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AFM images of G1-phase premature condensed chromosomes: Evidence for 30 nm changed to 50 nm chromatin fibers
Applied Surface Science 254 (2008) 1676–1683 www.elsevier.com/locate/apsusc
AFM images of G1-phase premature condensed chromosomes: Evidence for 30 nm changed to 50 nm chromatin fibers Yihui Fan a,1, Renfang Mao a,1, Jing Bai a,d,1, Xiaohong Zhang c, Qingquan Lei c, Songbin Fu a,b,* a
Laboratory of Medical Genetics, Harbin Medical University; Harbin 150081, China Bio-pharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081, China c The Institute of Electrical and Electronic Engineering, Harbin University of Science and Technology, Harbin 150040, China d Key Laboratory for Medical Cell Biology Engineering of Heilongjiang Higher Education Institutions, Harbin 150081, China b
Received 8 June 2007; received in revised form 25 June 2007; accepted 12 July 2007 Available online 22 July 2007
Abstract To gain evidence for 30 nm changed to 50 nm chromatin fibers, we used atomic force microscopy (AFM) to study the ultrastructural organization of G1-phase premature condensed chromosomes (PCC). The surface of early G1-phase PCC is smooth and fibrous structures exist around the chromatids. The height of early G1-phase PCC is about 410 nm and the width is 1.07 0.11 mm (n = 30). At late G1-phase, the surface becomes globular. The height of late G1-phase PCC is about 370 nm and the width is 845.04 82.84 nm (n = 30). Phase image reveals that early G1-phase PCC is composed of 50 nm (48.91 6.63 nm, n = 30) chromatin fibers and these 50 nm chromatin fibers tangle together, while late G1phase PCC is composed of 30 nm (30.96 4.07 nm, n = 30) chromatin fibers. At high magnification, fibers existing around the chromatids become clear in early G1-phase PCC. Chromatin fibers revealed by closer view of the end of chromatid are about 50 nm. In late G1-phase PCC, the surface presents globular structures. The shape of these globular structures is regular and the diameter is 118.96 11.70 nm (n = 30). Our results clearly show that 30 nm chromatin fibers change to 50 nm chromatin fibers in G1-phase PCC and suggest that 50 nm chromatin fibers are the basic component of the mitotic chromosomes. # 2007 Elsevier B.V. All rights reserved. Keywords: Premature condensed chromosomes (PCC); Atomic force microscopy (AFM); 50 nm Chromatin fibers; 30 nm Chromatin fibers
1. Introduction Chromosomes are complex structures containing DNA, histones, RNA and non-histone proteins, which appear during cell division. Its higher-order structure plays a critical role in many aspects of gene regulation [1], perhaps extending even to complex processes such as aging [2]. The first order of organization is nucleosome, which is considered to be the fundamental unit of chromosome structure [3,4]. Nucleosomes further pack into 30 nm chromatin fibers as the second order of organization. However, despite intense efforts the following condensation steps involving long range chromatin
* Corresponding author at: Harbin Medical University, Laboratory of Medical Genetics, Harbin 150081, China. Fax: +86 451 86632768. E-mail address: [email protected] (S. Fu). 1 They contributed equally to this paper. 0169-4332/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2007.07.142
fiber interactions, ultimately resulting in metaphasic chromosome formation remains poorly understood. To elucidate how 30 nm chromatin fibers form the structure of the higher order is still a fundamental problem in studies on mitotic chromosomes. Premature condensed chromosomes (PCC) is a powerful experimental tool to study cell cycle-specific changes in the higher-order arrangement of chromatin fibers. Chromosomes decondense gradually throughout G1-phase, reach a maximum level of dispersion at the time of replication during S-phase, and then begin a recondensation process that culminates in the formation of the maximally condensed metaphase chromosomes [5]. Therefore, precise investigation of this process will provide valuable information for understanding the higherorder organization of chromosomes. Is 30 nm or thicker fiber the basic unit of higher-order organization of mitotic chromosomes? It is a pivotal question to thoroughly understand the structure of chromosomes. Hoshi
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Fig. 1. Light microscopy image of G1-phase PCC: (A) early G1-phase; (B) late G1-phase.
Fig. 2. AFM image of early G1-phase PCC: (A) Height image of early G1-phase PCC; (B) bearing analyze of (A). The height of early G1-phase PCC is about 410 nm; (C) three-dimensional image of (A).
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Fig. 3. AFM image of late G1-phase PCC: (A) height image of late G1-phase PCC; (B) bearing analyze of (A). The height of late G1-phase PCC is about 370 nm; (C) three-dimensional image of A.
and Ushiki used atomic force microscopy (AFM) to study structure of G-banded human metaphase chromosomes and found that G-positive ridges were produced by an aggregation of fibrous structures about 50–100 nm in diameter [6]. Liu et al. used AFM to investigate the surface structure of barley chromosome and found granular structures with a diameter of ca. 50 nm on the surface of metaphase chromosomes [7]. Tamayo used AFM to study structure of human chromosomes treated with RNAase and pepsin, and found a granular structure with a grain size from 50 to 100 nm [8]. Fukushi and Ushiki used AFM to examine the structure of C banded human metaphase chromosomes and 50 nm thick chromatin fibers clearly shown in the entire length of the chromosomes [9]. As for the structure of native chromosomes, Hoshi et al. proposed
globular or fibrous structures about 50 nm thick on the surface of each chromatid [10]. In our previous study, we found S-phase PCC is composed of 30 nm chromatin fibers [11]. Taking these results together, we infer that there might be a state that 30 nm changing to 50 nm chromatin fibers between S-phase and Mphase. The current study was carried out in G1-phase PCC to find the evidence. 2. Materials and methods 2.1. Cell culture and cell synchronization HeLa cells were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO) and
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Fig. 4. Phase image of early G1-phase PCC: (A) early G1-phase PCC is composed of 50 nm chromatin fibers. The width of chromatin fibers is noted by black box; (C) line profile of (A), the section is indicated by the white line in (B); (D) closer view of (A); (F) line profile of one fiber, the section is indicated by the white line in (E). Red arrows are the measure points and the width of the chromatin fiber is 51.084 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
maintained in a humidified 5% CO2 air incubator at 37 8C. To synchronize cells into G1-phase, we cultured cells in RPMI1640 medium without FCS for 24 h.
onto glass slides, followed by air-drying. These glass slides were treated with 0.025% trypsin at 4 8C for 120 s and stained with a Giemsa solution.
2.2. Induction of premature condensed chromosomes
2.4. Observation by atomic force microscopy (AFM)
Calyculin A (Wako Chemicals) were dissolved in dimethylsulfoxide (DMSO) as a stock solution and stored at 20 8C. It was added at concentration of 50 nM/L to the medium, and the cells were incubated at 37 8C 1 h before harvest.
Glass slides were first observed by light microscopy, and the region containing an ideal karyotype of early G1-phase or late G1-phase PCC was marked, then AFM (Nanoscopy IIIa) imaging was carried out using a dynamic force mode. This nanoscopy was equipped with a piezo translator with a maximum xy scan range of 14 mm width and a z range of about 1.2 mm. Cantilevers were rectangular, with a constant force of 35 N/m and a resonance frequency of 360 KHz. All images were collected simultaneously as constant force images and variable deflection images in a dynamic force
2.3. Preparation of chromosome spreads Cell suspension was exposed to 75 mM KCl and fixed with a mixture of methanol and acetic acid (3:1). Spreads of chromosomes were made by dropping the cell suspension
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mode in air at room temperature. The constant force images were usually displayed by a computer using ‘‘gradation mode’’ that represents the height of specimens as color gradations. 3. Results and discussion 3.1. Light microscopy image of G1-phase PCC Fig. 1A shows the light microscopic morphology of PCC from HeLa cells in early G1-phase. The characteristic features are relatively short and thick single chromatids. As cells progress to late G1-phase, the PCC appears attenuated (Fig. 1B).
3.2. AFM image of G1-phase PCC Fig. 2A shows the surface of early G1-phase PCC is smooth. Chromatin fibers exist around the chromatids and these fibrous structures show more clearly at the end of chromatids. The height of early G1-phase PCC is about 410 nm (Fig. 2B) and the width is 1.07 0.11 mm (n = 30). In late G1-phase, the surface becomes globular (Fig. 3A). The height of late G1-phase PCC is about 370 nm (Fig. 3B) and the width is 845.04 82.84 nm (n = 30). Phase image reveals that early G1-phase PCC is composed of 50 nm (48.91 6.63 nm, n = 30) chromatin fibers (Fig. 4), while late G1-phase PCC is composed of 30 nm (30.96 4.07 nm, n = 30) chromatin fibers (Fig. 5). Section analysis shows more clearly profiles of chromatin fibers
Fig. 5. Phase image of late G1-phase PCC: (A) late G1-phase PCC is composed of 30 nm chromatin fibers. The width of chromatin fibers is noted by black box; (C) line profile of (A), the section is indicated by the white line in (B); (D) closer view of (A). (F) Line profile of one fiber, the section is indicated by the white line in E. Red arrows are the measure points and the width of the chromatin fiber is 29.529 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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Fig. 6. High magnification of AFM image of early G1-phase PCC: (A) Height image. The surface of early G1-phase PCC is smooth. Around the chromatid, chromatin fibers exist. (B) Three-dimensional image of (A). (C) Closer view of (A). At the end of chromatid, 50 nm chromatin fibers clearly revealed. (D) Three-dimensional image of (C).
(Fig. 4C and F; Fig. 5C and F) and red arrows are measure points (Fig. 4F and Fig. 5F). In early G1-phase, 50 nm chromatin fibers tangle together and some 50 nm chromatin fibers loop out of the axis (Fig. 4A). In late G1-phase, several 30 nm chromatin fibers arrange in parallel or twist together (Fig. 5A). AFM image shows a very distinct structure between early G1-phase and late G1-phase. It suggests that the organization of chromosome has a vigorous dynamic change in cell cycle. The atomic force microscope (AFM), invented by Binnig et al. [12], is a new device among the scanning probe microscopes (SPM). Scientific efforts in the past few years indicated that AFM could be a potential powerful tool for biological and biomedical research [13–15]. Sample can be imaged directly using AFM, requiring little or no sample pretreatment. Such advantages make AFM more suitable for studying physiologic ultrastructure of chromosomes. Because spatial resolution is lower than vertical resolution of AFM, mitotic chromosome is too dense to suit for AFM scanning. Therefore, we select more dispersed state of high-order organization of mitotic chromosomes for AFM scanning. Our present and previous studies imply that height image is fit
for revealing surface feature of chromosomes and phase image is fit for revealing internal fiber organization. 3.3. High magnification image of G1-phase PCC Fig. 6A presents sharper images of fibers existing around the chromatid in early G1-phase PCC. Chromatin fibers revealed by closer view of the end of chromatids are about 50 nm (Fig. 6C). It is consistent with our phase image finding. In early G1-phase PCC, the surface feature is similar to mitotic chromosomes revealed by AFM [10,16], but the fibrous structures around the chromatids are more clearly observed especially at the end of chromatids. At the end of chromatids, chromatin fibers about 50 nm are demonstrated in high magnification of height image. It proves our phase image finding that 50 nm chromatin fibers constitute early G1-phase PCC. We propose that these fibers may be the basic component of the chromosomes and probably produced by a twisting of 30 nm chromatin fibers. Investigators used X-ray to prove the two start helical crossed linker model of 30 nm chromatin fiber [17,18]. According to this model, the 50 nm chromatin fibers may be a more compact pattern of two 10 nm chromatin fibers.
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Fig. 7. High magnification of AFM image of late G1-phase PCC: (A) Height image. Late G1-phase PCC shows globular structures. Each globe has a regular feature and the diameter is 118.96 11.70 nm (n = 30). (B) Three-dimensional image of (A); (C) closer view of (A). (D) Three-dimensional image of (C).
Fig. 7 presents the surface as globular in late G1-phase PCC at high magnification. The shape of these globular structures is regular and the diameter is 118.96 11.70 nm (n = 30). Biochemical analysis of chromatin domains suggests that interphase chromatin is organized in about 50 kb domains [19]. A widely used estimate results from the compaction of the 1200 bp associated with six nulcleosomes into one 10 nm thick turn of helical chromatin fiber. 50 kb dsDNA would compact into 417 nm length of the 30 nm chromatin fiber. In our present study, the diameter of globe is 118.96 11.70 nm. Three or four 30 nm chromatin fibers unify together and the total length is about 450 nm. It suggests that the globular structure might be the functional unit of chromosomes in interphase. 4. Conclusions We used AFM to study the organization of G1-phase PCC. The height of early G1-phase PCC is about 410 nm and the width is 1.07 0.11 mm. The early G1-phase PCC is composed of 50 nm (48.91 6.63 nm, n = 30) chromatin fibers. The surface of late G1-phase PCC presents globular structures and the diameter is 118.96 11.70 nm (n = 30). The height of late G1-phase is about 370 nm and the width is 845.04 82.84 nm
(n = 30). The late G1-phase PCC is composed of 30 nm chromatin fibers. Our results evidently show that 30 nm (30.96 4.07 nm, n = 30) chromatin fibers change to 50 nm chromatin fibers in G1-phase. Acknowledgments Research was supported by the Hi-Tech Research, Development Program (No. 2002BA711A08) of China; the National Natural Science Foundation (No. 30370783); and the Ph.D. Programs Foundation (No. 20040226001) of MOE to Dr. S.B. Fu. References [1] [2] [3] [4] [5] [6] [7]
A.P. Wolffe, D. Guschin, J. Struct. Biol. 129 (2000) 102–122. J. Campisi, Science 289 (2000) 2062–2063. R.D. Kornberg, Annu. Rev. Biochem. 46 (1977) 931–954. J.B. Rattner, C.C. Lin, Cell 42 (1985) 291. H.K. Steven, Chromosoma 88 (1983) 333–342. O. Hoshi, T. Ushiki, Arch. Histol. Cytol. 64 (2001) 475–482. X.Q. Liu, S. Sugiyamaa, Q.Y. Xua, T. Koboria, S. Hagiwaraa, T. Ohtania, Ultramicroscopy 94 (2003) 217–223. [8] J. Tamayo, J. Struct. Biol. 141 (2003) 198–207.
Y. Fan et al. / Applied Surface Science 254 (2008) 1676–1683 [9] D. Fukushi, T. Ushiki, Arch. Histol. Cytol. 68 (2005) 81–87. [10] O. Hoshi, M. Shigeno, T. Ushiki, Arch. Histol. Cytol. 69 (2006) 73–78. [11] Y.H. Fan, X.H. Zhang, J. Bai, R.F. Mao, C.Y. Zhang, Q.Q. Lei, S.B. Fu, Appl. Surf. Sci. 253 (2007) 5281–5286. [12] G. Binnig, C.F. Quate, C. Gerber, Phys. Rev. Lett. 56 (1986) 930– 933. [13] J.H. Hoh, P.K. Hansma, Trends Cell Biol. 2 (1992) 208–213.
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[14] E. Henderson, Prog. Surf. Sci. 46 (1994) 39–60. [15] R. Lal, S.A. John, Am. J. Physiol. 266 (1994) C1–C21. [16] O. Hoshi, R. Owen, M. Milesb, T. Ushikia, Cytogenet. Genome Res. 107 (2004) 28–31. [17] S. Thomas, Nature 436 (2005) 138–141. [18] J.J. Philip, R. Daniela, Curr. Opin. Struct. Biol. 16 (2006) 336–343. [19] D.A. Jackson, P. Dickinson, P.R. Cook, EMBO J. 9 (1990) 567–571.
AFM images of G1-phase premature condensed chromosomes: Evidence for 30 nm changed to 50 nm chromatin fibers Yihui Fan a,1, Renfang Mao a,1, Jing Bai a,d,1, Xiaohong Zhang c, Qingquan Lei c, Songbin Fu a,b,* a
Laboratory of Medical Genetics, Harbin Medical University; Harbin 150081, China Bio-pharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081, China c The Institute of Electrical and Electronic Engineering, Harbin University of Science and Technology, Harbin 150040, China d Key Laboratory for Medical Cell Biology Engineering of Heilongjiang Higher Education Institutions, Harbin 150081, China b
Received 8 June 2007; received in revised form 25 June 2007; accepted 12 July 2007 Available online 22 July 2007
Abstract To gain evidence for 30 nm changed to 50 nm chromatin fibers, we used atomic force microscopy (AFM) to study the ultrastructural organization of G1-phase premature condensed chromosomes (PCC). The surface of early G1-phase PCC is smooth and fibrous structures exist around the chromatids. The height of early G1-phase PCC is about 410 nm and the width is 1.07 0.11 mm (n = 30). At late G1-phase, the surface becomes globular. The height of late G1-phase PCC is about 370 nm and the width is 845.04 82.84 nm (n = 30). Phase image reveals that early G1-phase PCC is composed of 50 nm (48.91 6.63 nm, n = 30) chromatin fibers and these 50 nm chromatin fibers tangle together, while late G1phase PCC is composed of 30 nm (30.96 4.07 nm, n = 30) chromatin fibers. At high magnification, fibers existing around the chromatids become clear in early G1-phase PCC. Chromatin fibers revealed by closer view of the end of chromatid are about 50 nm. In late G1-phase PCC, the surface presents globular structures. The shape of these globular structures is regular and the diameter is 118.96 11.70 nm (n = 30). Our results clearly show that 30 nm chromatin fibers change to 50 nm chromatin fibers in G1-phase PCC and suggest that 50 nm chromatin fibers are the basic component of the mitotic chromosomes. # 2007 Elsevier B.V. All rights reserved. Keywords: Premature condensed chromosomes (PCC); Atomic force microscopy (AFM); 50 nm Chromatin fibers; 30 nm Chromatin fibers
1. Introduction Chromosomes are complex structures containing DNA, histones, RNA and non-histone proteins, which appear during cell division. Its higher-order structure plays a critical role in many aspects of gene regulation [1], perhaps extending even to complex processes such as aging [2]. The first order of organization is nucleosome, which is considered to be the fundamental unit of chromosome structure [3,4]. Nucleosomes further pack into 30 nm chromatin fibers as the second order of organization. However, despite intense efforts the following condensation steps involving long range chromatin
* Corresponding author at: Harbin Medical University, Laboratory of Medical Genetics, Harbin 150081, China. Fax: +86 451 86632768. E-mail address: [email protected] (S. Fu). 1 They contributed equally to this paper. 0169-4332/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2007.07.142
fiber interactions, ultimately resulting in metaphasic chromosome formation remains poorly understood. To elucidate how 30 nm chromatin fibers form the structure of the higher order is still a fundamental problem in studies on mitotic chromosomes. Premature condensed chromosomes (PCC) is a powerful experimental tool to study cell cycle-specific changes in the higher-order arrangement of chromatin fibers. Chromosomes decondense gradually throughout G1-phase, reach a maximum level of dispersion at the time of replication during S-phase, and then begin a recondensation process that culminates in the formation of the maximally condensed metaphase chromosomes [5]. Therefore, precise investigation of this process will provide valuable information for understanding the higherorder organization of chromosomes. Is 30 nm or thicker fiber the basic unit of higher-order organization of mitotic chromosomes? It is a pivotal question to thoroughly understand the structure of chromosomes. Hoshi
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Fig. 1. Light microscopy image of G1-phase PCC: (A) early G1-phase; (B) late G1-phase.
Fig. 2. AFM image of early G1-phase PCC: (A) Height image of early G1-phase PCC; (B) bearing analyze of (A). The height of early G1-phase PCC is about 410 nm; (C) three-dimensional image of (A).
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Fig. 3. AFM image of late G1-phase PCC: (A) height image of late G1-phase PCC; (B) bearing analyze of (A). The height of late G1-phase PCC is about 370 nm; (C) three-dimensional image of A.
and Ushiki used atomic force microscopy (AFM) to study structure of G-banded human metaphase chromosomes and found that G-positive ridges were produced by an aggregation of fibrous structures about 50–100 nm in diameter [6]. Liu et al. used AFM to investigate the surface structure of barley chromosome and found granular structures with a diameter of ca. 50 nm on the surface of metaphase chromosomes [7]. Tamayo used AFM to study structure of human chromosomes treated with RNAase and pepsin, and found a granular structure with a grain size from 50 to 100 nm [8]. Fukushi and Ushiki used AFM to examine the structure of C banded human metaphase chromosomes and 50 nm thick chromatin fibers clearly shown in the entire length of the chromosomes [9]. As for the structure of native chromosomes, Hoshi et al. proposed
globular or fibrous structures about 50 nm thick on the surface of each chromatid [10]. In our previous study, we found S-phase PCC is composed of 30 nm chromatin fibers [11]. Taking these results together, we infer that there might be a state that 30 nm changing to 50 nm chromatin fibers between S-phase and Mphase. The current study was carried out in G1-phase PCC to find the evidence. 2. Materials and methods 2.1. Cell culture and cell synchronization HeLa cells were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO) and
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Fig. 4. Phase image of early G1-phase PCC: (A) early G1-phase PCC is composed of 50 nm chromatin fibers. The width of chromatin fibers is noted by black box; (C) line profile of (A), the section is indicated by the white line in (B); (D) closer view of (A); (F) line profile of one fiber, the section is indicated by the white line in (E). Red arrows are the measure points and the width of the chromatin fiber is 51.084 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
maintained in a humidified 5% CO2 air incubator at 37 8C. To synchronize cells into G1-phase, we cultured cells in RPMI1640 medium without FCS for 24 h.
onto glass slides, followed by air-drying. These glass slides were treated with 0.025% trypsin at 4 8C for 120 s and stained with a Giemsa solution.
2.2. Induction of premature condensed chromosomes
2.4. Observation by atomic force microscopy (AFM)
Calyculin A (Wako Chemicals) were dissolved in dimethylsulfoxide (DMSO) as a stock solution and stored at 20 8C. It was added at concentration of 50 nM/L to the medium, and the cells were incubated at 37 8C 1 h before harvest.
Glass slides were first observed by light microscopy, and the region containing an ideal karyotype of early G1-phase or late G1-phase PCC was marked, then AFM (Nanoscopy IIIa) imaging was carried out using a dynamic force mode. This nanoscopy was equipped with a piezo translator with a maximum xy scan range of 14 mm width and a z range of about 1.2 mm. Cantilevers were rectangular, with a constant force of 35 N/m and a resonance frequency of 360 KHz. All images were collected simultaneously as constant force images and variable deflection images in a dynamic force
2.3. Preparation of chromosome spreads Cell suspension was exposed to 75 mM KCl and fixed with a mixture of methanol and acetic acid (3:1). Spreads of chromosomes were made by dropping the cell suspension
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mode in air at room temperature. The constant force images were usually displayed by a computer using ‘‘gradation mode’’ that represents the height of specimens as color gradations. 3. Results and discussion 3.1. Light microscopy image of G1-phase PCC Fig. 1A shows the light microscopic morphology of PCC from HeLa cells in early G1-phase. The characteristic features are relatively short and thick single chromatids. As cells progress to late G1-phase, the PCC appears attenuated (Fig. 1B).
3.2. AFM image of G1-phase PCC Fig. 2A shows the surface of early G1-phase PCC is smooth. Chromatin fibers exist around the chromatids and these fibrous structures show more clearly at the end of chromatids. The height of early G1-phase PCC is about 410 nm (Fig. 2B) and the width is 1.07 0.11 mm (n = 30). In late G1-phase, the surface becomes globular (Fig. 3A). The height of late G1-phase PCC is about 370 nm (Fig. 3B) and the width is 845.04 82.84 nm (n = 30). Phase image reveals that early G1-phase PCC is composed of 50 nm (48.91 6.63 nm, n = 30) chromatin fibers (Fig. 4), while late G1-phase PCC is composed of 30 nm (30.96 4.07 nm, n = 30) chromatin fibers (Fig. 5). Section analysis shows more clearly profiles of chromatin fibers
Fig. 5. Phase image of late G1-phase PCC: (A) late G1-phase PCC is composed of 30 nm chromatin fibers. The width of chromatin fibers is noted by black box; (C) line profile of (A), the section is indicated by the white line in (B); (D) closer view of (A). (F) Line profile of one fiber, the section is indicated by the white line in E. Red arrows are the measure points and the width of the chromatin fiber is 29.529 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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Fig. 6. High magnification of AFM image of early G1-phase PCC: (A) Height image. The surface of early G1-phase PCC is smooth. Around the chromatid, chromatin fibers exist. (B) Three-dimensional image of (A). (C) Closer view of (A). At the end of chromatid, 50 nm chromatin fibers clearly revealed. (D) Three-dimensional image of (C).
(Fig. 4C and F; Fig. 5C and F) and red arrows are measure points (Fig. 4F and Fig. 5F). In early G1-phase, 50 nm chromatin fibers tangle together and some 50 nm chromatin fibers loop out of the axis (Fig. 4A). In late G1-phase, several 30 nm chromatin fibers arrange in parallel or twist together (Fig. 5A). AFM image shows a very distinct structure between early G1-phase and late G1-phase. It suggests that the organization of chromosome has a vigorous dynamic change in cell cycle. The atomic force microscope (AFM), invented by Binnig et al. [12], is a new device among the scanning probe microscopes (SPM). Scientific efforts in the past few years indicated that AFM could be a potential powerful tool for biological and biomedical research [13–15]. Sample can be imaged directly using AFM, requiring little or no sample pretreatment. Such advantages make AFM more suitable for studying physiologic ultrastructure of chromosomes. Because spatial resolution is lower than vertical resolution of AFM, mitotic chromosome is too dense to suit for AFM scanning. Therefore, we select more dispersed state of high-order organization of mitotic chromosomes for AFM scanning. Our present and previous studies imply that height image is fit
for revealing surface feature of chromosomes and phase image is fit for revealing internal fiber organization. 3.3. High magnification image of G1-phase PCC Fig. 6A presents sharper images of fibers existing around the chromatid in early G1-phase PCC. Chromatin fibers revealed by closer view of the end of chromatids are about 50 nm (Fig. 6C). It is consistent with our phase image finding. In early G1-phase PCC, the surface feature is similar to mitotic chromosomes revealed by AFM [10,16], but the fibrous structures around the chromatids are more clearly observed especially at the end of chromatids. At the end of chromatids, chromatin fibers about 50 nm are demonstrated in high magnification of height image. It proves our phase image finding that 50 nm chromatin fibers constitute early G1-phase PCC. We propose that these fibers may be the basic component of the chromosomes and probably produced by a twisting of 30 nm chromatin fibers. Investigators used X-ray to prove the two start helical crossed linker model of 30 nm chromatin fiber [17,18]. According to this model, the 50 nm chromatin fibers may be a more compact pattern of two 10 nm chromatin fibers.
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Fig. 7. High magnification of AFM image of late G1-phase PCC: (A) Height image. Late G1-phase PCC shows globular structures. Each globe has a regular feature and the diameter is 118.96 11.70 nm (n = 30). (B) Three-dimensional image of (A); (C) closer view of (A). (D) Three-dimensional image of (C).
Fig. 7 presents the surface as globular in late G1-phase PCC at high magnification. The shape of these globular structures is regular and the diameter is 118.96 11.70 nm (n = 30). Biochemical analysis of chromatin domains suggests that interphase chromatin is organized in about 50 kb domains [19]. A widely used estimate results from the compaction of the 1200 bp associated with six nulcleosomes into one 10 nm thick turn of helical chromatin fiber. 50 kb dsDNA would compact into 417 nm length of the 30 nm chromatin fiber. In our present study, the diameter of globe is 118.96 11.70 nm. Three or four 30 nm chromatin fibers unify together and the total length is about 450 nm. It suggests that the globular structure might be the functional unit of chromosomes in interphase. 4. Conclusions We used AFM to study the organization of G1-phase PCC. The height of early G1-phase PCC is about 410 nm and the width is 1.07 0.11 mm. The early G1-phase PCC is composed of 50 nm (48.91 6.63 nm, n = 30) chromatin fibers. The surface of late G1-phase PCC presents globular structures and the diameter is 118.96 11.70 nm (n = 30). The height of late G1-phase is about 370 nm and the width is 845.04 82.84 nm
(n = 30). The late G1-phase PCC is composed of 30 nm chromatin fibers. Our results evidently show that 30 nm (30.96 4.07 nm, n = 30) chromatin fibers change to 50 nm chromatin fibers in G1-phase. Acknowledgments Research was supported by the Hi-Tech Research, Development Program (No. 2002BA711A08) of China; the National Natural Science Foundation (No. 30370783); and the Ph.D. Programs Foundation (No. 20040226001) of MOE to Dr. S.B. Fu. References [1] [2] [3] [4] [5] [6] [7]
A.P. Wolffe, D. Guschin, J. Struct. Biol. 129 (2000) 102–122. J. Campisi, Science 289 (2000) 2062–2063. R.D. Kornberg, Annu. Rev. Biochem. 46 (1977) 931–954. J.B. Rattner, C.C. Lin, Cell 42 (1985) 291. H.K. Steven, Chromosoma 88 (1983) 333–342. O. Hoshi, T. Ushiki, Arch. Histol. Cytol. 64 (2001) 475–482. X.Q. Liu, S. Sugiyamaa, Q.Y. Xua, T. Koboria, S. Hagiwaraa, T. Ohtania, Ultramicroscopy 94 (2003) 217–223. [8] J. Tamayo, J. Struct. Biol. 141 (2003) 198–207.
Y. Fan et al. / Applied Surface Science 254 (2008) 1676–1683 [9] D. Fukushi, T. Ushiki, Arch. Histol. Cytol. 68 (2005) 81–87. [10] O. Hoshi, M. Shigeno, T. Ushiki, Arch. Histol. Cytol. 69 (2006) 73–78. [11] Y.H. Fan, X.H. Zhang, J. Bai, R.F. Mao, C.Y. Zhang, Q.Q. Lei, S.B. Fu, Appl. Surf. Sci. 253 (2007) 5281–5286. [12] G. Binnig, C.F. Quate, C. Gerber, Phys. Rev. Lett. 56 (1986) 930– 933. [13] J.H. Hoh, P.K. Hansma, Trends Cell Biol. 2 (1992) 208–213.
1683
[14] E. Henderson, Prog. Surf. Sci. 46 (1994) 39–60. [15] R. Lal, S.A. John, Am. J. Physiol. 266 (1994) C1–C21. [16] O. Hoshi, R. Owen, M. Milesb, T. Ushikia, Cytogenet. Genome Res. 107 (2004) 28–31. [17] S. Thomas, Nature 436 (2005) 138–141. [18] J.J. Philip, R. Daniela, Curr. Opin. Struct. Biol. 16 (2006) 336–343. [19] D.A. Jackson, P. Dickinson, P.R. Cook, EMBO J. 9 (1990) 567–571.