Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.     

Fission yeast Pcp1 links polo kinase‐mediated mitotic entry to γ‐tubulin‐dependent spindle formation

The centrosomal pericentrin‐related proteins play pivotal roles in various aspects of cell division; however their underlying mechanisms remain largely elusive. Here we show that fission‐yeast pericentrin‐like Pcp1 regulates multiple functions of the spindle pole body (SPB) through recruiting two critical factors, the γ‐tubulin complex (γ‐TuC) and polo kinase (Plo1). We isolated two pcp1 mutants (pcp1‐15 and pcp1‐18) that display similar abnormal spindles, but with remarkably different molecular defects. Both mutants exhibit defective monopolar spindle microtubules that emanate from the mother SPB. However, while pcp1‐15 fails to localise the γ‐TuC to the mitotic SPB, pcp1‐18 is specifically defective in recruiting Plo1. Consistently Pcp1 forms a complex with both γ‐TuC and Plo1 in the cell. pcp1‐18 is further defective in the mitotic‐specific reorganisation of the nuclear envelope (NE), leading to impairment of SPB insertion into the NE. Moreover pcp1‐18, but not pcp1‐15, is rescued by overproducing nuclear pore components or advancing mitotic onset. The central role for Pcp1 in orchestrating these processes provides mechanistic insight into how the centrosome regulates multiple cellular pathways.     

Comparative 5-doxylstearoyllecithin and 3-doxylcholestane EPR spin labeling study of phospholipid bilayer perturbation by different oxidized lecithin species

A 3-doxylcholestane spin label was employed in addition to 5-doxylstearoyl lecithin for a more detailed study of the different effects exerted by variously oxidized lecithins on fatty acid alignment in phospholipid planar bilayers. Either spin label was enclosed in oriented PLPC planar samples also containing in turn a variety of conjugated-dienes lecithins and cleaved chain lecithins, in order to monitor EPR spectral angular dependence loss. Data obtained by use of arachidonoyl-hydroxystearoyl-PC and palmitoyl-hydroxylinoleoyl-PC confirm that the 5-DSPC nitroxide ring almost completely retains its orientation in CD-PCs-containing planar membranes, in contrast with angular dependence loss observed in the presence of the CC-PC molecular species palmitoyl-oxononanoyl-PC and palmitoyl-oxovaleroyl-PC, already seen with cleaved-chain palmitoyl-glutaroyl-PC and palmitoyl-azelaoyl-PC. However, the 3-DC nitroxide ring also loses its orientation with CD-PCs, in addition to being disoriented by cleaved chain-lecithins, similarly to 5DSPC. Joint information from the two spin labels will help to clarify whether OXPC-related disordering involved the whole bilayer structure or only the hydrophobic core. In addition, the propensity of different OXPCs to form bilayer vesicles in water suspension was also determined by Sepharose 4B gel-chromatography. The results suggest that CD-PCs might yield SPB bilayer structures with a disordered hydrophobic core, while pure CC-PC more probably forms non-bilayer disordered structures, possibly micelles or mixed micelle/bilayers.     

The fission yeast MO25 protein functions in polar growth and cell separation

Proteins of the MO25 family are widely conserved but their function has not been characterized in detail. Human MO25 is a cofactor of LKB1, a conserved protein kinase with roles in cell polarity in nematodes, flies and mammalian cells. Furthermore, the budding yeast MO25 homologue, Hym1, is important for cell separation and morphogenesis. We have characterized Pmo25p, the MO25 homologue in the fission yeast Schizosaccharomyces pombe. Pmo25p is an essential protein required for polar growth; in its absence the actin cytoskeleton becomes depolarized and cells adopt a round morphology. In addition, pmo25 mutants are defective in cell separation. Both functions of Pmo25p appear to be mediated by the Orb6p–Mob2p kinase complex. Pmo25p shows no distinct localization during interphase, but it is recruited to one of the two spindle pole bodies during anaphase and to the division site during cytokinesis. The septation initiation network (SIN) regulates the localization of Pmo25p, suggesting that it regulates Pmo25p function during cell division.     

The C-terminal region of the meiosis-specific protein kinase Ime2 mediates protein instability and is required for normal spore formation in budding yeast

The cyclin-dependent kinase Cdk1 and the related kinase Ime2 act in concert to trigger progression of the meiotic cell cycle in the yeast Saccharomyces cerevisiae. These kinases share several functions and substrates during meiosis, but their regulation seems to be clearly different. In contrast to Cdk1, no cyclin seems to be involved in the regulation of Ime2 activity. Ime2 is a highly unstable protein, and we aimed to elucidate the relevance of Ime2 instability. We first determined the sequence elements required for Ime2 instability by constructing a set of deletions in the IME2 gene. None of the small deletions in Ime2 affected its instability, but deletion of a 241 amino acid C-terminal region resulted in a highly stabilized protein. Thus, the C-terminal domain of Ime2 is important for mediating protein instability. The stabilized, truncated Ime2 protein is highly active in vivo. Replacement of the IME2 gene with the truncated IME2ΔC241 in diploid strains did not interfere with meiotic nuclear divisions, but caused abnormalities in spore formation, as manifested by the appearance of many asci with a reduced spore number such as triads and dyads. The truncated Ime2 caused a reduction of spore number in a dominant manner. We conclude that downregulation of Ime2 kinase activity mediated by the C-terminal domain is required for the efficient production of normal four-spore asci. Our data suggest a role for Ime2 in spore number control in S. cerevisiae.     

Three-dimensional electron microscopy analysis of ndc10-1 mutant reveals an aberrant organization of the mitotic spindle and spindle pole body defects in Saccharomyces cerevisiae

Kinetochore components play a major role in regulating the transmission of genetic information during cell division. Ndc10p, a kinetochore component of the essential CBF3 complex in budding yeast is required for chromosome attachment to the mitotic spindle. ndc10-1 mutant was shown to display chromosome mis-segregation as well as an aberrant mitotic spindle (Goh and Kilmartin, 1993). In addition, Ndc10p localizes along the spindle microtubules (Muller-Reichert et al., 2003). To further understand the role of Ndc10p in the mitotic apparatus, we performed a three-dimensional electron microscopy (EM) reconstruction of mitotic spindles from serial sections of cryo-immobilized ndc10-1 mutant cells. This analysis reveals a dramatic reduction in the number of microtubules present in the half-spindle, which is connected to the newly formed spindle pole body (SPB) in ndc10-1 cells. Moreover, in contrast to wild-type (WT) cells, ndc10-1 cells showed a significantly lower signal intensity of the SPB components Spc42p and Spc110p fused with GFP, in mother cell bodies compared with buds. A subsequent EM analysis also showed clear defects in the newly formed SPB, which remains in the mother cell during anaphase. These results suggest that Ndc10p is required for maturation of the newly formed SPB. Intriguingly, mutations in other kinetochore components, ndc80-1 and spc24-1, showed kinetochore detachment from the spindle, similar to ndc10-1, but did not display defects in SPBs. This suggests that unattached kinetochores are not sufficient to cause SPB defects in ndc10-1 cells. We propose that Ndc10p, alongside its role in kinetochore–microtubule interaction, is also essential for SPB maturation and mitotic spindle integrity.     

Platelets do not express the oxidized or reduced forms of tissue factor

Background

Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms.

Methods

Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results.

Results

Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography.

Conclusion and significance

Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF.     

Calmodulin as a protein linker and a regulator of adaptor/scaffold proteins

Calmodulin (CaM) is a universal regulator for a huge number of proteins in all eukaryotic cells. Best known is its function as a calcium-dependent modulator of the activity of enzymes, such as protein kinases and phosphatases, as well as other signaling proteins including membrane receptors, channels and structural proteins. However, less well known is the fact that CaM can also function as a Ca^2 +-dependent adaptor protein, either by bridging between different domains of the same protein or by linking two identical or different target proteins together. These activities are possible due to the fact that CaM contains two independently-folded Ca^2 + binding lobes that are able to interact differentially and to some degree separately with targets proteins. In addition, CaM can interact with and regulates several proteins that function exclusively as adaptors. This review provides an overview over our present knowledge concerning the structural and functional aspects of the role of CaM as an adaptor protein and as a regulator of known adaptor/scaffold proteins.     

Design and evaluation of a continuous semipartition bioreactor for in situ liquid‐liquid extractive fermentation

Extractive fermentation (or in situ product removal (ISPR)) is an operational method used to combat product inhibition in fermentations. To achieve ISPR, different separation techniques, modes of operation and physical reactor configurations have been proposed. However, the relative paucity of industrial application necessitates continued investigation into reactor systems. This article outlines a bioreactor designed to facilitate in situ product extraction and recovery, through adapting the reaction volume to include a settler and solvent extraction and recycle section. This semipartition bioreactor is proposed as a new mode of operation for continuous liquid‐liquid extractive fermentation. The design is demonstrated as a modified bench‐top fermentation vessel, initially analysed in terms of fluid dynamic studies, in a model two‐liquid phase system. A continuous abiotic simulation of lactic acid (LA) fermentation is then demonstrated. The results show that mixing in the main reaction vessel is unaffected by the inserted settling zone, and that the size of the settling tube effects the maximum volumetric removal rate. In these tests the largest settling tube gave a potential continuous volumetric removal rate of 7.63 ml/min; sufficiently large to allow for continuous product extraction even in a highly productive fermentation. To demonstrate the applicability of the developed reactor, an abiotic simulation of a LA fermentation was performed. LA was added to reactor continuously at a rate of 33ml/h, while continuous in situ extraction removed the LA using 15% trioctylamine in oleyl alcohol. The reactor showed stable LA concentration of 1 g/L, with the balance of the LA successfully extracted and recovered using back extraction. This study demonstrates a potentially useful physical configuration for continuous in situ extraction.