Visualization of the chromosome scaffold and intermediates of loop domain compaction in extracted mitotic cells

A novel extraction protocol for cells cultured on coverslips is described. Observations of the extraction process in a perfusion chamber reveal that cells of all mitotic stages are not detached from coverslips during extraction, and all stages can be recognized using phase contrast images. We studied the extracted cell morphology and distribution of a major scaffold component - topoisomerase IIalpha, in extracted metaphase and anaphase cells. An extraction using 2M NaCl leads to destruction of chromosomes at the light microscope level. Immunogold studies demonstrate that the only residual structure observed is an axial chromosome scaffold that contains topoisomerase IIalpha. In contrast, mitotic chromosomes are swelled only partially after an extraction using dextran sulphate and heparin, and it appears that this treatment does not lead to total destruction of loop domains. In this case, the chromosome scaffold and numerous structures resembling small rosettes are revealed inside extracted cells. The rosettes observed condense after addition of Mg2+-ions and do not contain topoisomerase IIalpha suggesting that these structures correspond to intermediates of loop domain compaction. We propose a model of chromosome structure in which the loop domains are condensed into highly regular structures with rosette organization.     

SMC and the bactofilin/PadC scaffold have distinct yet redundant functions in chromosome segregation and organization in Myxococcus xanthus

In bacteria, ParABS systems and structural maintenance of chromosome (SMC) condensin-like complexes are important for chromosome segregation and organization. The rod-shaped Myxococcus xanthus cells have a unique chromosome arrangement in which a scaffold composed of the BacNOP bactofilins and PadC positions the essential ParB∙parS segregation complexes and the DNA segregation ATPase ParA in the subpolar regions. We identify the Smc and ScpAB subunits of the SMC complex in M. xanthus and demonstrate that SMC is conditionally essential, with Δsmc or ΔscpAB mutants being temperature sensitive. Inactivation of SMC caused defects in chromosome segregation and organization. Lack of the BacNOP/PadC scaffold also caused chromosome segregation defects but this scaffold is not essential for viability. Inactivation of SMC was synthetic lethal with lack of the BacNOP/PadC scaffold. Lack of SMC interfered with formation of the BacNOP/PadC scaffold while lack of this scaffold did not interfere with chromosome association by SMC. Altogether, our data support that three systems function together to enable chromosome segregation in M. xanthus. ParABS constitutes the basic and essential machinery. SMC and the BacNOP/PadC scaffold have different yet redundant roles in chromosome segregation with SMC supporting individualization of daughter chromosomes and BacNOP/PadC making the ParABS system operate more robustly.     

Analysis of DNA attached to the chromosome scaffold

Two different methods have been described to investigate whether any specific DNA sequences are intimately associated with the metaphase chromosome scaffold. The chromosome scaffold, prepared by dehistonization of chromosomes with 2 M NaCl, is a nonhistone protein complex to which many looped DNA molecules are attached (Laemmli et al., 1977, Cold Spring Harbor Symp. Quant. Biol. 42:351--360). Chromosome scaffold DNA was prepared from dehistonized chicken MSB chromosomes by restriction endonuclease EcoRI digestion followed by removal of the looped DNA by sucrose gradient sedimentation. Alternatively, the scaffold DNA was prepared from micrococcal nuclease-digested intact chromosomes using sucrose gradients containing 2M NaCl. Solution hybridization of the radioactively labeled scaffold DNA with a large excess of total nuclear DNA revealed that, in either case, the scaffold DNA is not a unique sequence class of genomic DNA. Southern-blotting hybridization also showed that the scaffold DNA prepared from EcoRI-digested dehistonized chromosomes was not enriched (or depleted) in the ovalbumin gene sequences. The possibility of a dynamic interaction of protein and DNA in the chromosome scaffold and the possibility that the scaffold is a preparative artifact are discussed.     

Macromolecular and ultrastructural organization of the mitotic chromosome scaffold

Using electron microscopy (EM), we have examined three structural domains of the mitotic chromosome scaffold of mouse erythroleukemia (MEL) Friend cells with different morphologic organization: centromeric, intermediate, and telomeric. The intermediate, most extensive, domain exhibited a specific fibrogranular structure representing tightly packed granular bodies with diameters between 20 and 60 nm. The chromosome scaffold contained three main components: proteins (81%), RNA (12%), and DNA (7%). The residual DNA extracted from the scaffold represented short fragments, 300 bp on average, belonging to the class of tandemly arranged repetitive DNA. In situ hybridization experiments confirmed its typical centromeric location. Scaffold RNA represented three fractions: a major RNA fraction with an electrophoretic mobility corresponding to that of 5S RNA and two minor fractions with electrophoretic mobilities somewhat lower than that of 18S RNA. Scaffold RNA was localized mainly in the centromeric region. We show that the newly synthesized protein component of the chromosome scaffolds migrates slowly to the chromosomes, reaching a maximum specific radioactivity 12 h from the onset of the chase period.     

Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression

Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.     

Chromosome scaffold and structural integrity of mitotic chromosomes

Chromosome scaffold represents a continuous protein substructure revealed in isolated metaphase chromosomes after harsh extraction. According to postulates of the widespread radial loop model the scaffold plays an important role in the formation and maintenance of structural integrity of the mitotic chromosomes. Here, the data concerning the structure and major components of the chromosome scaffold are presented. The experiments suggesting that the scaffold represents a system of discrete linker proteins and the data about high mobility of scaffolding proteins are discussed. Furthermore, the data about higher-level chromatin structures (elementary chromonema and 200-250 nm fibers) and behavior of scaffolding proteins are compared. The results presented agree with the idea that at the present stage it is possible to discriminate chromatin complexes, whose structural integrity is not maintained by the chromosome scaffold.     

Chromosome scaffold and structural integrity of mitotic chromosomes

Chromosome scaffold represents a continuous protein substructure revealed in isolated metaphase chromosomes after harsh extraction. According to postulates of the widespread radial loop model the scaffold plays an important role in the formation and maintenance of structural integrity of the mitotic chromosomes. Here, the data concerning the structure and major components of the chromosome scaffold are presented. The experiments suggesting that the scaffold represents a system of discrete linker proteins and the data about high mobility of scaffolding proteins are discussed. Furthermore, the data about higher-level chromatin structures (elementary chromonema and 200–250 nm fibers) and behavior of scaffolding proteins are compared. The results presented agree with the idea that at the present stage it is possible to discriminate chromatin complexes, whose structural integrity is not maintained by the chromosome scaffold.     

Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells

Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporated later into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculated M(r) of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs.     

SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA

SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.     

Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii~1

Dinoflagellate is one of the primitive eukaryotes,whosenucleus may represent one of the transition stages fromprokaryotic nucleoid to typical eukaryotic nucleus.Usingselective extraction together with embeddment-free sectionand whole mount electron microscopy,a delicate nuclearmatrix filament network was shown,for the first time,indinoflagellate Crypthecodinium cohnii nucleus.Chromosomeresidues are connected with nuclear matrix filaments to forma complete network spreading over the nucleus.Moreover,we demonstrated that the dinoflagellate chromosome retainsa protein scaffold after the depletion of DNA and solubleproteins.This scaffold preserves the characteristic mor-phology of the chromosome.Two dimensional elec-trophoreses indicated that the nuclear matrix and chromo- some scaffold are mainly composed of acidic proteins.Ourresults demonstrated that a framework similar to the nuclearmatrix and chromosome scaffold in mammalian cells appearsin this primitive eukaryote,suggesting that these structuresmay have been originated from the early stages of eukaryoteevolution.     

Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum.The nuclear matrix and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl.SD-PAGE analyses revealed that the nuclear matrix and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight.Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence,suggesting the presence of the antigen in them.Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold.Immunoelectron microscopic observations further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.     

Identification and Analysis of a Novel Dimerization Domain Shared by Various Members of c-Jun N-terminal Kinase (JNK) Scaffold Proteins

Mitogen-activated protein kinases (MAPKs) form a kinase tier module in which MAPK, MAP2K, and MAP3K are held by scaffold proteins. The scaffold proteins serve as a protein platform for selective and spatial kinase activation. The precise mechanism by which the scaffold proteins function has not yet been fully explained. WDR62 is a novel scaffold protein of the c-Jun N-terminal kinase (JNK) pathway. Recessive mutations within WDR62 result in severe cerebral cortical malformations. One of the WDR62 mutant proteins found in a patient with microcephaly encodes a C-terminal truncated protein that fails to associate efficiently with JNK and MKK7β1. The present article shows that the WDR62 C-terminal region harbors a novel dimerization domain composed of a putative loop-helix domain that is necessary and sufficient for WDR62 dimerization and is critical for its scaffolding function. The loop-helix domain is highly conserved between orthologues and is also shared by the JNK scaffold protein, JNKBP1/MAPKBP1. Based on the high sequence conservation of the loop-helix domain, our article shows that MAPKBP1 homodimerizes and heterodimerizes with WDR62. Endogenous WDR62 and MAPKBP1 co-localize to stress granules following arsenite treatment, but not during mitosis. This study proposes another layer of complexity, in which coordinated activation of signaling pathways is mediated by the association between the different JNK scaffold proteins depending on their biological function.     

Scaffold-like structures in mouse chromosomes revealed by restriction endonuclease digestion and electron microscopy

A scaffold-like structure is observed under the electron microscope when mouse chromosomes are digested with the restriction endonuclease Hae III. This structure, located in the inner part of chromatids, may correspond to those fragments of chromatin loops anchored to the chromosome scaffold and is obtained when chromosomes are treated either in suspension or attached to grids. The width of the structure is correlated with the extent of digestion in chromosomes treated in suspension. Those treated on grids show this structure whenever chromatids do not collapse. These results agree with the model of chromosome organization based on a non-histone protein scaffold.     

A search for protein cores in chromosomes: Is the scaffold an artifact?

A protein chromosome scaffold structure has been proposed that acts as a structural framework for attachment of chromosomal DNA. There are several troubling aspects of this concept: (1) such structures have not been seen in many previous thin-section and whole-mount electron microscopy studies of metaphase chromosomes, while they are readily seen in leptotene and zygotene chromosomes; (2) such a structure poses problems for sister chromatid exchanges; and (3) the published photographs show a marked variation in the amount of scaffold in different whole-mount preparations. An alternative explanation is that the scaffold in whole-mount preparations represents incomplete dispersion of the high concentration of chromatin in the center of chromosomes, and when the histones are removed and the DNA dispersed, the remaining nonhistone proteins (NHPs) aggregate to form a chromosome-shaped structure. Two studies were done to determine if the scaffold is real or an artifact: (1) Chinese hamster mitotic cells and isolated chromosomes were examined using two protein stains -EDTA-regressive staining and phosphotungstic acid (PTA) stain. The EDTA-regressive stain showed ribonucleoprotein particles at the periphery of the chromosomes but nothing at the center of the chromosomes. The PTA stain showed the kinetochore plates but no central structures; and (2) isolated chromosomes were partially dispersed to decrease the high concentration of chromatin in the center of the chromosome, then treated with 4 M ammonium acetate or 2 M NaCl to dehistonize them and disperse the DNA. Under these circumstances, no chromosome scaffold was seen. We conclude that the scaffold structure is an artifact resulting from incomplete dispersion of central chromatin and aggregation of NHPs in dehistonized chromosomes.     

Nonhistone scaffold of mitotic chromosomes in situ

Based on the example of giant nuclei of Chironomus plumosus, we have shown (Makarov, Chentsov, 2010) that it is possible to identify the body of the polytene chromosome after its treatment in situ with 2 M NaCl and DNase in the presence of 2 mM CuCl₂. The Cu₂₊ ions stabilize bonds between the nonhistone components that form a nonhistone scaffold that repeats the general morphology and disc pattern of polytene chromosome. The goal of this work was to repeat the procedure for revealing the stabilized nonhistone scaffold in situ in the usual mitotic chromosomes of cells of the SPEV culture. After the extraction of all histones and DNA in the presence of 2 mM CuCl₂, we managed to reveal the residual body of mitotic chromosome (its nonhistone scaffold) at all stages of mitosis both in incident light and after using antibodies to proteins of topoisomerase IIα and SMC 1. Topoisomerase IIα and SMC 1 are distributed uniformly throughout the chromosome body and do not form any axial structures.     

Silver staining of histone-depleted metaphase chromosomes

To investigate a possible relationship between the core-like structures seen in silver-stained chromosomes (prepared by standard cytogenetic methods) and the scaffolds observed in histone-depleted chromosomes, the ability of the scaffold to stain with silver has been examined. Isolated chromosomes were histone-depleted by washing in ammonium acetate or by spreading the chromosomes on an ammonium acetate hypophase. The residual chromosome structures were carbon-platinum shadowed or stained with silver, and then examined by electron microscopy. The results provide clear evidence that the scaffold structure has a high affinity for silver and is therefore similar in its silver-staining potential to the core structure in standard chromosomes. This suggests that the silver core in standard chromosomes may represent the scaffold visualized by histone depletion. The peripherally dispersed DNA radiating from the scaffold also proved to be silver-reactive, and additional experiments demonstrated that purified DNA is capable of binding silver. This result indicates that cytological silver staining is not simply a matter of staining protein, as has previously been thought, but may also involve the staining of chromosomal DNA. In the ammonium acetate-treated and carbon-platinum-shadowed preparations, the scaffold structure was highly variable in its morphology and appeared to be composed of undispersed or incompletely dehistonized chromatin fibers. The silver-stained scaffold reflected this variability. Taken together with other evidence, these findings lead to a questioning of the reality of chromosome core structures.     

MukEF Is required for stable association of MukB with the chromosome

MukB is a bacterial SMC(structural maintenance of chromosome) protein required for correct folding of the Escherichia coli chromosome. MukB acts in complex with the two non-SMC proteins, MukE and MukF. The role of MukEF is unclear. MukEF disrupts MukB-DNA interactions in vitro. In vivo, however, MukEF stimulates MukB-induced DNA condensation and is required for the assembly of MukB clusters at the quarter positions of the cell length. We report here that MukEF is essential for stable association of MukB with the chromosome. We found that MukBEF forms a stable complex with the chromosome that copurifies with nucleoids following gentle cell lysis. Little MukB could be found with the nucleoids in the absence or upon overproduction of MukEF. Similarly, overproduced MukEF recruited MukB-green fluorescent protein (GFP) from its quarter positions, indicating that formation of MukB-GFP clusters and stable association with the chromosome could be mechanistically related. Finally, we report that MukE-GFP forms foci at the quarter positions of the cell length but not in cells that lack MukB or overproduce MukEF, suggesting that the clusters are formed by MukBEF and not by its individual subunits. These data support the view that MukBEF acts as a macromolecular assembly, a scaffold, in chromosome organization and that MukEF is essential for the assembly of this scaffold.     

cDNA cloning and mapping of a novel islet-brain/JNK-interacting protein

IB1/JIP-1 is a scaffold protein that regulates the c-Jun NH(2)-terminal kinase (JNK) signaling pathway, which is activated by environmental stresses and/or by treatment with proinflammatory cytokines including IL-1beta and TNF-alpha. The JNKs play an essential role in many biological processes, including the maturation and differentiation of immune cells and the apoptosis of cell targets of the immune system. IB1 is expressed predominantly in brain and pancreatic beta-cells where it protects cells from proapoptotic programs. Recently, a mutation in the amino-terminus of IB1 was associated with diabetes. A novel isoform, IB2, was cloned and characterized. Overall, both IB1 and IB2 proteins share a very similar organization, with a JNK-binding domain, a Src homology 3 domain, a phosphotyrosine-interacting domain, and polyacidic and polyproline stretches located at similar positions. The IB2 gene (HGMW-approved symbol MAPK8IP2) maps to human chromosome 22q13 and contains 10 coding exons. Northern and RT-PCR analyses indicate that IB2 is expressed in brain and in pancreatic cells, including insulin-secreting cells. IB2 interacts with both JNK and the JNK-kinase MKK7. In addition, ectopic expression of the JNK-binding domain of IB2 decreases IL-1beta-induced pancreatic beta-cell death. These data establish IB2 as a novel scaffold protein that regulates the JNK signaling pathway in brain and pancreatic beta-cells and indicate that IB2 represents a novel candidate gene for diabetes.