Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Scaffold attachment of DNA loops in metaphase chromosomes

We have examined the higher-order loop organization of DNA in interphase nuclei and metaphase chromosomes from Drosophila Kc cells, and we detect no changes in the distribution of scaffold-attached regions (SARs) between these two phases of the cell cycle. The SARs, previously defined from experiments with interphase nuclei, not only are bound to the metaphase scaffold when endogenous DNA is probed but also rebind specifically to metaphase scaffolds when added exogenously as cloned, end-labeled fragments. Since metaphase scaffolds have a simpler protein pattern than interphase nuclear scaffolds, and both have a similar binding capacity, it appears that the population of proteins required for the specific scaffold-DNA interaction is limited to those found in metaphase scaffolds. Surprisingly, metaphase scaffolds isolated from Drosophila Kc cells contain both the lamin protein and a pore-complex protein, glycoprotein (gp) 188. To study whether lamin contributes to the SAR-scaffold interaction, we have carried out comparative binding studies with scaffolds from HeLa metaphase chromosomes, which are free of lamina, and from HeLa interphase nuclei. All Drosophila SAR fragments tested bind with excellent specificity to HeLa interphase scaffolds, whereas a subset of them bind to HeLa metaphase scaffolds. The maintenance of the scaffold-DNA interaction in metaphase indicates that lamin proteins are not involved in the attachment site for at least a subset of Drosophila SARs. This evolutionary and cell-cycle conservation of scaffold binding sites is consistent with a fundamental role for these fragments in the organization of the genome into looped domains.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Lectin-mediated uptake of lysosomal hydrolases by genetically deficient human fibroblasts.

A protein factor named S-II that stimulates RNA polymerase II was previously purified from Ehrlich ascites tumor cells [1]. In this work using an antibody prepared against purified S-II, the localization of S-II in the cell was investigated by an indirect immunofluorescence technique. In 3T3 cells, specific immunofluorescence was detected only in the nucleoplasm where RNA polymerase II is located, and not in the nucleoli where RNA polymerase I is present. In Ehrlich ascites tumor cells fluorescence was detected mainly in the nucleoplasm, although some fluorescence was also detectable in the cytoplasm, possibly due to leak of S-II from the nuclei during preparation of the immunofluorescent samples. In metaphase cells fluorescent was not found on chromosomes but throughout the cytoplasm. These findings suggest that S-II is a nuclear protein and that it spreads into the cytoplasm without being attached to chromosomes in metaphase, but is reassembled into the nucleoplasm in the interphase. Specific immunofluorescence was also detected in the nuclei of HeLa cells and salivary glands cells of flesh-fly larvae, suggesting that the nucleoplasm of these heterologous cells contains proteins immunologically cross-reactive with the antibody against S-II.     

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.     

A potential role for human cohesin in mitotic spindle aster assembly

The cohesin multiprotein complex containing SMC1, SMC3, Scc3 (SA), and Scc1 (Rad21) is required for sister chromatid cohesion in eukaryotes. Although metazoan cohesin associates with chromosomes and was shown to function in the establishment of sister chromatid cohesion during interphase, the majority of cohesin was found to be off chromosomes and reside in the cytoplasm in metaphase. Despite its dissociation from chromosomes, however, microinjection of an antibody against human SMC1 led to disorganization of the metaphase plate and cell cycle arrest, indicating that human cohesin still plays an important role in metaphase. To address the mitotic function of human cohesin, the subcellular localization of cohesin components was reexamined in human cells. Interestingly, we found that cohesin localizes to the spindle poles during mitosis and interacts with NuMA, a spindle pole-associated factor required for mitotic spindle organization. The interaction with NuMA persists during interphase. Similar to NuMA, a significant amount of cohesin was found to associate with the nuclear matrix. Furthermore, in the absence of cohesin, mitotic spindle asters failed to form in vitro. Our results raise the intriguing possibility that in addition to its well demonstrated function in sister chromatid cohesion, cohesin may be involved in spindle assembly during mitosis.     

Factors influencing ADP-ribosylation differences between chromosomal proteins of interphase and metaphase HeLa cells

Fundamental differences were previously discovered in the ADP-ribosylation of proteins from metaphase chromosomes and interphase nuclei of HeLa cells. The number of modified nonhistone species was found to be dramatically reduced for metaphase chromosomes. An investigation has therefore been made of factors which could influence, and therefore be responsible for, this change in ADP-ribosylation during the cell cycle. Modified proteins were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing mitotic and interphase samples from permeabilized cells that had been incubated with [32P]NAD. Whole cells showed a difference between interphase and metaphase similar to that for isolated nuclei and chromosomes. Chromosome expansion, disruption of chromosomes or nuclei, DNA nicking, and cellular growth activity significantly changed the incorporation of 32P label. Inhibitors of protein, RNA, and DNA synthesis did not, however, greatly affect ADP-ribosylation. The pattern of labeled species was not altered by the presence of nonradioactive NAD, though the extent of labeling declined. The results were not artifactually due to the procedure used to arrest cells in mitosis. Similar results were found with Novikoff rat hepatoma cells, demonstrating that the difference between metaphase and interphase is not confined to HeLa cells.     

F-actin is a Nuclear and Chromosomal Component of the Meristematic Cells of Allium sativum

It is known that actin functionates in the form of F-actin. However, the presence of Factin in eukaryotic nuclei and chromosomes has not been well established. The authors labeled meristematic cells of Allium sativum L. with rabbit anti-chicken actin antibody and FITC-conjugated goat anti-rabbit IgG antibody and observed with fluorescence microscopy. Both the nuclei and chromosomes showed prominent yellow-green fluorescence, indicating the presence of actin in them. Fluorescence examination with TR1TC-conjugated phalloidin demonstrated prominent red fluorescence in the intact interphase cells, cytoplasm-free interphase nuclei, prophase and metaphase chromosomes as well as the daughter nuclei at telophase indicating the presence of F-actin; but the fluorescence was absent or very weak in the cells exposed to cytochalasin D before fixation. When double labeling of the anti-actin antibody and phalloidin was applied, the same nuclei and chromosomes were found to emanate yellow-green fluorescence representing actin at the excitation wavelength of F1TC, and red fluorescence representing F-actin at the excitation wavelength of TRITC, respectively. The FITC fluorescence and TRITC fluorescence shared the same distribution among the nuclei and chromosomes. These results indicate that F-actin is a component of the nuclei and chromosomes of the meristematic cells of A. sativum. It also suggests that F-actin may be the major existing form of actin in them.     

Immunofluorescent studies of human chromosomes with antibodies against phosphorylated H1 histone

One of the prominent cell cycle-related modifications of histone proteins whose function remains unresolved is the phosphorylation of linker histone H1. In this work we have used indirect immunofluorescence on human cells with antibodies that are specific for phosphorylated histone H1 to examine the cellular distribution and chromosome association patterns of this protein. With confocal microscopy on whole cells, strong immunofluorescence was seen in association with mitotic chromosomes as well as a prominent punctate pattern of labeling throughout the mitotic cell, whereas interphase cells showed very little, if any, specific fluorescence. Multiple patterns of fluorescence distribution were detected with metaphase chromosomes, ranging from apparent tight colocalization with the DNA to expanded ”puffy” mitotic figures to an amorphous network of staining. It was also shown that the ability to label chromosomes could vary drastically with different fixation procedures, adding further complications to interpretation of the potentially complex role of phosphorylated histone H1 in chromatin condensation or decondensation. Received: 8 September 1999; in revised form: 14 September 1999 / Accepted: 17 September 1999     

C- and Q-chromatin of the experimentally condensed human interphase chromosomes]

Condensed interphase chromosomes of the cultured human lymphocytes obtained by the fusion of interphase and metaphase cells were studied using C- and Q-bands techniques. The appearance and localization of the constitutive heterochromatin blocks on condensed chromosomes at G1-period were the same as on the metaphase ones. These characters were used for a group and individual identification of some chromosomes condensed at G1-period and for a study of the association of the constitutive heterochromatin blocks in the interphase nuclei. The fluorescent analysis of the chromosomes condensed at G1-period detected some bright fluorescent blocks of the constitutive heterochromatin.     

Cell-cycle-dependent dissociation of histone H1 from chromatin in nuclei of P. polycephalum.

The dissociation curves of histone H1 from chromatin in interphase and metaphase nuclei from Physarum polycephalum have been determined using CaCl2 as dissociating agent. H1 is less strongly bound to metaphase chromosomes than to interphase chromatin. However, no differences could be detected in the binding of Hl to early S, late S or G2 phase chromatin. The number of CaCl2 molecules involved in binding one H1 molecule to chromatin was reduced from 5 in interphase to 4 in metaphase. The non-electrostatic contribution to the free-energy of binding was small in both cases. A comparison of the binding properties of H1 to sheared chromatin, native chromatin and metaphase chromosomes suggests that the electrostatic binding functions of H1 are completely satisfied within the nucleosome and that further electrostatic interactions are not involved in folding the nucleosomal fibre into the 300 A "solenoid" or the more tightly folded metaphase chromosome.     

Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclin-dependent kinase

Cyclin-dependent kinases (CDKs) are involved in the control of cell cycle progression. Plant A-type CDKs are functional homologs of yeast Cdc2/Cdc28 and are expressed throughout the cell cycle. In contrast, B-type CDK (CDKB) is a family of mitotic CDKs expressed during the S/M phase, and its precise function remains unknown. Here, we identified two B2-type cyclins, CycB2;1 and CycB2;2, as a specific partner of rice CDKB2;1. The CDKB2;1-CycB2 complexes produced in insect cells showed a significant level of kinase activity in vitro, suggesting that CycB2 binds to and activates CDKB2. We then expressed green fluorescent protein (GFP)-fused CDKB2;1 and CycB2;2 in tobacco BY2 cells to investigate their subcellular localization during mitosis. Surprisingly, the fluorescence signal of CDKB2;1-GFP was tightly associated with chromosome alignment as well as with spindle structure during the metaphase. During the telophase, the signal was localized to the spindle midzone and the separating sister chromosomes, and then to the phragmoplast. On the other hand, the CycB2;2-GFP fluorescence signal was detected in nuclei during the interphase and prophase, moved to the metaphase chromosomes, and then disappeared completely after the cells passed through the metaphase. Co-localization of CDKB2;1-GFP and CycB2;2-GFP on chromosomes aligned at the center of the metaphase cells suggests that the CDKB2-CycB2 complex may function in retaining chromosomes at the metaphase plate. Overexpression of CycB2;2 in rice plants resulted in acceleration of root growth without any increase in cell size, indicating that CycB2;2 promoted cell division probably through association with CDKB2 in the root meristem.     

Proteins of metaphase chromosomes and interphase chromatin.

Metaphase chromosomal and interphase chromatin proteins from cells of two species have been compared by polyacrylamide gel electrophoresis. Consistent, common changes in the quantitative distribution of the nonhistone chromosomal proteins are observed in both species. Proteins of ca. 65,000 and 68,000 MW are enriched in interphase chromatin while proteins of ca. 50,000 and 200,000 are more prominent components of metaphase chromosomes. A group of proteins of 90,000-100,000 are also increased in metaphase chromosomes compared to interphase chromatin. By two dimensional gel analysis, the most abundant proteins from chromosomes of both cell types are similar, suggesting a structural role for these nonhistone proteins (1).     

Association of chromatin proteins high mobility group box (HMGB) 1 and HMGB2 with mitotic chromosomes

High mobility group box (HMGB) 1 and 2 are two abundant nonhistone nuclear proteins that have been found in association with chromatin. Previous studies based on immunofluorescence analysis indicated that HMGB1 dissociates from chromosomes during mitosis. In the present work, HMGB1 and 2 subcellular localization was reinvestigated in living cells by using enhanced green fluorescent protein- and Discosome sp. red fluorescent protein-tagged proteins. Contrary to previous reports, HMGB1 and 2 were shown to be present under two forms in mitotic cells, i.e., free and associated with the condensed chromatin, which rapidly exchange. A detailed analysis of HMGB2 interaction with mitotic chromosomes indicated that two sites encompassing HMG-box A and B are responsible for binding. Importantly, this interaction was rapidly inactivated when cells were permeabilized or exposed to chemical fixatives that are widely used in immunodetection techniques. A comparable behavior was also observed for two proteins of the HMG-nucleosome binding (HMGN) group, namely, HMGN1 and HMGN2.     

The plant-specific family of DNA-binding proteins containing three HMG-box domains interacts with mitotic and meiotic chromosomes

? The high mobility group (HMG)-box represents a DNA-binding domain that is found in various eukaryotic DNA-interacting proteins. Proteins that contain three copies of the HMG-box domain, termed 3 × HMG-box proteins, appear to be specific to plants. The Arabidopsis genome encodes two 3 × HMG-box proteins that were studied here. ? DNA interactions were examined using electrophoretic mobility shift assays, whereas expression, subcellular localization and chromosome association were mainly analysed by different types of fluorescence microscopy. ? The 3 × HMG-box proteins bind structure specifically to DNA, display DNA bending activity and, in addition to the three HMG-box domains, the basic N-terminal domain contributes to DNA binding. The expression of the two Arabidopsis genes encoding 3 × HMG-box proteins is linked to cell proliferation. In synchronized cells, expression is cell cycle dependent and peaks in cells undergoing mitosis. 3 × HMG-box proteins are excluded from the nuclei of interphase cells and localize to the cytosol, but, during mitosis, they associate with condensed chromosomes. The 3 × HMG-box2 protein generally associates with mitotic chromosomes, while 3 × HMG-box1 is detected specifically at 45S rDNA loci. ? In addition to mitotic chromosomes the 3 × HMG-box proteins associate with meiotic chromosomes, suggesting that they are involved in a general process of chromosome function related to cell division, such as chromosome condensation and/or segregation.     

多聚甲醛固定对荧光蛋白分子间FRET效率的影响

目的：研究多聚甲醛固定对利用荧光共振能量转移(fluorescence resonance energy transfer, FRET)检测细胞中蛋白质相互作用的影响,解决运动能力较强的细胞中FRET效率检测的问题。方法：选用两个已知能够相互作用的蛋白分子TRA和TRB,将荧光蛋白ECFP和EYFP的编码基因通过融合PCR分别标记在其C端;将两个融合基因共转染靶细胞,一组细胞经低浓度（0.5%）多聚甲醛短时（0.5~1h）固定,另一组不固定,利用激光共聚焦扫描显微镜检测两个融合蛋白之间的FRET效率,比较其在两组细胞之间的差异情况。结果：经过统计学分析,在活细胞和经低浓度多聚甲醛短时间固定的细胞中,ECFP与EYFP之间的FRET效率没有显著差异。结论：低浓度短时间的多聚甲醛固定对于荧光蛋白分子之间的相互作用没有显著的影响,因此对于运动能力过强的细胞可以固定后再进行FRET检测。     

多头绒泡菌间期细胞核和中期染色体中的银染蛋白分析*

电镜原位观察结合图象分析研究了多头绒泡菌Physarum polycephalum Schw间期细胞核和中期染色体中银染蛋白的形状、大小和分布。结果看到,银染蛋白主要呈颗粒状存在于间期细胞核和中期染色体中。银粒的大小不一,分布不均匀。间期细胞核中存在众多直径在5~15nm的银粒,其中10nm以上的较大银粒主要分布于核仁,集缩染色质和核基质部分10nm以上银粒不多。中期细胞核内10nm以上的较大银粒主要分布于染色体中。染色体中除含有一些较大银粒外,多数银粒的直径为5~10nm。本文结果提示,构成染色体骨架的嗜银蛋白可能来自间期细胞核的染色质、核基质和核仁。