上皮细胞分裂过程中中等纤维的变化

Immunofluorescence microscopy was used to follow the rearrangement of keratin filaments and vimentin filaments during mitosis in Vero and HeLa cell lines. The experiment results showed that the three dimensional organization and structure of intermediate filaments changed drastically during mitosis. The behavior of intermediate filaments was different in these two epithelial cell lines. In mitotic Vero cells the keratin filaments and vimentin filaments maintained their filamentous structure and formed a cage around the mitotic apparatus. In mitotic HeLa cells the keratin filaments and vimentin filaments reorganized extensively and formed granular cytoplasmic bodies. The ratio of granular cytoplasmic body formation changed in different mitotic phase. The interphase intermediate filament network was reconstructed after mitosis. It is proposed that the state of intermediate filament network in these cells is cell cycle-dependent and intermediate filaments may have some skeletal role in mitosis.     

上皮细胞分裂过程中中等纤维的变化

应用制备的血清抗体,采用免疫细胞化学方法观察了两株培养上皮细胞的分裂过程中IF的动态变化过程。实验结果显示,在上皮细胞分裂过程中,IF形态结构及空间分布发生了显著变化,不同细胞之间存在差异,分裂的Vero细胞中角蛋白纤维和波形纤维都维持纤维形态,围绕分裂器形成纤维网罩或纤维束环,随着细胞分裂的进行,IF网的空间组织结构和外观发生动态变化;分裂的HeLa细胞中,角蛋白纤维和波形纤维广泛重组形成颗粒状胞质小体,分裂结束后重建IF网。实验结果表明,IF变化具有细胞周期依赖性和一定的细胞特异性。本文对IF在细胞分裂过程中的功能意义作了讨论。     

The regulation of intermediate filament reorganization in mitosis. p34cdc2 phosphorylates vimentin at a unique N-terminal site

The disassembly of vimentin-containing intermediate filament (IF) networks during mitosis in BHK-21 cells is accompanied by increased phosphorylation of vimentin (Chou, Y.-H., Rosevear, E., and Goldman, R. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1885-1889). We have recently identified p34cdc2 as the catalytic subunit of one of the two endogenous vimentin kinases in mitotic baby hamster kidney cells (Chou, Y.-H., Bischoff, J. R., Beach, D., and Goldman, R. D. (1990) Cell 62, 1063-1071). To begin to characterize the biochemical basis of the p34cdc2-mediated IF disassembly process, we have purified and sequenced the 32P-labeled tryptic peptides derived from in vitro-phosphorylated vimentin. The results demonstrate that Ser-55, in the N-terminal non-alpha-helical domain of vimentin, is the most favored phosphorylation site. This finding supports the idea that the N-terminal domain of type III IF protein plays a crucial role in regulating IF structure and supramolecular organization.     

Pathway of incorporation of microinjected lamin A into the nuclear envelope

When microinjected into the cytoplasm of 3T3 cells, biotinylated human lamin A rapidly enters the nucleus and gradually becomes incorporated into the nuclear lamina region as determined by immunofluorescence. The incorporation of the microinjected material takes several hours and progresses through a series of morphologically identifiable stages. Within minutes after microinjection, lamin A is found in spots distributed throughout the nucleus, except in nucleolar regions. Over a time course of up to 6 h, these spots appear to decrease in size and number as the biotinylated lamin A becomes associated with the endogenous nuclear lamina. Eventually, the typical nuclear rim staining pattern normally revealed by immunofluorescence with nuclear lamin antibodies is seen with antibiotin. This latter rim staining property is passed on to daughter cells following mitosis. These results indicate that the microinjected biotinylated nuclear lamin A retains those properties required for its integration into the lamina, as well as those necessary for the disassembly and subsequent reassembly of the nuclear lamina during cell division. The initial rapid accumulation into foci and the subsequent slower incorporation into the nuclear lamina appear to be analogous to the stages of incorporation following the microinjection of cytoskeletal intermediate filament proteins such as vimentin and keratin (Vikstrom, K., G. G. Borisy, and R. D. Goldman. 1989. Proc. Natl. Acad. Sci. USA. 86:549-553; Miller, R. K., K. Vikstrom, and R. D. Goldman. 1991. J. Cell Biol. 113:843-855). Foci are also observed in some uninjected cells using nuclear lamin antibodies, indicating that these features are a genuine component of nuclear substructure. Evidence is presented that shows the appearance of these nuclear structures is cell cycle dependent.     

A 62-kD protein required for mitotic progression is associated with the mitotic apparatus during M-phase and with the nucleus during interphase

A protein of 62 kD is a substrate of a calcium/calmodulin-dependent protein kinase, and both proteins copurify with isolated mitotic apparatuses (Dinsmore, J. H., and R. D. Sloboda. 1988. Cell. 53:769-780). Phosphorylation of the 62-kD protein increases after fertilization; maximum incorporation of phosphate occurs during late metaphase and anaphase and correlates directly with microtubule disassembly as determined by in vitro experiments with isolated mitotic apparatuses. Because 62-kD protein phosphorylation occurs in a pattern similar to the accumulation of the mitotic cyclin proteins, experiments were performed to determine the relationship between cyclin and the 62-kD protein. Continuous labeling of marine embryos with [35S]methionine, as well as immunoblots of marine embryo proteins using specific antibodies, were used to identify both cyclin and the 62-kD protein. These results clearly demonstrate that the 62-kD protein is distinct from cyclin and, unlike cyclin, is a constant member of the cellular protein pool during the first two cell cycles in sea urchin and surf clam embryos. Similar results were obtained using immunofluorescence microscopy of intact eggs and embryos. In addition, immunogold electron microscopy reveals that the 62-kD protein associates with the microtubules of the mitotic apparatus in dividing cells. Interestingly, the protein changes its subcellular distribution with respect to microtubules during the cell cycle. Specifically, during mitosis the 62-kD protein associates with the mitotic apparatus; before nuclear envelope breakdown, however, the 62-kD protein is confined to the nucleus. After anaphase, the 62-kD protein returns to the nucleus, where it resides until nuclear envelope disassembly of the next cell cycle.     

The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes

Actin, keratin, vinculin and desmoplakin organization were studied in primary mouse keratinocytes before and during Ca2+-induced cell contact formation. Double-label fluorescence shows that in cells cultured in low Ca2+ medium, keratin-containing intermediate filament bundles (IFB) and desmoplakin-containing spots are both concentrated towards the cell center in a region bounded by a series of concentric microfilament bundles (MFB). Within 5-30 min after raising Ca2+ levels, a discontinuous actin/vinculin-rich, submembranous zone of fluorescence appears at cell-cell interfaces. This zone is usually associated with short, perpendicular MFB, which become wider and longer with time. Later, IFB and the desmoplakin spots are seen aligned along the perpendicular MFB as they become redistributed to cell-cell interfaces where desmosomes form. Ultrastructural analysis confirms that before the Ca2+ switch, IFB and desmosomal components are found predominantly within the perimeter defined by the outermost of the concentric MFB. Individual IF often splay out, becoming interwoven into these MFB in the region of cell-substrate contact. In the first 30 min after the Ca2+ switch, areas of submembranous dense material (identified as adherens junctions), which are associated with the perpendicular MFB, can be seen at newly formed cell-cell contact sites. By 1-2 h, IFB-desmosomal component complexes are aligned with the perpendicular MFB as the complexes become redistributed to cell-cell interfaces. Cytochalasin D treatment causes the redistribution of actin into numerous patches; keratin-containing IFB undergo a concomitant redistribution, forming foci that coincide with the actin-containing aggregates. These results are consistent with an IF-MF association before and during desmosome formation in the primary mouse epidermal keratinocyte culture system, and with the temporal and spatial coordination of desmosome and adherens junction formation.     

Microtubule rearrangements during mitosis in multinucleate cells

The peroxidase-antiperoxidase (PAP) method for the detection of polymerized tubulin has been used to study the microtubule rearrangements during mitosis in PtK1 and HeLa multinucleate cells obtained by polyethyleneglycol (PEG)-mediated fusion. We demonstrate here that the transition of the microtubular cytoskeleton from interphase to mitosis is an inducible event and independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. However, for the induction of the microtubule rearrangements nuclear envelope breakdown is required. At midprophase, cytoskeletal microtubule rearrangements start for multinucleate PtK1 cells, whereas in HeLa cells such changes are delayed, and a more abrupt transition is observed here. After complete nuclear envelope breakdown (prometaphase) mitotic asters and spindles but no cytoplasmic (interphase) microtubuli can be observed in both systems. Metaphase is characterized by an interaction between the different mitotic poles which show the form of bipolar spindles, but individual separated mitotic poles far removed from the chromatin can also be seen.     

Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.     

Interaction of antibodies against nuclear envelope-associated proteins from rat liver nuclei with rodent and human cells

Antibodies have been prepared against the three major polypeptides of the nuclear pore complex-lamina fraction from rat liver nuclei. The three antisera prepared in chickens give similar results in indirect immunofluorescence microscopy. In rat embryo fibroblasts we observe bright fluorescence at the level of the nuclear envelope, with no fluorescence of the nuclear interior and little or no fluorescence of the cytoplasm. The nuclear envelope regions of rat hepatoma cells, mouse A9 cells, HeLa cells and rat liver nuclei also fluoresce brightly. HeLa nucleoids, which are depleted of nuclear envelope components, still exhibit specific fluorescence when reacted with these antibodies. Distribution of the antigens changes during mitosis. Fluorescence in the cytoplasm is observed following the breakdown of the nuclear envelope at prometaphase. The antigens appear to progressively accumulate at the periphery of the chromosomes until telophase. In late telophase fluorescence occurs predominantly at the periphery of the chromosomes where the new nuclear envelope is formed.     

Fission yeast cdc31p is a component of the half-bridge and controls SPB duplication

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461-1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Deltacdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.