary Fig. 4b). Because KIF4 knockdown also reduced hCAP-E level on the chromosomes (Fig. 3b, Supplementary Fig. 4b). These results indicate the interdependency of KIF4 and condensin for localization to the chromosome scaffold5. In Topo IIα-depleted cells, chromosomes had an elongated shape. Although, a single dotted line liked distribution of hCAP-E and KIF4 was observed on the chromatid axes, no double strands were detected (Fig. 3d). These results signify that condensin is primarily important for construction of the chromosome scaffold10, and that Topo IIα should be responsible for construction the DCS, because, without Topo IIα, although the chromosome scaffold could be established, double strands could not be observed (Fig. 3d).

Bottom Line: This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes.We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity.Our model provides new insights into chromosome higher order structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, JAPAN.

ABSTRACT
Chromosome higher order structure has been an enigma for over a century. The most important structural finding has been the presence of a chromosome scaffold composed of non-histone proteins; so-called scaffold proteins. However, the organization and function of the scaffold are still controversial. Here, we use three dimensional-structured illumination microscopy (3D-SIM) and focused ion beam/scanning electron microscopy (FIB/SEM) to reveal the axial distributions of scaffold proteins in metaphase chromosomes comprising two strands. We also find that scaffold protein can adaptably recover its original localization after chromosome reversion in the presence of cations. This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes. We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity. Our model provides new insights into chromosome higher order structure.

No MeSH data available.


Related in: MedlinePlus