MnSi nanostructures obtained from epitaxially grown thin films: magnetotransport and Hall effect.

We present a comparative study of the (magneto)transport properties, including Hall effect, of bulk, epitaxially grown thin film and nanostructured MnSi. In order to set our results in relation to published data we extensively characterize our materials, this way establishing a comparatively good sample quality. Our analysis reveals that in particular for thin film and nanostructured material, there are extrinsic and intrinsic contributions to the electronic transport properties, which by modeling the data we separate out. Finally, we discuss our Hall effect data of nanostructured MnSi under consideration of the extrinsic contributions and with respect to the question of the detection of a topological Hall effect in a skyrmionic lattice.

Self-electroporation as a model for fusion pore formation.

The creation of a small opening called the fusion pore is a necessary prerequisite for neurotransmitter release from synaptic vesicles. It is known that high intensity electric fields can create pores in vesicles by a process called electroporation. Due to the presence of charged phosphatidylserine (PS) molecules on the inner leaflet of the cell membrane, an electric field that is strong enough to cause electroporation of a synaptic vesicle might be present. It was shown by K. Rosenheck [K. Rosenheck. Biophys J 75, 1237-1243 (1998)] that in a planar geometry, fields sufficient to cause electroporation can occur at intermembrane separations of less than approximately 3 nm. It is frequently found, however, that the cell membrane is not planar but caves inward at the locations where a vesicle is close to it. Indentation of the cell membrane in the fusion region was modelled as a hemisphere and a theoretical study of the electric field in the vicinity of the cell membrane taking into account the screening effect of dissolved ions in the cytoplasm was performed. It was discovered that fields crossing the electroporation threshold occurred at a distance of 2 nm or less, supporting the claim that electroporation could be a possible mechanism for fusion pore formation.

Cdc2-independent induction of premature mitosis by okadaic acid in HeLa cells.

The induction of premature mitosis by okadaic acid (OA) in HeLa cells in S-phase or in G2-phase has been studied using light microscopy, immunofluorescence, and immunochemical techniques. The observations indicate an involvement of a cdc2-independent pathway in these cells. It has been claimed that inhibition of an OA-sensitive phosphatase, possibly of PP1, induces activation of a kinase which is sensitive to staurosporine and Zn2+. This kinase brings about mitosis-specific cytoskeletal rearrangements, chromosome condensation, and nuclear envelope breakdown, inducing a mitosis-like state. However, other mitotic events do not follow. The possibility that this kinase may be a NIMA-like Nek2 kinase is discussed.

Induction of premature mitosis in cells blocked in S-phase: possibilities of its use in cancer therapy.

Oncogenesis leads to weak regulations of many cell cycle checkpoints. This could be useful for cancer therapy. To test this assumption, five human malignant cell lines were used to induce premature mitosis by overriding the S-phase control, which ultimately leads to cell death. Two cell lines showed a significantly high frequency of premature mitotic cells induced by okadaic acid; whereas the others did not. One cell line, although having a high cyclin B level in cells blocked in S-phase, failed to show premature mitosis. The cyclin B level in cells blocked in S-phase alone could not explain the differential behavior of the cell lines. It was observed that in addition, the activation of cdc25 in these cells could also play an important role in the induction of premature mitosis in such cells.

Effects of low concentrations of okadaic acid in HeLa cells.

HeLa cells treated for prolonged period with okadaic acid (OA; 5-10nM) inhibiting protein phosphatase 2A (PP2A) and also protein phosphatase 1 (PP1) partially showed prolonged effects on mitotic progression. In the presence of OA cells progressed normally in mitosis almost upto 4 hr, then a progressive accumulation of mitotic cells could be noticed. Most of the mitotic cells seemed to be arrested at the metaphase-anaphase transition point. In arrested mitotic cells the chromosomes remained arranged at the equiatorial plate, but with prolonged treatment the chromosomes got either scattered or clumped. However, a slow release into anaphase could also be observed after 15 hr treatment. Immunofluorescence studies for microtubules and electron microscope investigations indicated the dearrangement of spindle fibres, and a prolonged treatment led to the formation of multipolarity. This was also confirmed by spread preparations of chromosomes and the formation of multinucleate cells in preparations released from the mitotic block. Chromosomes became highly condensed showing mostly nondisjunction, but separation of sister chromatids could be observed in many cells. Immunoblot assays indicated a degradation of cyclin A, but the cyclin B1 level was significantly higher in the arrested mitotic cells after 12 hr treatment. After 24 hr of treatment the cyclin B1 level was slightly lower in arrested cells. Possible roles of protein phosphatase 2A inhibition and a prolonged partial inhibition of PP1 on the mitotic progression and the cyclin degradation at the metaphase-anaphase transition have been discussed.

Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome.

HeLa cells growing in vitro were treated with the peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the proteasome N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). Immunofluorescence studies of treated cells revealed the formation of massive perinuclear aggregates rich in ubiquitin and proteasomal antigens, which on the ultrastructural level appeared as perinuclear aggregates of electron-dense material, usually in the vicinity of Golgi cisternae. Histochemical studies disclosed that these cells contained protein-rich perinuclear aggregates detected by amido black staining, while unusual accumulations of lipids, carbohydrates, or nucleic acids were not present. Inhibition of protein synthesis by cycloheximide prevented the formation of aggregates, whereas microtubule disruption by nocodazole induced a dispersion of the aggregates. We hypothesize that aggregates induced by PSI treatment correspond to accumulations of proteasome-substrate complexes in a well-defined region, where the proteolytic processes of the ubiquitin-proteasome pathway seem to be somehow centered. We propose to call this region the proteolysis center.

Are proteasomes involved in the formation of the kinetochore?

Proteasomes catalyse the degradation of proteins responsible for the regulation of mitosis enabling the cell to complete cell division. We have studied the effect of an inhibitor of the chymotrypsin-like activity of the proteasome on the trilaminar structure of the kinetochore in HeLa cells. Whereas a role for the proteasome in the degeneration of the kinetochore was predicted, we found instead that the inhibitor strongly regarded kinetochore development. We observed different 'developmental' stages of the kinetochore from the fibrous ball of a 'prekinetochore' to the 'mature' kinetochore in one cell. The data presented here support the proposition that proteasomes are involved in kinetochore formation.

Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.

Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control.

Okadaic acid overrides the S-phase check point and accelerates progression of G2-phase to induce premature mitosis in HeLa cells.

Okadaic acid has been shown to induce premature mitosis in HeLa cells belonging to both S-phase and G2-phase. The response is dependent on concentration and perhaps also on the differential susceptibility of cells to okadaic acid (OA). OA resembles caffeine in its ability to override the DNA-replication check point but differs in being capable of inducing premature mitosis in cells in G2-phase, thereby accelerating mitosis. Chromosomes from mitotic cells induced in G2-phase show a typical banding pattern similar to G-banding. From immunoblot experiments and survey of the relevant literature it has been argued that OA may modulate both a tyrosine kinase and a tyrosine phosphatase activity to override the S-phase checkpoint and accelerate the activation of MPF in cells in G2-phase, respectively.

An inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces arrest in G2-phase and metaphase in HeLa cells.

HeLa cells were treated with different concentrations of an inhibitor of the proteasome chymotrypsin-like activity, the peptidyl aldehyde N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). A detailed analysis, which included flow cytometry, cell counting and morphological assessment, was performed. PSI treatment induces a significant reduction of mitotic activity, accompanied by metaphase arrest of the mitotic cells. DNA flow cytometry shows an accumulation of the cells in G2+M phases of the cell cycle, which indicates the existence of a proteasome-mediated step in the G2-phase of the cell cycle. After removal of the inhibitor and supplementation with fresh medium, the cell cycle is resumed, but the mitotic cells show increased misalignment of chromosomes in the metaphase plate. PSI also induces HeLa cells to acquire a fibroblastoid phenotype.

Localization of proteasomal antigens during different phases of the cell cycle in HeLa cells.

We localized two different proteasome-associated epitopes with the use of two specific monoclonal antibodies, MCP21 and anti-p25, in interphase and mitotic HeLa cells cultured in vitro. We provide evidence for an association of those antigens with the spindle poles in metaphase and with the midbody region in anaphase and telophase. Triton-nonextractable structures are also labeled in interphase. Moreover, the labeling pattern of interphase cells obtained with the monoclonal antibodies MCP21 and anti-p25 differs considerably, and in the case of MCP21 displays clear cell cycle-specific changes: nuclear labeling is absent or very weak in G1 and S phases of the cell cycle, whereas it is strong in G2 phase. This might reflect the existence of different subpopulations of proteasomes in the cell.

Immunoelectron microscopic studies on centromere-kinetochore complexes detached from chromosomes.

The centromere-kinetochore complexes of Chinese hamster ovary (CHO) cells were detached and separated from the condensed chromatin by treatment with hydroxyurea and caffeine. By labelling the complex for immunoelectron microscopy (immuno-EM) with a mixture of antibodies against centromere proteins (anti-CENP-A, -B, -C) in some cells, we could demonstrate complete detachment of the complexes. No remnants were left at the bulk of condensed chromatin in these cells. In some mitotic cells complex and chromatin were found side by side. It could be shown that the fine structure of the separated material of the complex differs significantly from that of the rest of chromatin. The complex consists of proteins and DNA. This leads us to suppose that the organization of chromatin in the centromere-kinetochore complex is different.

A critical appraisal of synchronization methods applied to achieve maximal enrichment of HeLa cells in specific cell cycle phases.

Synchronization of mammalian cell cultures is a prerequisite for studies of molecular mechanisms of cell cycle control. Many researchers routinely use widely spread tumor cell lines like HeLa for these purposes, and a great variety of synchronization protocols has been described. Generally, they have been developed for monolayer cultures, usually with satisfactory results. However, we found that is not necessarily the case for cells cultivated in suspension. A critical appraisal of different standardized methods for selective enrichment of HeLa cells in suspension in all phases of the cell cycle has been undertaken. Our results reveal that only a few of the applied procedures can really yield high numbers of synchronized cells in G1, S, G2, and M phases, working with suspension cultures.

Comparative analysis of the polypeptide pattern of cumulus cells during maturation of porcine cumulus oocyte complexes in vivo and in vitro.

The protein patterns of porcine cumuli oophori matured as intact cumulus oocyte complexes either in vivo or in vitro with or without FSH and LH for 46 h were investigated. In in vivo-matured cumuli oophori, a 53 kDa band disappeared after 24 h maturation, but reappeared at 46 h. Furthermore, the production of a polypeptide with a relative molecular mass of 44,000 ceased and the appearance of 2 other proteins with relative molecular masses of 38,000 and 28,000 was observed. In cumuli oophori matured in vitro with or without addition of FSH and LH the 53 kDa band ceased after a culture period of 12 h. This band was produced again after a culture period of 46 h. In contrast, the polypeptide with the relative molecular mass of 44,000 ceased only in cumuli oophori cultures supplemented with FSH and LH, and the 2 proteins of M(r) 38,000 and 28,000 were detected only in the protein profiles of mature cumuli cultured with FSH and LH. It is concluded that the addition of FSH and LH to the culture medium is necessary for cumuli oophori to synthesize a protein pattern, which corresponds closely to that produced by cumuli oophori matured in vivo.

Making first contacts between the spindle and the chromosomes in HeLa cells.

To guarantee an ordered bipartition of the genetic material during mitosis, the chromosomes must be incorporated into the mitotic spindle. In HeLa cells, this process starts early in prophase when the nuclear envelope is still nearly complete, but only a few small holes in the double membranes offer access to the chromosomes for individual microtubules growing out from the poles. Inside the nuclear domain, these microtubules make contact with the kinetochore/centromere complexes which can be found in the vicinity of the hole. These complexes seem to be distributed at random during early prophase until early prometaphase. Therefore, the chromosomes become incorporated in a sequential order. No accumulation of the complexes in the nucleus near the centrosomes can be recognized. The individual microtubules attach tangentially to the kinetochores. This contact can already take place before the kinetochore is fully developed.

Failure of sister chromatid separation leads to formation of diplochromosomes in colcemid treated PtK1 cells.

The origin of diplochromosomes has been traced in multinucleate rat kangaroo cells (PtK1) obtained after colcemid treatment. In these cells the diplochromosomes were shown to originate from restitution nuclei, indicating that they were formed due to the omission or failure of sister chromatid separation and not due to endoreduplication. In this context the mechanism of sister chromatid separation has been discussed. The independence of this mitotic event from other associated processes, such as chromosome condensation, nuclear envelope breakdown or spindle formation has been stressed.

Failure of centromere separation leads to formation of diplochromosomes in next mitosis in okadaic acid treated HeLa cells.

High concentrations of okadaic acid, sufficient to inhibit phosphatase 1 and 2A activities, induces formation of diplochromosomes in HeLa cells. It has been shown that this is due to a failure of sister chromatid separation in earlier mitosis in the presence of okadaic acid in the medium and not due to bypassing of mitosis (endoreduplication). Moreover, it has been demonstrated that the sister chromatid adherence does not depend on any under-replicated chromatin segment shared by the sister chromatids which might happen in okadaic acid induced premature mitosis, but due to the failure of the centromeres to separate at metaphase-anaphase transition. The role of phosphatase 1 in sister chromatid separation has been discussed.

Formation of dicentrics with variable lengths in transformed cells.

Several dicentric chromosomes in transformed cells exhibit drastic variations in the length of the region between the two centromeres. In these dicentric chromosomes, the position of the centromeres from the proximal end remains unaltered. The increase or decrease in the length of the intercentromeric segment is attributed to interchromatid reorganization so that one chromatid gains a piece of the sister chromatid. This is achieved simply through two chromatid breaks in the interchromosomal loop, followed by nonreciprocal rejoining. These observations provide new credence to Revell's hypothesis.

Effects of okadaic acid on mitotic HeLa cells.

Mitotic HeLa cells were treated with different concentrations of okadaic acid (OA), known to inhibit phosphatase 1 and 2A activities. The cytological effects on the course of mitosis were studied at the light microscopic, immunoflourescence and electron microscopic levels. At the lowest concentration used (1 nM), OA did not show any effect on mitosis, but at higher concentrations it showed pronounced effects. The mitotic chromosomes became scattered, the mitotic spindle became deranged and the cells failed to enter anaphase. At the electron microscopic level formation of isolated microtubules and regular trilaminar kinetochores were observed. An extensive growth of the endoplasmic reticulum could be noted in these cells. Decondensation of chromatin and nuclear envelope re-formation could be seen only after withdrawal of OA. A high frequency of multinucleate cells could be found after 24 h of recovery. Cells treated with 100 nM OA for 3 hours showed diplochromosomes in over 50% of mitotic cells after 24 h recovery. These were presumably formed due to the failure of sister chromatid separation in the earlier mitosis in the presence of OA. At the electron microscopic level the diplochromosomes showed a quadruplet structure. The role of phosphatase 1 in controlling some late mitotic events, i.e. sister chromatid separation, MPF-inactivation and nuclear envelope re-formation etc., is discussed.

Failure of kinetochore development and mitotic spindle formation in okadaic acid-induced premature mitosis in HeLa cells.

The mitotic events associated with okadaic acid (OA)-induced premature chromosome condensation (PCC) in S-phase-blocked HeLa cells were studied at the light microscope, immunofluorescence, and electron microscope level. The development of PCC in these cells has been compared with that in multinucleate cells and also in uninucleate hamster cells induced by caffeine. In OA-induced PCC, the nuclear envelope breaks down and chromosomes condense, but the mitotic spindle and trilaminar kinetochores fail to develop. In S-phase PCC in multinucleate cells, only the mitotic spindle does not develop, whereas in caffeine-induced PCC, all these events are found to be associated. The possible difference in their pathways of induction and, in this connection, the dissociability of the early mitotic events have been discussed.