Dieter Schroeter

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.

Sequence of centromere separation: kinetochore formation and DNA replication in dicentric chromosomes showing premature centromere separation in rat cerebral cells.

A subpopulation of rat cerebral endothelial cells, designated B1, exhibits an array of multicentric chromosomes. Because of the formation of bridges at anaphase, this cell population produced new types of multicentrics at every cell division. These chromosomes showed kinetochore proteins at every centromeric site and all centromeric regions replicated their DNA at the end of the S phase, more or less simultaneously. A new subpopulation of cells, designated B2, obtained from the original sample frozen at Wayne State University displayed several dicentrics. In contrast to B1 these chromosomes exhibit premature centromere separation as reported for mouse and human cell lines. These B2 dicentrics show only one site of kinetochore protein deposition. The timing of DNA replication around the centric region of prematurely separating centromere is also changed similar to the earlier reported premature DNA synthesis for mouse dicentrics. These observations suggest a universality of relationship between premature centromere separation, a lack of kinetochore formation, and early replication of the centric/pericentric DNA associated with these centromeres. The cause of sudden change from activity to inactivity of these chromosomes, though interesting, is not clear.

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 phophatase 1 in sister chromatid separation has been discussed

Pig sperm tail tubulin: Its extraction and characterization

Axonemal tubulin extracted from pig sperm tails has been characterized by one- and two-dimensional electrophoresis and by one-dimensional peptide mapping. The electrophoretic mobilities of its subunits after reduction and carboxymethylation were similar to those of the major subunits of pig brain tubulin. Sperm tail tubulin subunits also had roughly the same isoelectric points as pig brain tubulin subunits, except that they appeared to have a relatively larger tailing effect. The proteolytic cleavage pattern of the pig sperm tail beta-tubulin closely resembled those of both the tunicate (Ciona intestinalis) sperm beta-tubulin and pig brain beta-tubulin. The peptide pattern of pig sperm tail alpha-tubulin, however, was more similar to that of tunicate sperm tail alpha-tubulin than to that of pig brain alpha-tubulin. This supports the hypothesis put forward in a previous investigation [1] that functionally similar tubulins from taxonomically distant species can be more related than functionally dissimilar tubulins from the same species.

Development of Kinetochores During Early Mitosis in HeLa Cells and the Stability of a Trilaminar Kinetochore Structure

The ultrastructure of the kinetochore varies during the course of mitosis. This variation is considered to be a progressive maturation process that may correlate with kinetochore function. We examined the development of kinetochore structures on condensing prophase chromosomes in HeLa cells. Usually, one accepts the three laminar structure of the kinetochore of a condensed metaphase chromosome to reflect the general state and to be a prerequisite for microtubule attachment. The study of developing kinetochores in mitotic cells revealed unquestionable exceptions from this dogma.

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 retarded 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.

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.