Biomedical subjects
Publications and source records attributed to D Mazia.
Aortic arch injections centred on the head have been performed routinely in patients with cerebral ischaemia. Digital angiograms with modified windowing (low and narrow) have been used. This "cerebral" arch injection allows much improved analysis of the cerebral parenchymal vascularization, giving better understanding of hemispheric ischaemia and making the decision about revascularization more rational.
T-1 induces modifications in the shape of the centrosome at division in fertilized eggs of the North American sea urchin, Lytechinus pictus. Phase contrast microscopy observations of mitotic apparatus isolated from T-1-treated (1.7-8.5 microM) eggs at first division shows that the centrosomes already begin to spread or to separate by prophase and that the mitotic spindle is barrel-shaped. When eggs are fertilized with sperm that have been preteated with T-1, the centrosomes become flattened; the spindles are of normal length. Immunofluorescence microscopy using an anti-centrosomal monoclonal antibody reveals that T-1 modifies the structure of the centrosome so that barrel-shaped spindles with broad centrosomes are observed at metaphase, rather than the expected focused poles and fusiform spindle. Higher concentrations of T-1 induce fragmentation of centrosomes, causing abnormal accumulation of microtubules in polar regions. These results indicate that T-1 directly alters centrosomal configuration from a compact structure to a flattened or a spread structure. T-1 can be classified as a new category of mitotic drugs that may prove valuable in dissecting the molecular nature of centrosomes.
The mitotic apparatus of sea urchin embryos was isolated using a polyethylene glycol (PEG)/EGTA-medium. Such a procedure preserves the birefringence and the Ca2+ lability of the isolated mitotic apparatus. The method of isolation gives good preservation of the microtubules and of the intracellular Ca2+-transport system as visualized by a monoclonal antibody to a 46-kDa protein. Triple fluorescence studies allow a comparison of the relative locations of microtubules, Ca2+-sequestering membranes and chromatin (by Hoechst 33342) in the mitotic apparatus. We find that the Ca2+-sequestering membranes are concentrated mainly in the centers of the asters and do not follow the distribution of microtubules in the mitotic apparatus. Regulation of microtubules by Ca2+ may not depend on immediate contiguity of microtubules and the Ca2+-regulating sites.
Explore the source record for details and available documents.
A mouse monoclonal antibody generated against Drosophila intermediate filament proteins (designated Ah6/5/9 and referred to herein as Ah6) is found to cross-react specifically with centrosomes in sea urchin eggs and with a 68-kDa antigen in eggs and isolated mitotic apparatus. When preparations stained with Ah6 are counterstained with a human autoimmune serum whose anti-centrosome activity has been established, the immunofluorescence images superimpose exactly. A more severe test of the specificity of the antibody demands that it display all of the stages of the centrosome cycle in the cell cycle: the flattening and spreading of the compact centrosomes followed by their division and the establishment of two compact poles. The test was made by an experimental design that uses a period of exposure of the eggs to 2-mercaptoethanol. This treatment allows observation of the stages of the centrosome cycle--separation, division, and bipolarization--while the chromosomes are arrested in metaphase. Mitosis is arrested in the presence of 0.1 M 2-mercaptoethanol. Chromosomes remain in a metaphase configuration while the centrosomes divide, producing four poles perpendicular to the original spindle axis. Microtubules are still present in the mitotic apparatus, as indicated by immunofluorescence and transmission electron microscopy. When 2-mercaptoethanol is removed, the chromosomes reorient to the poles of a tetrapolar (sometimes tripolar) mitotic apparatus. During the following cycle, the blastomeres form a monopolar mitotic apparatus. The observations of the centrosome cycle with the Ah6 antibody display very clearly all the stages that have been seen or deduced from work with other probes. The 68-kDa antigen that reacts with the Ah6 monoclonal antibody to Drosophila intermediate filament proteins must be a constant component of sea urchin centrosomes because it is present at all stages of the centrosome cycle.
During fertilization the sperm brings two centrosomes into the egg. One centrosome contains a centriole of normal length originally seen as the basal body of the sperm flagellum. Characteristically, the proximal half is enwrapped in osmiophilic material. This centrosome is attached to the centrosomal fossa, a bowl-shaped depression of the nuclear envelope of the male pronucleus. Microtubules radiate out from the osmiophilic half characterizing this structure as a centrosome and microtubule organizing center (MTOC). The second centrosome which also acts as an MTOC is attached to the mitochondrion of the sperm. At the beginning it appears as an unstructured accumulation of osmiophilic material out of which later on centriolar microtubules grow. Though this centrosome is marked by an immature centriole it is capable of organizing microtubules and of reproducing itself. This centrosome becomes loosely associated with the female pronucleus by means of microtubules. Then it separates from the mitochondrion which finally is lost. When the two pronuclei fuse, the centrosome derived from the basal body remains firmly attached to the centrosomal fossa, which has persisted in the envelope of the zygote nucleus after pronuclear fusion. Using the fossa as a marker of the position of this centrosome on the nuclear surface, we conclude that it is a stationary centrosome in the process of bipolarization for the first mitosis.
After fusion of the two pronuclei the former sperm head centrosome is attached to the envelope of the zygote nucleus while the former mitochondrial centrosome is only loosely associated with it. These two centrosomes are not yet in opposite positions but are separated from each other by spreading centrosomal material. This spreading is mediated by microtubules. It is concluded that the attached centrosome remains stationary while the motile one is moved around the nuclear surface to an antipodal position, 180 degrees from the other. The first bipolarization process which occurs prior to the breakdown of the nuclear envelope is compared to the second and all other bipolarizations: Similarities and dissimilarities can be found, but similar or identical mechanisms for both processes are assumed.
The forms and locations of centrosomes in mouse oocytes and in sea urchin eggs were followed through the whole course of fertilization and first cleavage by immunofluorescence microscopy. Centrosomes were identified with an autoimmune antiserum to centrosomal material. Staining of the same preparations with tubulin antibody and with the DNA dye Hoechst 33258 allowed the correlation of the forms of the centrosomes with the microtubule structures that they generate and with the stages of meiosis, syngamy, and mitosis. The results with sea urchin eggs conform to Boveri's view on the paternal origin of the functional centrosomes. Centrosomes are seen in spermatozoa and enter the egg at fertilization. Initially, the centrosomes are compact, but as the eggs enter the mitotic cycle the forms of the centrosomes go through a cycle in which they spread during interphase, apparently divide, and condense into two compact poles by metaphase. In anaphase, they spread to form flat poles. In telophase and during reconstitution of the daughter nuclei, the centrosomal material is disposed as hemispherical caps around the poleward surfaces of the nuclei. Mouse sperm lack centrosomal antigen. In the unfertilized mouse oocyte, the meiotic spindle poles are displayed as broad-beaded centrosomes. In addition, centrosomal material is detected in the cytoplasm as particles, about 16 in number, which are foci of small aster-like arrays of microtubules. The length and number of astral microtubules correlate with the size of the centrosomal foci. After sperm incorporation, as the pronuclei develop and more cytoplasmic microtubules assemble, a few of the foci associate with the peripheries of the nuclei. The number of foci multiplies during the first cell cycle. At the end of interphase, all of the centrosomal foci have concentrated on the nuclear peripheries and the cytoplasmic microtubules have disappeared. At prophase, the centrosomes are seen as two irregular clusters, marking the poles which, at metaphase and anaphase, appear as rough bands with foci, and the spindle is typically barrel-shaped. At telophase, the centrosomes are seen as arcs that lie on the nuclear peripheries after cleavage. The ordering of microtubules in all the stages reflects the shapes of the centrosomes. The findings on the sea urchin confirm the classical theory of the paternal origin of centrosomes and contrast with observations tracing the mitotic poles of the mouse egg to maternal centrosomal material. This evidence strengthens the conclusion that mouse centrosomes derive from the oocyte.
When sea urchin eggs entering mitosis are exposed to an appropriate concentration of mercaptoethanol, the chromosome cycle is restrained while the centrosome cycle advances. The two poles of the mitotic apparatus separate into four poles, while the chromosomes remain in their metaphase arrangements until released by the removal of the mercaptoethanol. We follow the centrosomes through the stages of the generation of two poles by each original pole. In electron microscopic studies, the osmiophilic component of the centrosomes serves as an indicator of their changing forms as each pole generates two poles. In light microscopic studies, including observations of birefringence, the shapes of the polar ends of the spindles are taken as indicators of the shapes of the centrosomes. The successive stages of the centrosome cycle are (1) compact spherical centrosomes at the time of formation of the mitotic apparatus; (2) expansion and flattening of the centrosomes, leading to (3) formation of thin flat plates, perpendicular to the spindle axis. Corresponding to the extended flat shape of the centrosomes, the spindle poles are flat; microtubules 'point' to the centrosomal plate and not the centrioles. The centrioles are separated in the flattening of the centrosomes. (4) The flat plate divides into two and each of the two halves becomes more compact, defining two separate poles. Our findings resurrect and update Boveri's [5] observations and interpretations of the centrosome. Centrosomes have shapes. The shapes may be imparted to the microtubular structures that they generate. The formation of two separate centrosomes from one, in the formation of mitotic poles, is describable as a sequence of changes in shape.
The original theory of the centrosome as the 'reproductive organ' of the cell provides a logical explanation of the mitotic poles and the accuracy of cell division. No alternative explanation has replaced it. The historical problem was the failure to identify centrosomes as compact physical bodies in a great many kinds of cells. In this essay, I consider the evidence that centrosomes are flexible bodies; they may take on alternative forms and their forms determine the shapes of mitotic poles and other organizers of microtubular structures. Compact corpuscular centrosomes are not necessary and would not be expected in cases where microtubules clearly do not originate from point sources. A model of the flexible centrosome is introduced and the speculation that the centrosome is a bearer of morphological information is considered.
Griseofulvin (4-6 X10-5 and 1 X 10-4 M) prevents the formation of any microtubule-based structures of sea urchin (Strongylocentrotus purpuratus, Lytechinus variegatus, Arbacia punctulata) eggs at fertilization. Sperm incorporation occurs, though the migrations of the pronuclei, dependent on the formation of the sperm aster, are arrested. Similarly in "streak" and the mitotic apparatus fail to assemble. Cycles of DNA synthesis, chromosome activity, nuclear breakdown and reconstitution, and even cleavage attempts occur on schedule in the absence of any mitotic movements. The action of griseofulvin, unlike that of colchicine, is readily reversible by the removal of the drug. Microtubules are formed, and the chromosome are separated. At 1 X 10-6 M, diminutive microtubule-based structures (e.g. sperm aster, mitotic apparatus) are observed though syngamy and division are arrested. These results demonstrate an independence of the cycle of microtubule-mediated events from other cyclical processes during the first cell cycles.
Unfertilized sea urchin eggs were parthenogenetically activated via a prolonged hypertonic treatment. The continuous subjection to the osmotic stress turned on the cell cycle and brought about the very slow development of mature centrioles. Within 1 h of the exposure, large osmiophilic aggregates were detected at the nuclear surface and were interpreted as early centriolar precursor forms. Ultrastructural examination of eggs during the 8 h of treatment revealed that the precursor forms systematically converted into mature centrioles which then produced daughter centrioles. All centriolar precursor bodies were associated with microtubules; cartwheel structures were the earliest morphological feature of centrioles detected within the osmiophilic bodies. Nascent centrioles appeared at the nuclear surface and, during their development into centrioles, continually maintained intimate associations with the nuclear envelope. We suggest that the resultant modification of the intracellular environment via the hypertonic stress promotes the activation of centriolar 'information-bearing-residues' or 'seeds' located at the nuclear envelope. The oocyte centrioles, before disappearing from the maturing egg, may have produced these 'seeds' and left them at the nuclear surface.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Monopolar mitotic apparatus can be produced in sea urchin eggs by a manoeuvre that distributes the four poles of the second mitosis into four separate blastomeres. The pole of the monopolar mitotic apparatus generates a half-spindle that is similar in structural details to the half-spindle of a normal bipolar mitotic apparatus, although the chromosomes are not as well aligned as in a normal metaphase plate. The chromosomes are oriented; one kinetochore faces the pole while its sister kinetochore faces away from the pole. The poleward kinetochore is connected to the pole by bundles of microtubules. No microtubules are seen on the sister kinetochore that faces away from the pole. Therefore, a single pole can direct most of the events in the establishment of a mitotic apparatus. Our interpretation examines the cooperation of kinetochores and poles in the formation of microtubules between them, stressing the half-spindle as the medium of cooperation and leaving open the question whether the kinetochores are origins or terminations of microtubules.
Explore the source record for details and available documents.
Unfertilized sea urchin eggs enter a mitotic chromosome cycle after treatment with sea water containing ammonia. Centrioles cannot be found but microtubules are formed in the later stages of the cycle. The microtubules are displayed in an astral arrangement centered on clusters of osmiophilic bodies. In early stages, distinct kinetochores on the condensed chromosomes show no attachments to microtubules. Later, a few microtubules may be attached to the kinetochores. The chromosomes and microtubules are contained in a "clear zone", a large compact accumulation of membranes which displaces yolk particles and mitochondria, but not ribosomes, from that region of the cell. No bipolar spindle is formed.
Explore the source record for details and available documents.