Completion of mitosis requires neither fzr/rap nor fzr2, a male germline-specific Drosophila Cdh1 homolog

Proteolysis of mitotic regulators like securins and cyclins requires Fizzy(FZY)/Cdc20 and Fizzy-related(FZR)/Hct1/Cdh1 proteins. Budding yeast Cdh1 acts not only during G1, but is also required for B-type cyclin degradation during exit from mitosis when Cdh1 is a target of the mitotic exit network controlling progression through late mitosis and cytokinesis. In contrast, observations in frog and Drosophila embryos have suggested that the orthologous FZR is not involved during exit from mitosis. However, the potential involvement of minor amounts of maternally derived FZR was not excluded in these studies. Similarly, the reported absence of severe mitotic defects in chicken Cdh1(-/-) cells might be explained by the recent identification of multiple Cdh1 genes [10]. Here, we have carefully analyzed the FZR requirement during exit from mitosis in Drosophila, which, apart from fzr, has only one additional homolog. We find that this fzr2 gene, although expressed in the male germline, is not expressed during mitotic divisions. Moreover, by characterizing fzr alleles, we demonstrate that completion of mitosis including Cyclin B degradation does not require FZR. However, fzr is an essential gene corresponding to the rap locus, and FZR, which accumulates predominantly in the cytoplasm, is clearly required during G1.     

Deletion of ghrelin impairs neither growth nor appetite

Pharmacological studies show that ghrelin stimulates growth hormone release, appetite, and fat deposition, but ghrelin's physiological role in energy homeostasis has not been established. Ghrelin was also proposed to regulate leptin and insulin release and to be important for the normal function of stomach, heart, kidney, lung, testis, and placenta. To help determine a definable physiological role for ghrelin, we generated ghrelin-null mice. In contrast to predictions made from the pharmacology of ghrelin, ghrelin-null mice are not anorexic dwarfs; their size, growth rate, food intake, body composition, reproduction, gross behavior, and tissue pathology are indistinguishable from wild-type littermates. Fasting produces identical decreases in serum leptin and insulin in null and wild-type mice. Ghrelin-null mice display normal responses to starvation and diet-induced obesity. As in wild-type mice, the administration of exogenous ghrelin stimulates appetite in null mice. Our data show that ghrelin is not critically required for viability, fertility, growth, appetite, bone density, and fat deposition and not likely to be a direct regulator of leptin and insulin. Therefore, antagonists of ghrelin are unlikely to have broad utility as antiobesity agents.     

Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis

We have used autoantibodies to probe the function of three human centromere proteins in mitosis. These antibodies recognize three human polypeptides in immunoblots: CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD). Purified anticentromere antibodies (ACA-IgG) disrupt mitosis when introduced into tissue culture cells during interphase. We have identified two execution points for antibody inhibition. Antibodies injected into the nucleus greater than or equal to 3 h before mitosis prevent the chromosomes from undergoing normal prometaphase movements in the subsequent mitosis. Antibodies injected in the nucleus during late G2 cause cells to arrest in metaphase. Surprisingly, antibodies introduced subsequent to the beginning of prophase do not block mitosis. These results suggest that the CENP antigens are involved in two essential interphase events that are required for centromere action in mitosis. These may include centromere assembly coordinate with the replication of alpha-satellite DNA at the end of S phase and the structural maturation of the kinetochore that begins at prophase.     

Dual functions of Nicotiana benthamiana Rae1 in interphase and mitosis

Rae1 performs multiple functions in animal systems, acting in interphase as an mRNA export factor and during mitosis as a mitotic checkpoint and spindle assembly regulator. In this study we characterized multiple functions of Rae1 in plants. Virus-induced gene silencing of Nicotiana benthamiana Rae1 , NbRae1 , which encodes a protein with four WD40 repeats, resulted in growth arrest and abnormal leaf development. NbRae1 was mainly associated with the nuclear envelope during interphase, and NbRae1 deficiency caused accumulation of poly(A) RNA in the nuclei of leaf cells, suggesting defective mRNA export. In the shoot apex, depletion of NbRae1 led to reduced mitotic activities, accompanied by reduced cyclin-dependent kinase (CDK) activity and decreased expression of cyclin B1, CDKB1-1, and histones H3 and H4. The secondary growth of stem vasculature was also inhibited, indicating reduced cambial activities. Differentiated leaf cells of NbRae1 -silenced plants exhibited elevated ploidy levels. Immunolabeling in BY-2 cells showed that NbRae1 protein localized to mitotic microtubules and the cell plate-forming zone during mitosis, and recombinant NbRae1 directly bound to microtubules in vitro . Inhibition of NbRae1 expression in BY-2 cells using a β-estradiol-inducible RNAi system resulted in severe defects in spindle organization and chromosome alignment and segregation, which correlated with delays in cell cycle progression. Together, these results suggest that NbRae1 plays a dual role in mRNA export in interphase and in spindle assembly in mitosis.     

Signalling of information that is neither cryptic nor private

It is commonly assumed that in order for animal signals to be advantageous, the information being signalled could not have been obtained otherwise, and is therefore ‘cryptic’ or ‘private’. Here, we suggest a scenario in which individuals can gain an advantage by signalling ‘public’ information that is neither cryptic nor private. In that scenario, signalling increases the efficiency with which that ‘public’ information is transmitted. We formalize our idea with a game in which offspring can signal their condition to their parents. Specifically, we consider a resource‐strapped parent who can only invest in one of its two offspring, and we allow offspring the chance to influence parental investment through a signal. A parent in the game seeks to invest in the higher‐quality offspring, which it could identify either through a publicly available cue, such as body size, or by relying on a signal provided by the offspring. We find that if the signal can convey information about offspring quality more efficiently than cues, then signalling of condition between offspring and parents can be favoured by selection, even though parents could potentially have acquired the same information from the cue. Our results suggest that the biological function of signals may be broader than currently considered, and provide a scenario where low cost signalling can be favoured. More generally, efficiency benefits could explain signalling across a range of biological and economic scenarios.     

Darwin's concept of final cause: neither new nor trivial

Darwin'suse of final cause accords with the Aristotelian idea of finalcauses as explanatory types – as opposed to mechanical causes, which arealways particulars. In Wright's consequence etiology, anadaptation is explained by particular events, namely, its past consequences;hence, that etiology is mechanistic at bottom. This justifies Ghiselin'scharge that such versions of teleology trivialize the subject, But a purelymechanistic explanation of an adaptation allows it to appear coincidental.Patterns of outcome, whether biological or thermodynamic, cannot be explainedbytracing causal chains, even were that possible. They are explicanda of aspecialkind. The form of their explanation, in statistical mechanics or by naturalselection, is not captured by statistical variants of the covering-law model orrelated models of explanation. In them as in classical teleology, types ofoutcome are cited to explain why there are outcomes of those types. But onlywhen types are explanatory by being selected for, as inexplanations of animal and human behavior as well as in Darwin's theory ofnatural selection, but not in statistical mechanics, is the explanationteleological. Darwin's theory is nontrivially teleological.     

Responses to neither exogenous nor endogenous endothelin-1 are altered in patients with hypercholesterolemia

There is some controversy regarding whether vascular responses to endothelin are altered in hypercholesterolemia. Studies performed to date have been compromised by the use of endothelin antagonists at inappropriate concentrations. In the current study, we examine the role of endothelin-1 in hypercholesterolemic patients using lower, more selective doses of specific endothelin antagonists. Twenty-two patients with hypercholesterolemia (total plasma cholesterol > 6.0 mmol/l) and 17 healthy controls were recruited. Forearm vascular responses to endothelin-1 (5 pmol/min), the endothelin A antagonist BQ-123 (10 nmol/min), and the endothelin B antagonist BQ-788 (1 nmol/min) were obtained. Endothelin-1 caused a significant vasoconstriction in both hypercholesterolemic and control subjects, an effect that was not significantly different between the two groups (P = 0.784). BQ-123 caused a significant vasodilatation that was not significantly different between the two groups (P = 0.899). Similarly, responses to BQ-788 (P = 0.774) and mean plasma endothelin-1 levels were not different (control vs. hypercholesterolemia, 1.16 +/- 0.18 vs. 1.06 +/- 0.15 fmol/ml; P = 0.64). Responses to neither exogenous nor endogenous endothelin are influenced by plasma cholesterol levels in humans. It is thus unlikely that the endothelin system contributes to early vascular disease pathology in patients with hypercholesterolemia.     

Bordered tug-of-war models are neither general nor predictive of reproductive skew

Models of reproductive skew assume reproductive shares are either conceded, competed over, or both. Previous mathematical evaluations found that simultaneous concessions and contests are evolutionarily unstable. Recently, Shen and Reeve (2010) challenged these conclusions and developed a series of sub-models they argued to be a unified approach to reproductive skew: the general bordered tug-of-war (BTOW). However, BTOW fails as a general model for two reasons: (1) the BTOW strategy cannot invade populations where individuals either only compete for or only concede reproductive shares and (2) contrary to Shen and Reeve’s assertion, BTOW populations are easily invaded by strategies with fewer or no concessions, but competing at lower levels. The failure of BTOW as a general model has major implications for interpreting experiments on reproductive skew. A large number of studies have measured the effects of genetic relatedness and competitive ability on reproductive skew, with a great majority finding no significant correlation between variation in within-group relatedness or competitive ability and across-group differences in skew. No model of reproductive skew except one variant of the BTOW predicts such results. With the rejection of BTOW as a valid general model, it is clear that these results are contradictory to reproductive skew theory rather than supportive of it.     

Timing of events in mitosis

BACKGROUND: Regulation of the major transitions in the cell cycle, such as G1/S, G2/M, and metaphase to anaphase, are increasingly well understood. However, we have a poor understanding of the timing of events within each phase of the cell cycle, such as S phase or early mitosis. Two extreme models of regulation are possible. A "regulator-controlled model" in which the order of events is governed by the activation of a series of cytoplasmic regulators, such as kinases, phosphatases, or proteases; or a "substrate-controlled model" in which temporal regulation is determined by the differential responses of the cellular machinery to a common set of activators. RESULTS: We have tried to distinguish between these two models by examining the timing of both biochemical and morphological events in Xenopus egg extracts during mitosis. Several proteins respond with different delays to the activation of Cdc2. We have found that the timing of phosphorylation is largely unchanged when these proteins are exposed to extracts that have been in mitosis for various periods of time. Similarly, when Xenopus interphase nuclei are added to extracts at different times after the G2/M transition, they undergo all the expected morphological changes in the proper sequence and with very similar kinetics. CONCLUSIONS: Our results suggest that during early mitosis (from prophase to metaphase) the timing of biochemical events (such as phosphorylation) and morphological events (such as structural changes in the nucleus) is at least partly controlled by the responses of the substrates themselves to a common set of signals.     

Effects of activated ADP-ribosylation factors on Golgi morphology require neither activation of phospholipase D1 nor recruitment of coatomer

Nine mutations in the switch I and switch II regions of human ADP-ribosylation factor 3 (ARF3) were isolated from loss-of-interaction screens, using two-hybrid assays with three different effectors. We then analyzed the ability of the recombinant proteins to (i) bind guanine nucleotides, (ii) activate phospholipase D1 (PLD1), (iii) recruit coatomer (COP-I) to Golgi-enriched membranes, and (iv) expand and vesiculate Golgi in intact cells. Correlations of activities in these assays were used as a means of testing specific hypotheses of ARF action, including the role of PLD1 activation in COP-I recruitment, the role of COP-I in Golgi vesiculation caused by expression of the dominant activating mutant [Q71L]ARF3, and the need for PLD1 activation in Golgi vesiculation. Because we were able to find at least one example of a protein that has lost each of these activities with retention of the others, we conclude that activation of PLD1, recruitment of COP-I to Golgi, and vesiculation of Golgi in cells are functionally separable processes. The ability of certain mutants of ARF3 to alter Golgi morphology without changes in PLD1 activity or COP-I binding is interpreted as evidence for at least one additional, currently unidentified, effector for ARF action at the Golgi.     

Integration of murine leukemia virus DNA depends on mitosis.

In synchronized rat or mouse cells infected with Moloney murine leukemia virus (MLV), integration of viral DNA and production of viral proteins occur only after the cells traverse mitosis. Integration is blocked when cells are prevented from progressing through mitosis. Viral nucleoprotein complexes isolated from arrested cells contain full-length viral DNA and can integrate this viral DNA in vitro, showing that the block to integration in arrested cells is not due to a lack of mature integration machinery. When infected cells traverse mitosis, there is a sharp increase in nuclear accumulation of viral DNA. The dependence of integration on mitosis may therefore be due to a requirement for mitosis and nuclear envelope breakdown for entry of the viral integration complex into the nucleus.     

Phosphorylation of the nuclear lamins during interphase and mitosis

The nuclear lamina is a polymeric protein assembly that is proposed to function as an architectural framework for the nuclear envelope. Previous work suggested that phosphorylation of the major polypeptides of the lamina (the "lamins") may induce disassembly of this structure during mitosis. To further investigate the possible involvement of phosphorylation in regulation of lamina structure, we characterized lamin phosphorylation occurring in mammalian tissue culture cells during interphase and mitosis. Phosphorylation occurs continuously throughout all interphase periods (coordinately with nuclear envelope growth), and takes place mainly on the assembled lamina. When the lamina is disassembled during cell division, the lamins are modified with approximately 1-2 molecules of associated phosphate. This level of mitotic phosphorylation is 4-7-fold higher than the average interphase level. Lamin phosphate occurs predominantly as phosphoserine, and is distributed over numerous tryptic peptides, many of which are modified during both interphase and mitotic periods. Significantly, phosphorylation is the only detectable charge-altering postsynthetic modification of the lamins that occurs specifically during mitosis. The results of this study support the notion that phosphorylation is important for regulation of interphase and mitotic lamina structure.     

Ase1p organizes antiparallel microtubule arrays during interphase and mitosis in fission yeast

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.     

Chromatin binding of RCC1 during mitosis is important for its nuclear localization in interphase

RCC1, a guanine nucleotide exchange factor of the small GTPase Ran, plays various roles throughout the cell cycle. However, the functions of RCC1 in biological processes in vivo are still unclear. In particular, although RCC1 has multifunctional domains, the biological significance of each domain is unclear. To examine each domain of RCC1, we established an RCC1 conditional knockout chicken DT40 cell line and introduced various RCC1 mutants into the knockout cells. We found that nuclear reformation did not occur properly in RCC1-deficient cells and examined whether specific RCC1 mutants could rescue this phenotype. Surprisingly, we found that neither the nuclear localization signal nor the chromatin-binding domain of RCC1 is essential for its function. However, codisruption of these domains resulted in defective nuclear reformation, which was rescued by artificial nuclear localization of RCC1. Our data indicate that chromatin association of RCC1 during mitosis is crucial for its proper nuclear localization in the next interphase. Moreover, proper nuclear localization of RCC1 in interphase is essential for its function through its nucleotide exchange activity.     

Immunocytochemical localization of casein kinase II during interphase and mitosis

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.     

Architecture of the Trypanosoma brucei nucleus during interphase and mitosis

The structural basis of mitosis, spindle organisation and chromosome segregation, in the unicellular parasite Trypanosoma brucei is poorly understood. Here, using immunocytochemistry, fluorescent in situ hybridisation and electron microscopy, we provide a detailed analysis of mitosis in this parasite. We describe the organisation of the mitotic spindle during different stages of mitosis, the complex ultrastructure of kinetochores and the identification of a potential spindle-organising centre in the mitotic nucleus. We investigate the dynamics of chromosome segregation using telomeric and chromosome-specific probes. We also discuss the problems involved in chromosome segregation in the light of the fact that the T. brucei karyotype has 22 chromosomes in the apparent presence of only eight ultrastructurally defined kinetochores. Received: 9 August 1999; in revised form: 15 October 1999 / Accepted: 10 November 1999