Abi mutants in Dictyostelium reveal specific roles for the SCAR/WAVE complex in cytokinesis.

Actin polymerization drives multiple cell processes involving movement and shape change. SCAR/WAVE proteins connect signaling to actin polymerization through the activation of the Arp2/3 complex. SCAR/WAVE is normally found in a complex with four other proteins: PIR121, Nap1, Abi2,and HSPC300 (Figure S1A available online) [1-3]. However,there is no consensus as to whether the complex functions as an unchanging unit or if it alters its composition in response to stimulation, as originally proposed by Edenet al. [1]. It also is unclear whether complex members exclusively regulate SCAR/WAVEs or if they have additional targets [4-6]. Here, we analyze the roles of the unique Dictyostelium Abi. We find that abiA null mutants show less severe defects in motility than do scar null cells, indicating--unexpectedly--that SCAR retains partial activity in the absence of Abi. Furthermore, abiA null mutants have a serious defect in cytokinesis, which is not seen in other SCAR complex mutants and is seen only when SCAR itself is present. Detailed examination reveals that normal cytokinesis requires SCAR activity, apparently regulated through multiple pathways.

Pollen ultrastructure in anther cultures of Datura innoxia. III. Incomplete microspore division

During the microspore division in Datura innoxia, the mitotic spindle is oriented in planes both perpendicular (PE) and oblique (OB) to the spore wall against which the nucleus is situated. However, irrespective of polarity, the usual type of hemispherical wall is laid down at cytokinesis and isolates the generative cell from the rest of the pollen grain (type A). In PE spores the vegetative nucleus initially occupies a central position in the pollen grain, whereas in OB spores the vegetative nucleus lies at the periphery of the grain close to the generative cell. In anther cultures initiated just before the microspore division is due to take place, no marked change can be observed in either orientation or symmetry of the mitotic spindle when the spores divide. In some, however, cytokinesis is disrupted and deposition of the hemispherical wall arrested. In the absence of a complete wall, differentiation of the generative cell cannot take place and binucleate pollen grains are formed having 2 vegetative-type nuclei (type B). The 2 nuclei in the B pollens are always situated against the pollen-grain wall, suggesting that the disruption phenomenon is related to the OB spores. The incomplete wall always makes contact with the intine on the intine-side of the spindle. Wall material may be represented merely as short stubs projecting out from the intine into the cytoplasm, in which event the 2 nuclei lie close to each other and are separated by only a narrow zone of cytoplasm. In other grains the wall is partially developed between the nuclei and terminates at varying distances from the tonoplast; in these, the nuclei are separated by a wider zone of cytoplasm. The significance of these binucleate grains in pollen embryogenesis is discussed.

Pollen ultrastructure in anther cultures of Datura innoxia. I. Division of the presumptive vegetative cell.

Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 degrees C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48-72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generative-cell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cells in Nicotiana tabacum.

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G(2)/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G(2) regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G(2)/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the Go phase or asynchronously replicating cells. Our data suggested that IBV induces a G(2)/M phase arrest in infected cells to promote favorable conditions for viral replication.

Regulation of cell cycle and stress responses to hydrostatic pressure in fission yeast

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress.

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.

Localization to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division

The subcellular localization of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) (group I and group II coronaviruses, respectively) nucleoproteins (N proteins) were examined by confocal microscopy. The proteins were shown to localize either to the cytoplasm alone or to the cytoplasm and a structure in the nucleus. This feature was confirmed to be the nucleolus by using specific antibodies to nucleolin, a major component of the nucleolus, and by confocal microscopy to image sections through a cell expressing N protein. These findings are consistent with our previous report for infectious bronchitis virus (group III coronavirus) (J. A. Hiscox et al., J. Virol. 75:506-512, 2001), indicating that nucleolar localization of the N protein is a common feature of the coronavirus family and is possibly of functional significance. Nucleolar localization signals were identified in the domain III region of the N protein from all three coronavirus groups, and this suggested that transport of N protein to the nucleus might be an active process. In addition, our results suggest that the N protein might function to disrupt cell division. Thus, we observed that approximately 30% of cells transfected with the N protein appeared to be undergoing cell division. The most likely explanation for this is that the N protein induced a cell cycle delay or arrest, most likely in the G2/M phase. In a fraction of transfected cells expressing coronavirus N proteins, we observed multinucleate cells and dividing cells with nucleoli (which are only present during interphase). These findings are consistent with the possible inhibition of cytokinesis in these cells.

The inositol-3-phosphate synthase biosynthetic enzyme has distinct catalytic and metabolic roles

Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in inositol auxotrophy that can only be partially rescued by exogenous inositol. Removal of inositol supplementation from the ino1- mutant results in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis and substrate adhesion. Inositol depletion also caused a dramatic generalised decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 independent of inositol biosynthesis. To characterise this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) was identified with homology to mammalian macromolecular complex adaptor proteins. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism.