Lessons from yeast: the spindle pole body and the centrosome

The yeast spindle pole body (SPB) is the functional equivalent of the centrosome. Most SPB components have been identified and their functions partly established. This involved a large variety of techniques which are described here, and the potential use of some of these in the centrosome field is highlighted. In particular, very useful structural information on the SPB was obtained from a reconstituted complex, the γ-tubulin complex, and also from a sub-particle, SPB cores, prepared by extraction of an enriched SPB preparation. The labelling of SPB proteins with GFP at the N or C termini, using GFP tags inserted into the genome, gave informative electron microscopy localization and fluorescence resonance energy transfer data. Examples are given of more precise functional data obtained by removing domains from one SPB protein, Spc110p, without affecting its essential function. Finally, a structural model for SPB duplication is described and the differences between SPB and centrosome duplication discussed.     

Endpoint of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa characterized by acrosomal H+ and Ca2+ permeability: Population and single cell kinetics

The acrosome reaction induced by the mouse egg's zona pcllucida in mouse sperm has been shown to proceed in two stages as characterized empirically by sequential changes in patterns of chlorletracycline fluorescence on the sperm plasma membrane surfaces. The chlortctracy-cline fluorescence pattern characteristic of fully intact sperm is designated B:in sperm bound to structurally intact zonae that induce the acrosome reaction, the B pattern changes first to an intermediate pattern S and then to a terminal pattern AR characteristic of the completed acrosome reaction. In the same study, it was shown, using a 9-amino acridine fluorescent pH probe, that completion of the first stage was characterized by increase in H⁺ permeability such that the H⁺ gradient between sperm head and medium was dissipated. In this study, we show that the fluorescent pH probe 9-N-dodecylamino acridine and the intracellular Ca2⁺ fluores cent probe fura-2 are both localized to the anterior part of the sperm head encompassing the acrosomal compartment in intact sperm, and the fluorescence associated with each probe is lost as the first stage of the acrosome reaction is completed. Loss of the pH probe fluorescence, pattern N, corresponds to onset of H⁺ permeability, and loss of fura-2 fluorescence, pattern F, corresponds to onset of Ca²⁺ permeability. Localization of intracellular fura-2 fluorescence to the acrosomal compartment required extracellular Mn²⁺ to quench surface-bound fura-2 AM, the tetra-acetoxymethyl ester of fura-2 used to load the cells. Loss of acrosomal fura-2 fluorescence is due to quenching by tracer Mn²⁺ accompanying Ca²⁺. Onset of membrane permeability to both H⁺ and Ca²⁺, asseenby loss of patterns N and F, occurred in synchrony in populations of sperm bound to isolated, structurally intact zonae, with an overall time coursfe of 210 min postbinding. The loss of pattern N in individual sperm cells bound to zonae was rapid, with a half time of 2.1 min. Concomitant with this rapid loss of pattern N was a shift in the amplitude of flagellar motion from large to small. The lag times to pattern N loss in 50 individual cells ranged from 30 to 140 min. The variable lag times determine the population kinetics; the rate of the endpoinl reaction seen in the individual cells is rapid and constant. Dissipation of the H⁺ gradient with immediate loss of pattern N was readily achieved by addition of nigericin with no change in the time course of the onset of Ca²⁺ permeability of the membranes enclcsing the acrasome. Onset of Ca²⁺ permeability was always accompanied by onset of H⁺ permeability, but the alkalinization caused by H⁺ permeability induced by nigericin had no effect on Ca²⁺ permeability in intact sperm. This indicates that the permeabilization of the membranes marking the endpoint reaction of the B-to-S transition is most likely due to pore formation induced by punctate fusion of the plasma and outer acrasomal membranes, as would be expected for an exocytotic reaction.     

The antigenicity of the rabbit sperm acrosomes and the reversible sterility induced in the immunized rabbits

Rabbits were immunized against their alloantigens extracted from the sperm acrosome. The sequential method of extraction, first by MgCl₂ then by detergents, provided the plasma and outer acrosomal membranes and their contents in the first step and the inner acrosomal membrane and its components in the next step. Both groups of antigens appear to have produced monospecific antisera. The MgCl₂ extract antiserum lost its sperm immobilization and agglutination activities on absorption to its antigen. The detergent extract, under similar treatment, remained unaffected. Therefore, the agglutination and immobilization factors resided on the plasma membrane. The decrease in sperm concentration suggested that the immunogen affected the germinal epithelium. The reduction in the semen volume and seminal enzymes seems to indicate that the accessory sex glands were also affected by the treatment. The antiserum blocked the dissolution of the hamster and rabbit zona pellucida by the MgCl₂ extract. The normal serum had no such effect. Both the normal and immune sera inhibited acrosin equally well while the antisera inhibited other proteinases, arylsulfatase, and β-N-acetylhexosaminidase to a significantly greater extent. Immunized rabbits failed to impregnate females as long as the antibody titer remained high. As the titer declined these animals became fertile and the females delivered normal litters.     

Dynamics of the spindle pole body of the pathogenic yeast Cryptococcus neoformans examined by freeze-substitution electron microscopy

Cryptococcus neoformans is an opportunistic human pathogen belonging to basidiomycetous fungi and has unique properties in cell cycle progression. In the present study, dynamics of the spindle pole body (SPB) during the cell cycle was examined using freeze-substitution and serial thin-sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar-shaped in G1 through G2 phases. At the beginning of prophase, globular elements of the SPB enlarged, associated with numerous cytoplasmic microtubules, and separated on the nuclear envelope. At prometaphase, the SPBs entered the nuclear region by breaking a part of the nuclear membrane, were located at the isthmus, and were associated with numerous nuclear microtubules. The nuclear division process was carried out in the daughter cell, though the nucleolus remained in the mother cell. At anaphase, one half of the nucleus returned to the mother cell. At telophase, the SPB element was extruded back to the cytoplasm from the nuclear region. By analyzing serial sections of 63 cells, duplication of the SPB was found to take place in the early G1 phase. Thus, the location, structure, and duplication cycle of the C. neoformans SPB are different from those of Saccharomyces cerevisiae , but have similarities to those of Schizosaccharomyces pombe .     

Sperm Penetration and in vitro Fertilization of the Egg of the House Cricket Acheta domesticus

Summary

A study of sperm penetration of the egg of the house cricket, Acheta domesticus, using a fluorescent microscope technique, showed that sperm penetration of the chorion, prior to fertilization, is not restricted to a specialised area of the egg surface, such as the micropyle. An acrosomal filament is seen on penetrating sperm. Polyspermy (multiple sperm attachment) is seen under normal conditions.

Eggs fertilized in vitro developed to the 4 day (pre-katatrepsis) stage, but did not undergo katatrepsis. Development was confirmed by cytogenetic studies. The percentage of eggs showing cleavage nuclei (i.e. initial development) was 59% after in vitro fertilization and 5% in ovarian eggs incubated in a hypotonic medium.     

Kinetics of leaping primates: Influence of substrate orientation and compliance

Our current knowledge about the forces leapers generate and absorb is very limited and based exclusively on rigid force platform measurements. In their natural environments, however, leapers take off and land on branches and tree trunks, and these may be compliant. We evaluated the influence of substrate properties on leaping kinetics in prosimian leapers by using a combined field and laboratory approach. Tree sway and the timing of takeoffs relative to the movements of trees were documented for animals under natural conditions in Madagascar. Field data collected on three species (Indriindri, Propithecus diadema, Propithecus verreauxi) indicate that in the majority of takeoffs, the substrate sways and the animals takeoff before the elastic rebound of the substrate. This implies that force is “wasted” to deform supports. Takeoff and landing forces were measured in an experimental setting with a compliant force pole at the Duke University Primate Center. Forces were recorded for 2 Propithecus verreauxi and 3 Hapalemur griseus. Peak takeoff forces were 9.6 (P. verreauxi) and 10.3 (H. griseus) times body weight, whereas peak landing forces were 6.7 (P. verreauxi) and 8.4 (H. griseus) times body weight. As part of the impulse generated does not translate into leaping distance but is used to deform the pole, greater effort is required to reach a given target substrate, and, consequently, takeoff forces are high. The landing forces, on the other hand, are damped by the pole/substrate yield that increases the time available for deceleration. Our results contrast with previous studies of leaping forces recorded with rigid platform measuring systems that usually report higher landing than takeoff forces. We conclude that 1) Leapers generate and are exposed to exceptionally high locomotory forces. The takeoff forces are higher than the landing forces when using compliant supports, indicating that the takeoff rather than the landing may be critical in interpreting leaping behavior and related aspects of muscu-loskeletal design. 2) Large-bodied vertical clingers and leapers do not usually take advantage of the elastic energy stored in substrates. Rather, force (and energy) is wasted to deform compliant supports. 3) A compliant force pole approximates the conditions faced by large-bodied vertical clingers and leapers in the wild more closely than do rigid force platforms. © 1995 Wiley-Liss, Inc.     

N-Hydroxides, aminotriazole, and cyanide activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus

Abstract The pole of the Gram-positive rod Bacillus subtilis is formed by the construction of a crosswall which is then split. The newly exteriorized wall comes under stress and stretches to form the developing pole. A model is proposed to account for the even bisection of the septum. It is based on an extension of our previous finding that autolysin action on living cells is increased when the protonmotive force is dissipated in any of a number of ways. The first site of enzymatic attack is that region of the peripheral wall that has become farther removed from the cytoplasmic membrane as the result of the envagination of the developing septum. Later, enzymatic action lead to the cleavage midway between the portions of cytoplasmic membrane delimiting the septum as this region is farthest removed from the source of protonmotive force.     

Energized membrane may regulate nucleoid conformation in Bacillus subtilis

Abstract It was shown in a previous study, using a temperature-sensitive initiation mutant of Bacillus subtilis that DNA synthesis at the origin and terminus of replication occurred at cell poles. In the current study, it is demonstrated that cells specifically labelled at the origin or near the terminus, show a re-distribution of radioactivity when treated with the uncoupling agent, sodium azide. Since it is most likely that the DNA-cell wall attachment is mediated through membrane interactions, we now propose that the maintenance of this attachment is dependent on the energized state of the membrane.     

A novel spermatozoan ultrastructure in the shrimp Hippolyte obliquimanus Dana, 1852 (Decapoda: Caridea: Hippolytidae)

The aim of this study was to describe and illustrate the morphology of the spermatozoon of the Western Atlantic shrimp, Hippolyte obliquimanus. Individuals were sampled from Itaguá Beach (Ubatuba, southern Brazil). The male reproductive system was dissected and morphological analysis was undertaken using a stereomicroscope, a light microscope, and transmission electron and scanning electron microscopes. When viewed from the nuclear or acrosomal poles, each spermatozoon has many translucent radiating arms (about 20) from a denser cell body, while laterally the cell body and arms resemble a “cnidarian medusa”, with all the arms projecting away from the bell-like cell body. This sperm morphology is distinct from the “thumbtack”-shaped spermatozoa observed in the majority of carideans but has similarities to the spermatozoa of Rhynchocinetes spp. The morphology of sperm of several species of the genus Hippolyte resembles the spermatozoon of H. obliquimanus with the presence of posterior nuclear arms, but it is necessary to study other Hippolyte species to place these arms in the context of the genus.