Ultrastructural studies of mutant spermatozoids in ferns. I. The mature nonmotile spermatozoid of mutation 230X in Ceratopteris thalictroides(L.)Brongn

Electron microscopy reveals that nonmotility in the spermatozoids of mutant 230X of the fern Ceratopteris thalictroides results from highly aberrant flagella. With respect to its mitochondrial complement, amyloplasts, condensed chromatin within the nucleus and the multilayered structure (MLS), the mutation is almost indistinguishable from the wild-type spermatozoids. In contrast to flagellar mutations in other organisms (man, mouse, Drosophila, Chlamydomonas), which principally affect the microtubules of the axoneme, the basal body cartwheel is lacking in 230X. In its absence, compound microtubules with shared walls are still present, but in highly disorganized arrays. Since the amount of polymerized tubulin in the spermatozoids of 230X is approximately the same as in the wild type, the mutation does not seem to affect microtubule synthesis or assembly. Centriolar cartwheels appear to be essential templates for the alignment of triplet and doublet tubules in regular radial arrays. The MLS in 230X is almost normal, whereas the flagella are aberrant, indicating that there are two distinct functional classes of microtubules in archegoniate spermatozoids. In contrast to the helix of 3½ gyres found in the wild type, nuclear morphology in 230X exhibits profound distortions ranging from deep channels and holes to supernumerary attenuated arms. Parts of nuclei associated with the MLS are almost normal, but malformations are in variably associated with the presence of microtubules of the aberrant flagella that are in close proximity t o the nuclear surface. The shapes of the teratologies are directly related to the number and configuration of the adjacent perinuclear tubules. From these findings, it is argued that microtubules have a crucial role in nuclear shaping in archegoniates; and that the precise form of the nucleus is closely related to the geometry and development of the MLS. On the other hand, it is difficult to envisage how microtubules growing in the chaotic arrays found in 230X could themselves generate shaping forces, More likely, the actual force-generating system, situated in or near the nuclear envelope, has become misaligned and severely restricted by the perinuclear arrays of flagellar tubules, which function as cytoskeletal elements additional to those of the normal MLS. Archegoniate plants are particularly advantageous for the detection of basal body mutants, since centrioles are absent from the mitotic apparatus. Cytological and hybridization studies of 230X affirm the nuclear basis of the mutation, and provide no support for the possible genetic autonomy of centrioles.     

Effects of RNase and RNA on in vitro aster assembly

RNase alters the in vitro assembly of spindle asters in homogenates of meiotically dividing surf clam (Spisula solidissima) oocytes. Some effects of RNase, such as reduced astral fiber length, appear nonenzymatic and probably result from RNase binding to tubulin. However, RNase-induced changes in the microtubule organizing center are also observed. Since other polycations can mimic RNase effects, the existence of an RNA component of the spindle organizing center remains uncertain. Effects of RNase and other polycations on astral fiber length can be prevented and reversed by the RNase inhibitor, polyguanylic acid. Polyguanylic acid can also augment astral fiber length in the absence of added RNase or other polycations. Augmentation by polyguanylic acid is favored by high ionic strength, and can be duplicated by polyuridylic acid and, with less efficiency, by polyadenylic acid. Polucytidylic acid and unfractionated yeast RNA, however, are unable to augment aster assembly. Polyguanylic acid can also augment the length of astral fibers on complete spindles isolated under polymerizing condition. These results demonstrate that specfic polyribonucleotides can alter spindle assembly in vitro. The presence of an inhibitor of microtubule assembly in Spisula oocytes, which can be inactivated by specific RNAs, is suggested.     

Identification and sequence analysis of six new members of the NIMA-related kinase family in Chlamydomonas

The NIMA kinases are an evolutionarily conserved protein family with enigmatic roles in the regulation of mitosis. We report six new members of this family in Chlamydomonas, in addition to the previously identified NIMA-related kinase, Fa2p. Chlamydomonas NIMA-related kinases (CNKs) 1-6 were sequenced from subclones generated by RT-PCR using information from EST libraries and the recently sequenced Chlamydomonas genome. Phylogenetic and bioinformatic approaches were used to determine the relationships of the six new members with known members of the NIMA-related kinase family. Although humans express at least eleven NIMA-related kinases, the eukaryotic microbes that have been studied to date express only one or two members of the family. Thus, the discovery that Chlamydomonas expresses a total of at least seven NIMA-related kinases is intriguing. Our analyses suggest that members of this family may play roles in the assembly and function of cilia.     

Selective disappearance of maternal centrioles after fertilization in the anisogamous brown alga Cutleria cylindrica (Cutleriales, Phaeophyceae): Paternal inheritance of centrioles is universal in the brown algae

The behavior of centrioles in zygotes and female gametes developing parthenogenetically in the anisogamous brown alga Cutieria cyiindrica Okamura was studied using electron and immunofluorescence microscopy. Two pairs of centrioles, detected using anti-centrin antibody, were observed in the vicinity of the male and female nuclei, respectively, just after plasmogamy. The fluorescence intensity of one of the two centrin foci became weak 6 h after plasmogamy and finally disappeared. It was impossible to determine whether the male- or female-derived centrioles disappeared in zygotes, because there was nothing to detect morphological differences between the two centrioles. However, a prominent anti-centrin staining focus was located at the condensed male nucleus in zygotes in which karyogamy had not occurred yet. As a result, it was considered that the maternally inherited centrioles had selectively disappeared during development in C. cylindrica. The paternal inheritance of centrioles in zygotes was also confirmed by electron microscopy. Considering previous observations from oogamous and isogamous species of brown algae, we concluded that the paternal inheriance of centrioles could be universal in the brown algae.     

Foamy virus capsid assembly occurs at a pericentriolar region through a cytoplasmic targeting/retention signal in Gag

Foamy viruses (FV) are unusual retroviruses that differ in many aspects of their life cycle from the orthoretroviruses such as human immunodeficiency virus. Similar to Mason–Pfizer monkey virus (MPMV), FV assemble into capsids intracellularly. The capsids are then transported to a cellular membrane for acquisition of envelope (Env) glycoproteins and budding. However, unlike MPMV, budding of FV is dependent upon the presence of Env. Previous work suggested that FV Env proteins are localized to the endoplasmic reticulum (ER) where budding takes place. However, very little was known about the details of FV assembly. We have used immunofluorescence and electron microscopy to visualize the intracellular location of FV assembly and budding. We have found that, as in the case of MPMV, FV capsids assemble at a pericentriolar site in the cytoplasm. Surprisingly, FV Env is mostly absent from this site and, contrary to expectations, FV capsid structural protein (Gag) is absent from the ER. Gag and Env only co-localize at the trans -Golgi network, suggesting that Env–Gag interactions that are required for viral egress from the cell, occurs at this site. Finally, inhibitor studies suggest an important role of microtubule networks for foamy viral assembly and budding.     

Ultrastructure of Amoebophrya sp. and its changes during the course of infection

Amoebophrya is a syndinian parasite that kills harmful bloom forming algae. Previously uncharacterized ultrastructural aspects of infection and development were elucidated. The biflagellate dinospore has two mitochondria, electron-dense bodies, striated strips, trichocysts, and a nucleus with peripherally condensed chromatin. After finding an Akashiwo sanguinea host and adhering to its surface, the parasite penetrates the host surface, apparently using a microfilament based motility and electron-dense bodies within a microtubular basket in the process of parasitophorous vacuole membrane formation. After entering the host nucleus, possibly by a similar mechanism used to enter the host cell, the parasite cytosol expanded substantially prior to mitosis. From 12-36 hours mitochondria were inconspicuous but present. Chromatin condensation was variable. By 36 hours post-infection, parasites had multiple nuclei, a microtubule-supported cytopharynx, and were beginning to form a fully internal mastigocoel. By 48 hours, the characteristic "beehive" appearance was apparent with flagella projecting into a fully developed mastigocoel. The cytoplasm contained trichocysts, elongated mitochondria, and nuclei with peripherally condensed chromatin. Although Amoebophrya lacks an apical complex, its electron-dense bodies show functional similarities to apicomplexan rhoptries. Its lack of permanently condensed chromosomes, but compact dinospore chromatin, supports the idea that dinoflagellate permanently condensed chromosomes may be a remnant of a parasitic ancestor with a compact dispersal stage.     

Centrosomes and cancer.

The centrosome functions as the major microtubule organizing center (MTOC) of the cell and as such it determines the number, polarity, and organization of interphase and mitotic microtubules. Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division are all dependent on the normal function of the centrosome and on its orderly duplication, once and only once, in each cell cycle. Malignant tumor cells show characteristic defects in cell and tissue architecture and in chromosome number that can be attributed to inappropriate centrosome behavior during tumor progression. In this review, we will summarize recent observations linking centrosome defects to disruption of normal cell and tissue organization and to chromosomal instability found in malignant tumors.     

The Rise of the Cartwheel: Seeding the Centriole Organelle

The cartwheel is a striking structure critical for building the centriole, a microtubule‐based organelle fundamental for organizing centrosomes, cilia, and flagella. Over the last 50 years, the cartwheel has been described in many systems using electron microscopy, but the molecular nature of its constituent building blocks and their assembly mechanisms have long remained mysterious. Here, we review discoveries that led to the current understanding of cartwheel structure, assembly, and function. We focus on the key role of SAS‐6 protein self‐organization, both for building the signature ring‐like structure with hub and spokes, as well as for their vertical stacking. The resemblance of assembly intermediates in vitro and in vivo leads us to propose a novel registration step in cartwheel biogenesis, whereby stacked SAS‐6‐containing rings are put in register through interactions with peripheral elements anchored to microtubules. We conclude by evoking some avenues for further uncovering cartwheel and centriole assembly mechanisms.     

A proteolytic enzyme, trypsin, for immunofluorescence observation of microtubules in algal cells

The usefulness of a proteolytic enzyme, trypsin, in immunofluorescence microscopy was confirmed in algal ceils. Flagella of Umspora penicilliformis zoo-spores were visible using an anti-β tubulin antibody after trypsin treatment, and the cortical microtubules of vegetative cells could also be clearly detected. Interestingly, centrioles that were not detected in the control observation appeared in gametophyte cells of Acrosiphonia duriuscula and Monostroma angicava using the trypsin treatment.     

COORDINATNE NUCLEAR AND CHLOROPLAST DIVISION IN UNILOCULAR SPORANGIA OF LAMINARIA ANGUSTATA (LAMINARIALES,PHAEOPHYCEAE)1

Changes in the number of nuclei and chloroplasts were examined during the process of unispore formation in unilocular sporangia of Laminaria angustata. Just before meiosis, eight chloroplasts were always present in unilocular sporangial mother cells. The number of chloroplasts remained constant through meiosis. After the resulting four nuclei divided again (third nuclear division), a close association between a nucleus and a chloroplast developed among each of the eight nuclei and eight chloroplasts. The eight chloroplasts divided ahost synchronously before the synchronous division of the eight nuclei. Following the 16 nucleate stage with 16 chloroplasts and the final 32 nucleate stage with 32 chloroplasts, 32 unispores, each with a nucleus and a chloroplast, were fomd in unilocular sporangza of L. angustata. Immunofluorescence microscopy using an anti-centrin antibody showed that two anticentrin-stained structures (as future mitotic poles) occurred adjacent to each of the premitotic four nuclei, and each spot was located near a chloroplast. Therefore, after the third division, each of the eight nuclei established close contact with a chloroplast presumably mediated by the centrosomes.