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• W2014758962 abstract "The structure of mitotic chromosomes is still poorly understood. Here we describe the use of a novel approach based on elasticity measurements of a single chromosome for studying the organization of these objects. The data reveal that mitotic chromosomes exhibit a non-homogenous structure consisting of rigid elastic axes surrounded by a soft chromatin envelope. The chemical continuity of DNA, but not RNA, was required for the maintenance of these axes. The axes show a modular structure, and the structural maintenance of chromosomes (SMC) proteins participate in their organization. Topoisomerase II was not involved in either the organization of the axes or the maintenance of the mitotic chromosomes. A model for the assembly and the structure of the mitotic chromosome is proposed. According this model, the chromosome axes are dynamic structures that assemble at the onset and disassemble the end of mitosis, respectively. The SMC proteins, in addition to maintaining axis elasticity, are essential for the determination of the rod-like chromosome shape. The extreme compaction of mitotic chromosomes is determined mainly by the high amount of bivalent ions bound to DNA at mitosis. The structure of mitotic chromosomes is still poorly understood. Here we describe the use of a novel approach based on elasticity measurements of a single chromosome for studying the organization of these objects. The data reveal that mitotic chromosomes exhibit a non-homogenous structure consisting of rigid elastic axes surrounded by a soft chromatin envelope. The chemical continuity of DNA, but not RNA, was required for the maintenance of these axes. The axes show a modular structure, and the structural maintenance of chromosomes (SMC) proteins participate in their organization. Topoisomerase II was not involved in either the organization of the axes or the maintenance of the mitotic chromosomes. A model for the assembly and the structure of the mitotic chromosome is proposed. According this model, the chromosome axes are dynamic structures that assemble at the onset and disassemble the end of mitosis, respectively. The SMC proteins, in addition to maintaining axis elasticity, are essential for the determination of the rod-like chromosome shape. The extreme compaction of mitotic chromosomes is determined mainly by the high amount of bivalent ions bound to DNA at mitosis. DNA is highly compacted ∼ 10,000–20,000 times in the mitotic chromosomes of a typical vertebrate cell. It is far from clear how this folding is accomplished. The 30-nm chromatin fiber accounts only for 40:1 of the compaction ratio, and the remaining ∼ 500-fold compaction is achieved through a largely unknown mechanism (1Woodcock C.L. Dimitrov S. Curr. Opin. Genet. Dev. 2001; 11: 130-135Crossref PubMed Scopus (220) Google Scholar, 2Nasmyth K. Annu. Rev. Genet. 2001; 35: 673-745Crossref PubMed Scopus (581) Google Scholar). The complexity of the mitotic chromosome structure has both fascinated and frustrated scientists for many decades. Structural analysis of mitotic chromosomes has been very difficult because of the extreme compaction of the chromatin fibers. A number of models for the chromosome structure have been proposed, ranging from a hierarchical folding of chromatin (3Manueledis L. Science. 1990; 250: 1533-1540Crossref PubMed Scopus (288) Google Scholar) to spaghetti-like disorder (4DuPraw E.J. Nature. 1966; 209: 577-581Crossref PubMed Scopus (83) Google Scholar). The favored textbook model describes the structure of the mitotic chromosome as an assembly of chromatin loop domains attached to a central protein scaffold (5Paulson J.R. Laemmli U.K. Cell. 1977; 12: 817-828Abstract Full Text PDF PubMed Scopus (775) Google Scholar). This scaffold showed the same shape as the mitotic chromosomes, and this shape was preserved even upon removal of >95% of the chromosomal proteins and 99% of the DNA and RNA (5Paulson J.R. Laemmli U.K. Cell. 1977; 12: 817-828Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 6Adolph K.W. Cheng S.M. Laemmli U.K. Cell. 1977; 12: 805-816Abstract Full Text PDF PubMed Scopus (199) Google Scholar).Topoisomerase II and ScII, a structural maintenance of chromosomes (SMC) 1The abbreviations used are: SMCstructural maintenance of chromosomespNpiconewton(s).1The abbreviations used are: SMCstructural maintenance of chromosomespNpiconewton(s). protein, were identified as the major components of the scaffold. About 70% of the total amount of topoisomerase II was found in the mitotic chromosome scaffold fraction (7Earnshaw W.C. Halligan B. Cooke C.A. Heck M.M.S. Liu L.F. J. Cell Biol. 1985; 100: 1706-1715Crossref PubMed Scopus (587) Google Scholar). Several reports have suggested that this enzyme might occupy discrete foci that could correspond to the base of the chromatin loop domains (7Earnshaw W.C. Halligan B. Cooke C.A. Heck M.M.S. Liu L.F. J. Cell Biol. 1985; 100: 1706-1715Crossref PubMed Scopus (587) Google Scholar, 8Gasser S.M. Laroche T. Falquet J. Boy de la Tour E. Laemmli U.K. J. Mol. Biol. 1986; 188: 613-629Crossref PubMed Scopus (423) Google Scholar). It was proposed that topoisomerase II, in addition to its important enzymatic function in chromosome assembly, was a part of the structural framework of the mitotic chromosome (7Earnshaw W.C. Halligan B. Cooke C.A. Heck M.M.S. Liu L.F. J. Cell Biol. 1985; 100: 1706-1715Crossref PubMed Scopus (587) Google Scholar, 8Gasser S.M. Laroche T. Falquet J. Boy de la Tour E. Laemmli U.K. J. Mol. Biol. 1986; 188: 613-629Crossref PubMed Scopus (423) Google Scholar).The SMC proteins exist in the cell as high molecular mass complexes, one class of which is termed condensins (9Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). The biochemical manipulations of extracts isolated from Xenopus eggs have identified two forms of condensins, 8 S and 13 S. The active form of condensin, 13 S condensin, consists of two SMC subunits (XCAP-C and -E) and three non-SMC subunits (XCAP-D2, -G, and -H). Depletion and rescue experiments in Xenopus egg extracts have demonstrated that 13 S condensin is required for both assembly and maintenance of mitotic chromosomes (9Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar).The models reported in the literature are derived mainly from experiments using microscopy techniques. Recently, a novel and complementary approach via monitoring the changes in the elastic response was applied to study the organization of mitotic chromosomes (10Houchmandzadeh B. Marko J.F. Chatenay D. Libchaber A. J. Cell Biol. 1997; 138: 1-12Crossref PubMed Scopus (118) Google Scholar, 11Houchmandzadeh B. Dimitrov S. J. Cell Biol. 1999; 145: 215-223Crossref PubMed Scopus (59) Google Scholar, 12Poirier M. Eroglu S. Chatenay D. Marko J.F. Mol. Biol. Cell. 2000; 11: 269-276Crossref PubMed Scopus (82) Google Scholar, 13Poirier M.G. Eroglu S. Marko J.F. Mol. Biol. Cell. 2002; 13: 2170-2179Crossref PubMed Scopus (52) Google Scholar). The elasticity is determined by the underlying structure of the studied materials and reflects the interactions holding them together. The knowledge of the elasticity properties of the studied object allowed its structure to be properly modeled. For example, the measurements of the bending rigidity and the force-extension curve showed that chromosomes assembled in Xenopus egg extract display a strong anomaly in their elastic response, i.e. their flexibility was found to be 2000 times higher than what could be expected from the measurement of their longitudinal deformability (11Houchmandzadeh B. Dimitrov S. J. Cell Biol. 1999; 145: 215-223Crossref PubMed Scopus (59) Google Scholar). This strongly suggests the presence of thin rigid axes inside these chromosomes, the diameter of which can be estimated, from elasticity calculation, to be <20 nm (11Houchmandzadeh B. Dimitrov S. J. Cell Biol. 1999; 145: 215-223Crossref PubMed Scopus (59) Google Scholar). The axes are surrounded by a soft chromatin envelope (11Houchmandzadeh B. Dimitrov S. J. Cell Biol. 1999; 145: 215-223Crossref PubMed Scopus (59) Google Scholar). However, no direct data for such organization are yet available.In this work, we report a detailed study on the structural organization of mitotic chromosomes assembled in Xenopus egg extract by using new approaches for investigating their elasticity. It is shown that chemical continuity of chromosomal DNA is essential for the maintenance of mitotic chromosomes. Direct experimental evidence is presented showing that the structure of the mitotic chromosome is heterogeneous and consists of a soft chromatin envelope and rigid elastic axes. SMC proteins, but not topoisomerase II, are involved in the organization of these axes. Interestingly, topoisomerase II was not found to contribute to the stiffness of the whole chromosome. The data are summarized in a model of the mitotic chromosome that explains their main structural and functional properties.EXPERIMENTAL PROCEDURESPreparation of Mitotic Extracts—Xenopus egg extracts were prepared mainly by using the protocol of Losada et al. (14Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (256) Google Scholar). Briefly, after removal of the jelly by cysteine treatment, the eggs were resuspended in XBE2 buffer (100 mm KCl, 2 mm MgCl2, 0.1 mm CaCl2, 10 mm potassium HEPES, pH 7.7, 5 mm potassium EGTA, and 0.05 m sucrose) supplemented with protease inhibitors (10 μg/ml of leupeptin and apoprotin) and 100 μg/ml of cytochalasin D. The eggs were crushed by centrifugation at 16 °C (20 min at 15,000 rpm in an SW41 rotor; Beckman Instruments). The cytoplasmatic fraction was collected by puncturing the side of the tube with a 1-ml syringe, and leupeptin, apoprotin, cytochalasin D (at final concentration of 10 μg/ml), and one-twentieth volume of 20× energy mix (20 mm phosphocreatine, 2 mm ATP, and 5 μg/ml creatine kinase; final concentration) were added to it. Then the crude extract was transferred to 2-ml tubes used for a TLS-55 rotor (Beckman Instruments) and spun at 52,000 rpm for 2 h at 4 °C. The lipid layer was removed carefully with a help of a vacuum, and the golden layer fraction was recentrifuged under the same conditions for 45 min. The supernatant was collected, aliquoted in 25-μl fractions, and frozen in liquid nitrogen. The extract was stored at –80 °C.Isolation of Demembranated Xenopus Sperm Nuclei and Chromosome Assembly—Xenopus demembranated sperm nuclei were prepared as described (15de la Barre A.-E. Robert-Nicoud M. Dimitrov S. Methods Mol. Biol. 1999; 119: 219-229PubMed Google Scholar). The demembranated sperm was stored at –80 °C and thawed immediately before use. The assembly of mitotic chromosomes was performed according to the described protocol (16de la Barre A.-E. Gerson V. Gout S. Creaven M. Allis C.D. Dimitrov S. EMBO J. 2000; 19: 379-391Crossref PubMed Scopus (98) Google Scholar).Chromosome Micromanipulations—The chromosome micromanipulations were carried out using the device described previously (11Houchmandzadeh B. Dimitrov S. J. Cell Biol. 1999; 145: 215-223Crossref PubMed Scopus (59) Google Scholar). After completion of the chromosome assembly reaction, 5 μl of the reaction were transferred in a small reservoir containing 300 μl of EB (80 mm β-glycerophosphate, pH 7.3, 20 mm EGTA, 15 mm MgCl2, and 1 mm dithiothreitol; in some experiments the MgCl2 was omitted). Then, the ends of a single chromosome were grabbed by two pipettes either through aspiration or by antibody adhesion. The micropipettes used had an inner diameter of 1 μm and were prepared by using a puller (Sutter P-97). One of the pipettes was fixed, and a motion was imposed on the second pipette. The simultaneous measurements of the end-to-end distance between the pipettes (the length of the extended chromosome) and the deflection of the fixed pipette allowed the determination of the stretch modulus, i.e. the force necessary to stretch the chromosome twice its initial length (11Houchmandzadeh B. Dimitrov S. J. Cell Biol. 1999; 145: 215-223Crossref PubMed Scopus (59) Google Scholar).Three different antibodies were used for the pipette's coating, namely anti-histone antibody (panhistone antibody, Roche Molecular Biochemicals), immunopurified anti-XCAP-E antibody generated against the C terminus peptide of XCAP-E (9Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar), and immunopurified polyclonal anti-Xenopus topoisomerase II antibody (17Hirano T. Mitchison T.J. J. Cell Biol. 1993; 120: 601-612Crossref PubMed Scopus (187) Google Scholar). The pipettes were incubated overnight at 4 °C in a solution of phospate-buffered saline and 0.02% NaN3 containing the respective antibody. The concentration of the antibodies in the different experiment was usually 1 μg/ml. It should be noted, however, that the same results were obtained when the concentration of the antibodies used for pipette coating was within the interval 0.1–20 μg/ml, i.e. the elasticity chromosome data did not depend on the concentration of the antibodies within more than two orders of magnitude. The specificity of the anchorage of the chromosome to the antibody-coated pipettes was tested by using either non-antibody-coated, anti-nucleoplasmin antibody-coated (nucleoplasmin is a very abundant protein present in the extract that does not associate with the mitotic chromosomes), or bovine serum albumin-coated pipettes. In all three cases, no attachment of the chromosomes to the pipettes was observed. We would like to note that the addition of either one of the antibodies used for pipette coating to the assembled chromosomes in the egg extract does not change their morphology and physicochemical properties.Topoisomerase II Depletion of Mitotic Chromosomes—The topoisomerase II depletion of the in vitro assembled mitotic chromosomes was carried out as described by Hirano and Mitchison (17Hirano T. Mitchison T.J. J. Cell Biol. 1993; 120: 601-612Crossref PubMed Scopus (187) Google Scholar). After completion of the chromosome assembly reaction, 5 μl of the reaction was transferred to the chromosome-stretching chamber, which contained 300 μl of EB solution and 150 mm NaCl, and the stretching was carried out. To test if the increase of the ionic strength removes topoisomerase II, a chromosome assembly reaction was brought to 100–200 mm NaCl, and the chromosomes were pelleted by centrifugation on a bench-top centrifuge. The pellet was washed with EB containing NaCl at 100–200 mm concentration and used for immunoblotting. The immunoblotting was carried out following a previously described procedure (16de la Barre A.-E. Gerson V. Gout S. Creaven M. Allis C.D. Dimitrov S. EMBO J. 2000; 19: 379-391Crossref PubMed Scopus (98) Google Scholar) and using an immunopurified anti-Xenopus topoisomerase II antibody (17Hirano T. Mitchison T.J. J. Cell Biol. 1993; 120: 601-612Crossref PubMed Scopus (187) Google Scholar).Enzymatic/Micromechanical Experiments—To study the role of nucleic acids or proteins in the maintenance of the mitotic chromosome structure, a series of combined enzymatic/micromechanical experiments was carried out. A typical experiment was performed as follows. A single chromosome was caught by the two pipettes through aspiration, and a small tension was applied. Then a third micropipette, which contained the enzyme, was moved near the chromosome, and the enzyme solution was sprayed onto it while maintaining the tension. Phase contrast and fluorescence images were acquired through a CCD camera and recorded on a VCR. In some experiments the elastic response of both the intact (before injection of the enzyme) and the digested chromosome was measured.Four different enzymes, namely DNase I (10 units/μl; Roche Molecular Biochemicals), RNase A (500 μg/ml; Sigma), proteinase K (1 mg/ml; Sigma), and trypsin (50 μg/ml; Sigma) were used for spraying. It should be noted that the actual concentration of the sprayed enzyme on the chromosome was decreased by a factor of roughly 2–3 in comparison with that within the pipette as judged by experiments with fluorescent dyes (data not shown). When the spraying onto the chromosome was stopped, the enzymatic reaction arrests immediately due to the diffusion-mediated dissipation of the enzyme. In the case of the spraying of trypsin, the changes in the morphology and the elastic response of the chromosome due to the trypsin digestion were correlated with the SMC protein cleavage. Because it was not possible to visualize the degree of protein cleavage on a single chromosome, the digestion of a multitude of chromosomes by trypsin in conditions similar to those for a single chromosome was studied. Briefly, a chromosome assembly reaction was appropriately diluted with EB (see the section above titled “Chromosome Micromanipulations”), trypsin was added at concentration of 50 μg/ml, and the digestion was allowed to proceed for the appropriate time at room temperature. The chromosomes were then pelleted for 30 s on a bench top centrifuge, and the pellet was washed very quickly with ice-cold EB and recentrifuged for 15 s. After removal of the EB solution, the trypsin-digested chromosomes were immediately resuspended in SDS electrophoresis loading buffer and heated at 96 °C for 5 min. The chromosomal proteins were separated on an 8% polyacrylamide gel containing SDS. The immunoblotting was carried out by using immunopurified anti-SMC protein (anti-XCAP-E; Ref. 9Hirano T. Kobayashi R. Hirano M. Cell. 1997; 89: 511-521Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar) antibody. It should be noted that, in the different trypsin digestion experiments, a complete cleavage of the SMC proteins was observed 2–4 min after the onset of digestion reaction.RESULTSThe Chemical Continuity of DNA Is Crucial for the Maintenance of Mitotic Chromosome Structure—Elasticity studies have shown that native (10Houchmandzadeh B. Marko J.F. Chatenay D. Libchaber A. J. Cell Biol. 1997; 138: 1-12Crossref PubMed Scopus (118) Google Scholar) or in vitro assembled (11Houchmandzadeh B. Dimitrov S. J. Cell Biol. 1999; 145: 215-223Crossref PubMed Scopus (59) Google Scholar) chromosomes could be elongated by close to two orders of magnitude of their initial length without breaking. This demonstrates that a mitotic chromosome contains a very large reservoir of length, which is released gradually as a function of the applied tension. It is difficult to imagine that this peculiar property could be determined by the chromosomal proteins only. Thus, DNA and some nucleoprotein structures should be involved in these specific properties of mitotic chromosomes. If this is correct, one could expect the continuity of DNA to be essential for the maintenance of the elastic properties of the chromosomes. Hence, cleavage of chromatin DNA by nucleases would result in perturbation of the overall structure of the mitotic chromosome and loss of its stability. Moreover, the application of a small tension on the cleaved chromosome should lead to its dissolution.To test this hypothesis, a single in vitro assembled chromosome was digested with deoxyribonuclease I, and the morphological changes resulting from the enzyme cutting of chromosomal DNA were observed. The experimental approach used is described on Fig. 1A. Briefly, demembranated Xenopus sperm nuclei were incubated in extracts isolated from Xenopus eggs. After ∼150 min of incubation, well separated mitotic chromosomes were formed (Fig. 1B). Because these chromosomes are free in the extract solution, they can be easily manipulated. Initially, one end of the chromosome was aspirated and fixed to a pipette. Then, the other chromosome end was fixed to an another pipette also by aspiration, and some small tension was applied to the chromosome (Fig. 1C). Next, a solution of DNase I was injected through a third pipette, and the structural alterations of the chromosome were visualized by both phase contrast and fluorescence microscopy (Fig. 1C). After some initial decrease of the overall diameter, the chromosome breaks into two halves (Fig. 1C). When a buffer solution alone was injected, no changes in the chromosome structure were observed (not shown, but see also Fig. 1E). This demonstrates that the chromosome integrity depends on the intactness of DNA.Chromosomal Proteins, but Not RNA, Participate in the Maintenance of Mitotic Chromosome Structure—It is accepted that the chromosomal proteins are important players in maintaining the mitotic chromosome structure. To investigate the degree of protein contribution in the maintenance of the structural integrity of mitotic chromosomes, an experiment similar to the one described above was carried out, substituting proteinase K for DNase I (Fig. 1D). After some initial decondensation, the chromosome “melts” and, after 25 s of digestion, completely disintegrates. The melted chromosome did not exhibit any detectable elastic response (not shown). Thus, as expected, the presence of chromosomal proteins is crucial for chromosome structural organization.Recent data have suggested a role for RNA in the maintenance of condensed heterochromatin (18Maison C. Bailly D. Peters A.H. Quivy J.P. Roche D. Taddei A. Lachner M. Jenuwein T. Almouzni G. Nat. Genet. 2002; 30: 329-334Crossref PubMed Scopus (550) Google Scholar). In addition, the high molecular mass Xist RNA is involved in the maintenance of the highly compact inactive X chromosome (19Clerc P. Avner P. Semin. Cell Dev. Biol. 2003; 14: 85-92Crossref PubMed Scopus (29) Google Scholar). This suggests a function for RNA in stabilizing the structure of interphase chromosomes. To test if RNA plays some role in the organization of mitotic chromosomes, we treated a single chromosome with RNase in an experiment similar to that with DNase I or proteinase K. In contrast to both proteinase K or DNase I, the injection of high concentration RNase for more than 30 min did not affect the integrity of the chromosome (Fig. 1E, and results not shown). We conclude that RNA is unlikely to participate in the organization of mitotic chromosomes.Mitotic Chromosomes Exhibit a Non-homogenous Structure— The above data demonstrate that both the DNA and chromosomal proteins are key factors in chromosome organization and structure. The chromosomal proteins and DNA might be assembled into a chromosome in different ways, thus forming either a non-homogenous or a homogenous structure. Most of the available models suggest that the chromosome exhibited a non-homogenous structure. However, recent elasticity experiments have modeled the chromosomes isolated from newt as solid, elastic rods (13Poirier M.G. Eroglu S. Marko J.F. Mol. Biol. Cell. 2002; 13: 2170-2179Crossref PubMed Scopus (52) Google Scholar). These data indicate an essentially homogenous structure in its connectivity across the chromosome cross-section (13Poirier M.G. Eroglu S. Marko J.F. Mol. Biol. Cell. 2002; 13: 2170-2179Crossref PubMed Scopus (52) Google Scholar, 20Poirier M.G. Marko J.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15393-15397Crossref PubMed Scopus (123) Google Scholar). On the other hand, our studies on the elastic properties of in vitro assembled chromosomes fitted well with a model of chromosome exhibiting a non-homogenous structure formed of few rigid axes surrounded by a soft envelope of chromatin (11Houchmandzadeh B. Dimitrov S. J. Cell Biol. 1999; 145: 215-223Crossref PubMed Scopus (59) Google Scholar). Hence, a controversy exists in the literature. To clarify this controversy, we developed a new antibody-based micropipette technique to stretch a single chromosome and investigate its elasticity. The classical approach uses aspiration to fix both ends of the chromosome to two micropipettes with different rigidity (Fig. 2A; see also Ref. 10Houchmandzadeh B. Marko J.F. Chatenay D. Libchaber A. J. Cell Biol. 1997; 138: 1-12Crossref PubMed Scopus (118) Google Scholar). Once the chromosomal ends are fixed, the chromosome is stretched by moving one of the pipettes (Fig. 2B). The aspiration technique allows us to study the elastic properties of the whole chromosome. From the force-relative extension dependence, the stretch modulus (the force necessary to elongate the chromosome twice its initial length, i.e. the slope of the force-relative extension curve) can be measured. The stretch modulus is a characteristic of the underlying structure of the object. A high stretch modulus reflects the fact that a high force is needed to elongate the object.Fig. 2A, schematic presentation of the two techniques used for catching an individual chromosome. The aspiration technique (diagram 1) allows us to catch the whole chromosome and, respectively, to measure the elasticity of the chromosome as a whole, whereas by antibody adhesion (diagram 2) the elasticity response of specific chromosomal domains containing the protein of interest can be studied. B, a typical force-extension cycle for a single chromosome. The ends of the chromosome were caught by aspiration using two different micropipettes. The upper pipette was flexible (with a spring constant within the pN · μm–1 range) and immobile, whereas the lower one was rigid (spring constant within the nN · μm–1 range) and mobile. The chromosome was stretched by moving the lower pipette to obtain the desired extension, and, after a pause of few seconds, the constraint was relaxed with the same velocity. Deflection of the upper pipette provoked by the motion of the lower one yielded the force applied on the chromosome.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The new technique uses antibody-coated pipettes, and the “catching” of the chromosome is carried out through an antibody (Fig. 2A). Because an antibody against specific proteins can be used, this technique allows us to study selectively the elastic properties of mitotic chromosome domains that are associated with these proteins. If the chromosome structure is non-homogenous, one might expect different elastic responses when different antibodies (raised against proteins associated with different chromosomal domains) are used. In addition, the elastic response of the whole chromosome (when caught by aspiration) might also differ from the elastic responses of the different domains.To study the elastic response of chromatin, we used highly specific anti-histone antibodies that allowed us to fix the chromatin fibers to the pipettes. A typical force-extension dependence is shown on Fig. 3A. As shown, the slope (the stretch modulus) of this curve is several times smaller than the slope of the force extension curve for the whole chromosome that is obtained when aspiration is used to fix the chromosomal ends to the pipettes (Fig. 3A). Histograms, presenting a summary of the stretching of 18 chromosomes with anti-histone antibodies and 11 chromosomes through aspiration, are presented on Fig. 3, B and C. The data show a peak of the stretch modulus of ∼30 pN when chromosome stretching was performed through the anti-histone antibodies (Fig. 3C). However, stretching through aspiration gave an average of the stretch modulus of ∼120 pN (Fig. 3B). Hence, higher forces are necessary to elongate the chromosome when it is fixed through aspiration to the micropipettes compared with the forces needed to stretch the chromosome when the fixation is carried out through the chromatin fibers. Therefore, the chromosomes exhibit a non-homogenous organization with a lower elastic response for the chromatin entity, i.e. mitotic chromatin shows a relatively soft structure. Bearing this in mind, one should expect the existence of chromosomal structures that are more rigid and show higher elastic response. The available data suggest that topoisomerase II and, more particularly, the SMC family of proteins might be associated with such structures (5Paulson J.R. Laemmli U.K. Cell. 1977; 12: 817-828Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 7Earnshaw W.C. Halligan B. Cooke C.A. Heck M.M.S. Liu L.F. J. Cell Biol. 1985; 100: 1706-1715Crossref PubMed Scopus (587) Google Scholar, 8Gasser S.M. Laroche T. Falquet J. Boy de la Tour E. Laemmli U.K. J. Mol. Biol. 1986; 188: 613-629Crossref PubMed Scopus (423) Google Scholar, 21Laemmli U.K. Cheng S.M. Adolph K.W. Paulson J.R. Brown J.A. Baumbach W.R. Cold Spring Harbor Symp. Quant. Biol. 1978; 42: 351-360Crossref PubMed Google Scholar, 22Saitoh N. Goldberg I.G. Wood E.R. Earnshaw W.C. J. Cell Biol. 1994; 127: 303-318Crossref PubMed Scopus (238) Google Scholar).Fig. 3Elasticity measurements reveal a non-homogenous structure of mitotic chromosomes.A, two typical force-to-deformation curves for individual mitotic chromosomes obtained through aspiration (circles) and anti-histone antibody adhesion (stars). ΔL/L is the elongation induced by the force. Note the large difference in the slope (the stretch modulus) of both curves. B, a histogram summarizing the measurements of the stretch modulus of 11 individual chromosomes by the aspiration technique. The ordinate designates the number of chromosomes studied, whereas on the abscissa are noted the measured stretch modulus. C, same as panel B, but for the measured stretch modulus of 18 individual chromosomes by the anti-histone antibody adhesion technique.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The SMC Proteins Are Associated with Chromosome Regions Exhibiting Higher Ela" @default.
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• W2014758962 title "The Mitotic Chromosome Is an Assembly of Rigid Elastic Axes Organized by Structural Maintenance of Chromosomes (SMC) Proteins and Surrounded by a Soft Chromatin Envelope" @default.
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• W2014758962 cites W1532642855 @default.
• W2014758962 cites W1968806977 @default.
• W2014758962 cites W1970398821 @default.
• W2014758962 cites W1973690768 @default.
• W2014758962 cites W2012689223 @default.
• W2014758962 cites W2018023572 @default.
• W2014758962 cites W2019155756 @default.
• W2014758962 cites W2030293291 @default.
• W2014758962 cites W2031077971 @default.
• W2014758962 cites W2038932046 @default.
• W2014758962 cites W2044142379 @default.
• W2014758962 cites W2059634393 @default.
• W2014758962 cites W2059991470 @default.
• W2014758962 cites W2068690942 @default.
• W2014758962 cites W2084250387 @default.
• W2014758962 cites W2086080917 @default.
• W2014758962 cites W2095558945 @default.
• W2014758962 cites W2099997590 @default.
• W2014758962 cites W2112824334 @default.
• W2014758962 cites W2113065553 @default.
• W2014758962 cites W2121083656 @default.
• W2014758962 cites W2142658562 @default.
• W2014758962 cites W2160731157 @default.
• W2014758962 cites W2160774940 @default.
• W2014758962 cites W2164938051 @default.
• W2014758962 cites W2170016329 @default.
• W2014758962 cites W2317229987 @default.
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