Relation between the mitosis-inhibitory activity of a chalone preparation of Ehrlich ascites tumor and its diurnal secretion

Mitosis inhibitory activity of chalone-containing preparations extracted from cells of Ehrlich's ascites carcinoma (EAC) at varying time of day (9, 13, 17, 21 1, 5 and 9 o'clock) was studied in a temporary suspension culture of EAC. The inhibitory action of the preparations depended on the time of their extraction. This may be indicative of rhythmical alterations of the production or activity of EAC chalone. The maximum activity was demonstrated by the preparations extracted at 5, 9 and 13 o'clock. The activity of the preparations, extracted at 17, 21 and 1 o'clock was considerably less. The degree of mitotic activity inhibition also depends on the dose of duration of action of the chalone-containing preparation.     

Changes in the mitotic activity of the corneal epithelium of white rats following coolong of the animals at different times of the day]

A study was made of the influence of moderate hypothermia on the mitotic activity of albino rat corneal epithelium. The animals were cooled by the contact method for one hour to 28 degrees C; such procedure was conducted at 6 a.m., at noon, and at 6 p.m.; the epithelial reaction to cooling proved to depend on the time, the greatest suppression of mitotic activity (14-fold) occurring at daytime 3 hours after the cooling. A tendency to normalization of cell division was observed 6 and 12 hours after the cooling. The number of mitoses decreased 3 hours after the evening cooling, no changes in the mitotic activity in 3 and 6 hours after the morning cooling; cell division was found to be suppressed in 12 hours.     

Resolution of two molecular forms of sucrose-phosphate synthase from maize,soybean and spinach leaves

Two forms of sucrose-phosphate synthase (EC 2.4.1.14) were resolved from leaves of three species, maize (Zea mays L. cv. Pioneer 3184), soybean (Glycine max (L.) Merr., cv. Ransom) and spinach (Spinacia oleracea L. cv. Resistoflay) by hydroxyapatite Ultrogel chromatography, using a 75-mM (designated peak 1) and 250-mM (peak 2) K-phosphate discontinuous-gradient elution. Rechromatography of the two forms showed that they were not readily interconvertible. The distribution of activity between the two forms differed among species and changed during purification of the enzyme. Recovery of peak-1 activity was specifically lowered when maize leaf extracts were prepared in the absence of magnesium, indicating that the two forms may differ in stability. In addition, the forms of the enzyme from maize differed in the extent of glucose-6-phosphate activation. These results provide evidence for the existence of multiple forms of sucrose-phosphate synthase in leaves of different species and that the forms differ in regulatory properties.Abbreviations Fru6P fructose 6-phosphate - Glc6P glucose 6-phosphate - HAU hydroxyapatite Ultrogel - Pi inorganic phosphate - SPS sucrose-phosphate synthase - UDP uridine 5-diphosphate - UDPG uridinediphosphate glucose Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10511 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. Supported in part by USDA Competitive Research Grant No. 85-CRCR-1-1568     

The effects of silver ions and silver nanoparticles on cell division and expression of <Emphasis Type="Italic">cdc2</Emphasis> gene in <Emphasis Type="Italic">Allium cepa</Emphasis> root tips

The effects of silver nanoparticles (AgNPs), silver ions (Ag⁺), and polyvinylpyrrolidone (PVP) on mitosis and expression of a gene encoding cyclin-dependent kinase 2 (cdc2) in onion roots were compared. Three concentrations (5, 10, and 15 mg dm^-3) were employed in combination with three incubation times (3, 6, and 9 h). PVP enhanced mitotic index and cdc2 expression. Both silver forms decreased mitotic index and cdc2 expression. Genotoxicity of both silver forms were indicated by three major distinguishable classes of chromosome aberrations: spindle disturbances, clastogenic aberrations, and chromosome stickiness. Concerning Ag⁺ treatments, significant enhancements in occurrence of any chromosome aberration type was associated with significant decrease in mitotic index. On the other hand, disturbed spindle in AgNPs treatments was observed even in absence of significant reduction in mitotic index suggesting that AgNPs inhibit cellular events occurring during mitosis to proceed normally rather than starting of cell division.     

Concentrating on the mitotic spindle

In eukaryotes, the microtubule-based spindle drives chromosome segregation. In this issue, Schweizer et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506107) find that the spindle area is demarcated by a semipermeable organelle barrier. Molecular crowding, which is microtubule independent, causes the enrichment and/or retention of crucial factors in the spindle region. Their results add an important new feature to the models of how this structure assembles and is regulated.Mitosis is marked by the assembly of the mitotic spindle, a microtubule-based structure that facilitates accurate chromosome segregation. Many biochemical reactions are coupled to spindle assembly, from tubulin polymerization itself to the mitotic checkpoint, which inhibits chromosome disjunction until all the chromosomes are properly attached and aligned (Cleveland et al., 2003). Interestingly, these reactions are virtually unaltered over a broad morphological range; large spindles and small spindles follow roughly the same biochemical rules despite quite distinct geometries (Brown et al., 2007; Wühr et al., 2008). In this issue, Schweizer et al. report that the mitotic spindle area is delineated by membrane-bound organelles, generating a “spindle envelope” with unique molecular constituents compared with the surrounding cytoplasm. Spindle envelope–based molecular crowding provides an enticing hypothetical solution to the broad problem of confining mitotic biochemistry to a specific cellular space irrespective of cell size.Schweizer et al. (2015) used FRAP and fluorescence correlation spectroscopy (FCS) to measure the mobility of specific proteins. The authors found that tubulin and the Mad2 spindle assembly checkpoint protein were enriched in the spindle area in a microtubule polymer-independent manner. Given that the mobility of these proteins outside and within the spindle area was the same, changes in local concentration were likely a result of a barrier effect. In support of this hypothesis, modeling their FCS data with a fenestrated barrier separating the spindle area from the cytoplasm reproduced the measured FCS results. To test if a barrier surrounding the spindle area was important for spindle function, the authors disrupted the envelope area by laser microsurgery and found chromosome segregation errors consistent with defects in spindle assembly and kinetochore attachment monitoring. Thus, spindle envelope–based concentration of basal components in two critical spindle reactions, spindle assembly and mitotic checkpoint signaling, could mechanistically catalyze cell division.Molecular crowding can catalyze reactions and stabilize proteins by altering the local concentration of one or more rate-limiting components and is best characterized by membrane-bound organelles. Crowding by aggregation can greatly increase biochemical reactions without the need for a contiguous membrane (Weber and Brangwynne, 2012; Brangwynne, 2013) and this phenomenon can regulate cell cycle states (Lee et al., 2013). Here, Schweizer et al. (2015) propose that by creating a membrane-bound organelle exclusion zone, a spindle envelope could cause the molecular crowding of important spindle proteins and thereby their enrichment in the spindle area.The mitotic spindle is known to scale with cell size: smaller cells have smaller spindles (Levy and Heald, 2012). Spindle size scaling is prominent during development when repeated cell division without embryonic growth results in cells that can be several orders of magnitude smaller than that of the zygote. Recently, cytoplasm volume and tubulin concentration was shown to be an important factor in spindle size scaling; however, a curious exception to the size scaling rule is that there seems to be an upper limit to spindle size, resulting in stable spindle size when a threshold cell size is reached (Wühr et al., 2008; Good et al., 2013; Hazel et al., 2013). A spindle envelope would provide mechanisms to maintain increased local tubulin concentration independent of the absolute amount available in the cell. The net effect would be that spindle size scales in very large cells to the spindle envelope size rather than cell size in a manner analogous to chromosome size scaling to nuclear size independently of cell size (Fig. 1 A). Clearly this is a more complex problem and factors such as tubulin protein production and polymerization cofactors (such as the Tog family of proteins) clearly play an important role (Slep, 2009). However, spindle envelope–based molecular crowding could provide an elegant solution to a biochemical problem.Open in a separate windowFigure 1.Molecular crowding in the mitotic spindle. Schematic view of how a spindle envelope could mitigate spindle size scaling during development (A) or cell cycle control (B) in a common cytoplasm. (A) A spindle envelope (black dotted line) that excludes large membrane-bound organelles (yellow) could locally increase the concentration of spindle proteins (depicted as a red background) such as tubulin to control spindle size independent of cell size during early development. (B) Neighboring nuclei in a common cytoplasm (e.g., the syncytial mitotic gonad in C. elegans) could have differing mitotic states by restricting the diffusion of important regulatory proteins such as Mad2 (similar coloring as in A).A spindle envelope could also provide a cell biological solution to another developmental problem—independent cell cycle control of separate nuclei within a single cytoplasm (syncytia). For example, the mitotic region of the Caenorhabditis elegans germline contains germ cell precursors that divide independently of one another in a common cytoplasm. In some cases, two neighboring dividing nuclei can have different biochemistry, one arrested in metaphase because of a kinetochore microtubule attachment defect while the other is progressing into anaphase (Gerhold et al., 2015). By restricting the diffusive radius of signaling molecules like Mad2, a steep threshold of checkpoint activity can be maintained, allowing independent cell cycle control even in a common cytoplasm (Fig. 1 B).The mitotic spindle has long been known to exclude large membrane-bound organelles, even in the absence of microtubule polymer, leading to a hypothesized nontubulin-based “spindle matrix.” A spindle matrix would be an excellent candidate to underlie the spindle envelope. The molecular nature of a spindle matrix, however, has never been agreed upon with candidate mechanisms ranging from nonprotein macromolecules to actin (Pickett-Heaps et al., 1984; Chang et al., 2004). A convincing argument can be made that the Skeletor/Megator/Chromator proteins first identified in Drosophila melanogaster constitute a spindle matrix (Walker et al., 2000; Qi et al., 2004; Rath et al., 2004; Schweizer et al., 2014). These proteins are large and are found in the nucleus in interphase and as microtubule-independent fibrous structures in and around the spindle in mitosis. Depletion of these proteins results in mitotic errors; however, these may or may not be caused by a role as the spindle matrix.Schweizer et al. (2015) evaluated Megator (as a representative member of the complex) as a possible basis for generating the spindle envelope. FRAP and FCS showed that, like tubulin and Mad2, Megator is concentrated in the spindle envelope region independent of microtubules. However, Megator in the spindle region had slower diffusive properties compared with that around the cell periphery. Thus, unlike tubulin and Mad2, the mobility of Megator within the spindle was altered, indicating that Megator likely forms a high molecular weight complex with its binding partners Skeletor and Chromator in the spindle area, which may help form the spindle envelope.The sum of these results lead to a possible model whereby the Skeletor/Megator/Chromator proteins complex together and subsequently support a spindle envelope independent of microtubules. The spindle envelope excludes large membrane-bound organelles, leading to increased concentration of mitotic reaction constituents and thus ultimately catalyzing cell division. It will be exciting in the future to determine if the spindle area is indeed subject to molecular crowding in the purest of forms (solvent exclusion) and how this effect drives cell division.     

Multipolar mitosis in procaine-treated polyspermic sea urchin eggs and in eggs fertilized with UV-irradiated spermatozoa with a computer model to simulate the positioning of centrosomes

Procaine-treated eggs can be penetrated by more than one spermatozoon. Supernumerary male pronuclei can fuse with the female one giving raise to multipolar spindles or remain isolated within the egg's cytoplasm forming their own spindle. In all types of multiple mitotic figures (asters and spindles) the distribution of asters is equidistant either uniplanar or at maximum distance like at the apices of a polyhedron. Astral rays are not different from spindle fibers: they can attach to and attract chromosomes of "foreign" mitotic figures. When several mitotic figures are present in one egg, the partner asters are always of the same size, and microtubules of one aster never interdigitate with those of others. The hypothesis that positioning of centrosomes is brought about by spreading of a centrosome organizer in the form of an expanding calotte on the surface of the nucleus (Mazia, D., Int. Rev. Cytol. 100, 49-92 (1987)) is supported by a computer model.     

Regulation of homoserine dehydrogenase in developing organs of soybean seedlings

The regulation of homoserine dehydrogenase (HSD) activity (EC 1.1.1.3) by L-threonine, L-cysteine and K⁺ was examined using extracts of organs of soybean seedlings harvested 3, 6, 11, and 19 days after germination. K⁺ stimulated HSD activity from each source at least 2-fold. HSD activity was completely inhibited by 10 mM L-cysteine while 10 mM D-cysteine was not inhibitory. A progressive decrease in sensitivity of NAD-dependent HSD to inhibition by 10 mM L-threonine occurred in all organs except the leaf during the sampling period. This progressive decrease in sensitivity of the HSD to threonine inhibition was detected only when K⁺ was present in the assay mixtures. Four major molecular forms, including one rapidly migrating form (form I) and three more slowly migrating forms (forms II, III, IV) of HSD, were identified in extracts of soybean organs by polyacrylamide electrophoresis. Chromatographic and electrophoretic data indicate that form I, which was not inhibited by threonine or stimulated by K⁺, was of lower MW than forms II, III and IV which were of similar MW. These latter 3 forms were inhibited by threonine and stimulated by K⁺. During soybean seedling development form II increased in amount and forms I and IV decreased in amount. This alteration in the amounts of the forms of HSD occurred during the same period as the decrease in the amount of threonine inhibition. Since K⁺ stimulation of HSD decreased during soybean organ development and K⁺ enhanced threonine inhibition, this might account for the observed decrease in threonine inhibition.     

Chronotoxicity of cyclophosphane under different lighting conditions

Study of the chronotoxicity of cyclophosphamide injected to mice at 18, 24, 6 and 12 o'clock has shown that animals kept under the conditions of natural changes of day and night showed the circadian rhythm of the drug toxicity with the maximal survival of the animals after injection at 24 and 6 o'clock and with the minimal survival after injection at 18 o'clock. In animals maintained under the conditions of artificial constant light the toxicity rhythm was perversed within the first hours after injection and improved on subsequent observation. Moreover the earlier death was marked in these animals.     

A new chromosome fluorescence banding technique combining DAPI staining with image analysis in plants

In this study, a new chromosome fluorescence banding technique was developed in plants. The technique combined 4,6-diamidino-2-phenylindole (DAPI) staining with software analysis including three-dimensional imaging after deconvolution. Clear multiple and adjacent DAPI bands like G-bands were obtained by this technique in the tested species including Hordeum vulgare L., Oryza officinalis, Wall & Watt, Triticum aestivum L., Lilium brownii, Brown, and Vicia faba L. During mitotic metaphase, the numbers of bands for the haploid genomes of these species were about 185, 141, 309, 456 and 194, respectively. Reproducibility analysis demonstrated that banding patterns within a species were stable at the same mitotic stage and they could be used for identifying specific chromosomes and chromosome regions. The band number fluctuated: the earlier the mitotic stage, the greater the number of bands. The technique enables genes to be mapped onto specific band regions of the chromosomes by only one fluorescence in situ hybridisation (FISH) step with no chemical banding treatments. In this study, the 45S and 5S rDNAs of some tested species were located on specific band regions of specific chromosomes and they were all positioned at the interbands with the new technique. Because no chemical banding treatment was used, the banding patterns displayed by the technique should reflect the natural conformational features of chromatin. Thus it could be expected that this technique should be suitable for all eukaryotes and would have widespread utility in chromosomal structure analysis and physical mapping of genes.     

Aurora A kinase activation: Different means to different ends

Aurora A is a serine/threonine kinase essential for mitotic entry and spindle assembly. Recent molecular studies have revealed the existence of multiple, distinct mechanisms of Aurora A activation, each occurring at specific subcellular locations, optimized for cellular context, and primed by signaling events including phosphorylation and oxidation.

IntroductionDuring mitosis, almost 30% of the proteome is modified by the transfer of phosphate to serine, threonine, or tyrosine residues. These phosphorylation events are responsible for the profound transformation of cellular architecture and physiology that occurs as cells progress through mitosis. Pivotal protein kinases responsible for the massive increase in protein phosphorylation as cells transit into mitosis include Aurora A kinase (AURKA), Aurora B kinase, Polo-like kinase 1 (Plk1), and Cyclin-dependent kinase 1 (Cdk1)/Cyclin A/B complexes. Their precise and coordinated activation critically defines the G2-M transition.Overexpression and aberrant activation of AURKA have been functionally linked to oncogenic transformation through centrosome amplification, aneuploidy, and chromosomal instability. Beyond its pivotal role in mitotic cell division, AURKA has numerous nonmitotic functions in tumorigenesis. AURKA thus represents a critical “druggable target” in cancer, controlling key oncogenic pathways associated with drug resistance and poor patient outcome.AURKA activation is unexpectedly complex, and a number of different mechanisms have been described, including autophosphorylation of its activation segment and binding to a variety of allosteric modulators, which recruit and locally activate AURKA at specific subcellular localizations (Fig. 1). Recent findings show that these allosteric modulators activate AURKA through surprisingly distinct mechanisms, each acting at different subcellular locations to trigger a unique event in response to different upstream signals. We summarize current views of these activation mechanisms and speculate on the reasons underlying this complexity.Open in a separate windowFigure 1.Model of AURKA activation during cell cycle progression. Left panel: AURKA is activated during the G2/M transition by binding to Bora phosphorylated on its M3 motif by Cyclin A-Cdk1 (CycA-Cdk1). Phospho-Bora (pBora) binds unphosphorylated inactive AURKA (in gray, N and C denote the N-lobe and the C-lobe, respectively) via its M1, M2 (dark blue), and phospho-M3 (green) motifs. Binding of phospho-Bora turns on the catalytic activity of AURKA (red) by substituting in trans the phosphoregulatory site on Thr288, leading to mitotic entry. P denotes phosphate. Middle panel: AURKA is activated at centrosomes via Cep192-dependent oligomerization and oxidation of Cys290 by ROS. NEDB, nuclear envelope breakdown. Right panel: AURKA is activated at spindle microtubules by Tpx2 binding through M1 and M2 motifs and by autophosphorylation at Thr288.Autophosphorylation of the activation segment and binding to the allosteric regulator Targeting protein for Xklp2 (Tpx2) synergize to locally activate AURKA at microtubulesIn eukaryotes, the transfer of phosphate from ATP to protein substrates is mediated by the protein kinase domain, a bilobal catalytic entity. The protein kinase domain possesses a complicated structure, with many flexible parts but a highly restricted catalytic mechanism (i.e., there is only one way to transfer phosphate). This affords great opportunity for the diversification of how each kinase turns on and off (Endicott et al., 2012). Like the majority of eukaryotic protein kinases, AURKA is regulated by phosphorylation of a conserved residue, Thr288, within a flexible element of the kinase domain termed the activation segment. This event leads to a reorganization of the active site that is required, but not sufficient, for full catalytic activation. This is in sharp contrast to many other protein kinases, where phosphorylation of the activation segment is sufficient for maximal catalytic activation. Similar to the closely related AGC family kinases, AURKA has evolved a dependency for its full activation on the binding of an allosteric modulator to its smaller N-terminal kinase lobe (Leroux et al., 2018). This event positions or stabilizes structural elements in the kinase active site that are not sufficiently aligned by activation segment phosphorylation alone. For most AGC family kinases, such as the exemplars PKA and AKT, the allosteric modulator represents a linear peptide sequence contained within the protein kinase itself, but distal to the protein kinase domain. In contrast, in the case of AURKA, the allosteric modulator is presented by an entirely separate protein.The best characterized allosteric modulator of AURKA is the microtubule-binding protein Txp2. Upon nuclear envelope breakdown, Tpx2 is released by RAN-GTP from importins, which then allows it to concurrently recruit and activate AURKA at microtubules to promote mitotic spindle assembly. Tpx2 uses its first N-terminal 43 amino acids to activate AURKA, in a manner synergistic with activation segment phosphorylation, by binding across the N-lobe of the AURKA kinase domain (Fig. 1). Notably, in the absence of Tpx2 binding and activation segment phosphorylation, AURKA retains marginal but detectable protein kinase activity. Binding of Tpx2 alone boosts AURKA catalytic function modestly (15-fold) while autophosphorylation alone boosts catalytic function substantially (157-fold). However, the action of both events translates into a 448-fold enhancement in activity relative to the fully repressed state (Dodson and Bayliss, 2012). This coordination of maximal activation with the recruitment of AURKA to microtubules may serve a double duty to minimize spurious phosphorylation of proteins elsewhere in the cell.Autophosphorylation and binding to Tpx2 trigger conformational changes in AURKA that are sufficiently large to be probed by time-resolved fluorescence energy transfer approaches (Ruff et al., 2018). Time-resolved fluorescence energy transfer revealed that the activation segment of AURKA adopts a wide range of conformations in solution. Notably, binding of Tpx2 to AURKA locks an inward conformation of a catalytic element termed the DFG motif (the Asp in the Asp-Phe-Gly motif coordinates a magnesium ion required for ATP binding) by rigidifying an inherently flexible helix αC (Ruff et al., 2018). In contrast, Thr288 phosphorylation promotes a large conformational change in the activation segment that enables the binding of peptide substrates. Both Tpx2 binding and autophosphorylation are required for AURKA function at microtubules. This transient “doubly activated” form of AURKA is not detectable at spindle microtubules in normal cells due to the action of the AURKA-directed protein phosphatase 6 (PP6), which specifically dephosphorylates Tpx2-bound AURKA on the activation segment.Phospho-Bora activates unphosphorylated AURKA in the cytoplasm to trigger mitotic entryIn a manner thematically similar to how Tpx2 binding and AURKA autophosphorylation synergize to activate AURKA at microtubules, recent work revealed that a phosphorylated form of Bora activates cytoplasmic AURKA during mitotic commitment (Tavernier et al., 2021).Commitment to mitosis is tightly coordinated with DNA replication to preserve genome integrity. Commitment is achieved by a tightly choreographed biochemical tug-of-war between mitotic kinases and phosphatases (PPases). To this end, AURKA activates Plk1 by phosphorylating its activation segment at Thr210. In turn, Plk1 promotes activation of the Cdk1/Cyclin B complex by phosphorylating both negative and positive regulators of Cdk1 to trigger mitotic entry. As AURKA lies at the top of this mitotic kinase cascade, the key question that arises is how is AURKA initially activated in G2?As noted above, AURKA can autophosphorylate its own activation segment at Thr288, but this form of the enzyme is rapidly dephosphorylated by counteracting PPases in G2. As dephosphorylation maintains AURKA in an inactive state, how then does AURKA overcome the repressive effect of PPases to activate Plk1?AURKA activation during mitotic commitment is critically dependent on the evolutionarily conserved protein Bora, following its own phosphorylation on a key regulatory site on Ser112. This event is essential for the phosphorylation of Plk1 on Thr210 by AURKA in vitro and for timely mitotic entry in vivo, both in Xenopus egg extracts (Vigneron et al., 2018) and in human cells (Tavernier et al., 2021). Remarkably, phospho-Bora binds to and potently activates AURKA lacking phosphorylation of its activation segment, suggesting the possibility that the phosphate on S112 of Bora may physically and/or functionally substitute for the phosphorylated activation segment on AURKA.Dissection of how Bora binds AURKA revealed at least two motifs in Bora, denoted M1 and M2, with weak similarity to AURKA-binding elements in Tpx2^1–43. Both motifs are required for the binding and activating function of Bora on AURKA, and notably, the essential Ser112 phospho-regulatory site (in the sequence Pro-Ser-Pro, denoted motif M3) lies immediately C-terminal to binding motif M2. By analogy to the mechanism of action of Tpx2, Bora motif M1 likely binds in an extended manner parallel to the top surface of helix αC, whereas motif M2 adopts a helical conformation and binds parallel to the bottom surface of helix αC. As Bora motif M3 is immediately adjacent to motif M2, this binding mode would orient the Ser112 phospho-moiety of motif M3 in close proximity to a constellation of positively charged residues that normally engage the phosphate moiety of the phosphorylated activation segment of AURKA (Fig. 1; Tavernier et al., 2021). This mode of action elegantly allows phospho-Bora to allosterically activate AURKA during mitotic commitment, when AURKA itself is catalytically repressed by dephosphorylation. The precise atomic details of how phospho-Bora binds and activates AURKA and whether other protein kinases use analogous mechanisms for activation remain to be determined.Since phosphorylation of Bora Ser112 is essential for its ability to activate AURKA and commit cells to mitosis, the upstream kinase responsible for this regulatory event represents a critical component of the AURKA activation puzzle. This function is performed by Cyclin A–Cdk1, which is active in S-G2 and known to promote mitotic entry. Consistent with this model, Bora phosphorylated on S112 is sufficient to promote mitotic commitment in Xenopus egg extracts depleted of Cyclin A (Vigneron et al., 2018). In human cells, Cyclin A–Cdk1 is confined to the nucleus during S phase, but at the S/G2 transition it is abruptly exported to the cytoplasm, allowing it to phosphorylate Bora (Silva Cascales et al., 2021). As such, Bora acts as a bridge linking Cyclin A–Cdk1 activity to the activation of the mitotic kinase cascade. Why phospho-Bora can persist under conditions that disfavor AURKA activation by autophosphorylation remains an open question worthy of further investigation. Possibilities include that the phospho-Ser112 residue is a suboptimal PPase substrate or that it is protected from dephosphorylation by cis-activating factors.Redox regulation of AURKA during mitosisRecent work indicates that AURKA activity is also regulated by oxidative signaling with both stimulatory and inhibitory outcomes. While autophosphorylation of AURKA on Thr288 is largely neutralized in the cytoplasm and at spindle microtubules by counteracting PPases, it is readily detected at centrosomes. Centrosomal AURKA autoactivation is stimulated as a consequence of Cep192-mediated oligomerization and AURKA autophosphorylation, but the underlying mechanism was poorly understood. New studies reveal that oxidative modification of a conserved cysteine residue, Cys290, located in the activation segment of the kinase domain promotes AURKA autophosphorylation during mitosis when AURKA is oligomerized.While biochemical studies using purified proteins in the absence of an oligomerizing agent revealed that oxidative modification of Cys290 inhibited AURKA kinase activity (Byrne et al., 2020; Tsuchiya et al., 2020), cell treatment with oxidizing agents such as H₂0₂ increased AURKA phosphorylation on Thr288 (Wang et al., 2017; Tsuchiya et al., 2020). This increase in Thr288 phosphorylation was accompanied by dimerization of AURKA in a manner sensitive to reducing agents such as DTT. This result hinted that disulfide bond formation between AURKA monomers might be involved in promoting AURKA trans-autophosphorylation. Consistent with this hypothesis, a crystal structure of an AURKA kinase domain obtained under disulfide bond–promoting conditions revealed a face-to-face dimer orientation of the kinase domain stabilized by a Cys290–Cys290 disulfide bond (Lim et al., 2020). In this configuration, the active site of the AURKA kinase domain adopts a productive conformation predicted to support substrate phosphorylation. Given the inherent flexibility of the activation segment itself, this face-to-face configuration of the kinase domain was also predicted to support AURKA trans-autophosphorylation on Thr288. Follow-up biochemical studies proved that the Cys290–Cys290 disulfide–linked configuration of the AURKA kinase domain is indeed compatible with trans-autophosphorylation on Thr288.An interesting feature of the Cys290-dependent activation mechanism of AURKA is the requirement for Cep192. Presumably, the ability of Cep192 to recruit and oligomerize AURKA favors the formation of the Cys290–Cys290 disulfide bond between kinase domains (Fig. 1). In support of this model, oxidation-induced Cys290–Cys290 disulfide cross-linking of AURKA could also be recapitulated in Xenopus extracts by the addition of bivalent antibodies directed at AURKA.At subcellular locations beyond centrosomes, where AURKA is not dimeric, oxidation of the activation segment would be expected to have an opposite effect on protein kinase activity. For instance, a crystal structure of monomeric AURKA covalently bound to Coenzyme A (CoAlation), a major regulator of cellular metabolism that contains both nucleotide and thiol moieties, revealed that AURKA CoAlation robustly inhibits kinase activity (Tsuchiya et al., 2020) through an ATP-competitive mechanism. Competitive binding is achieved by the nucleotide moiety of Coenzyme A engaging the nucleotide-binding pocket of ATP, while the reactive pantetheine thiol moiety forms a disulfide bond with Cys290. This dual anchoring of Coenzyme A to AURKA imparts not only affinity but also specificity toward kinase inhibition. Supporting the possibility that Coenzyme A can exert a potent inhibitory effect on AURKA under physiological conditions, microinjection of CoA into mouse oocytes caused abnormal spindles and chromosome misalignment, phenotypes typically observed upon AURKA inactivation.Interestingly, when bound to AURKA, Tpx2 exerts a protective effect against inhibition by CoAlation. Given that Bora is predicted to bind AURKA similar to Tpx2 (Tavernier et al., 2021), this could allow Bora to also protect AURKA from inhibition by CoAlation. Together, these results suggest that each distinct cellular pool of AURKA will respond differently to oxidation signals.Reactive oxygen species (ROS) are emerging as important signaling molecules. ROS and oxidative stress have been shown to increase during G2 and M phases in an otherwise unperturbed asynchronous cell cycle (Patterson et al., 2019), suggesting that oxidative modification of biomolecules, including AURKA, might regulate mitotic progression. Likewise, H₂O₂ locally released by mitochondria, where a pool of AURKA has been recently shown to localize (Bertolin et al., 2018), is implicated in symmetry breaking and polarity establishment in early Caenorhabditis elegans embryos (De Henau et al., 2020). It will be particularly exciting to determine whether AURKA, which also plays a role in setting up embryo polarity, is regulated by redox signaling in this specific context.Concluding remarksAURKA is activated by a growing list of mechanisms, with each acting at specific stages of the cell cycle and subcellular location. The ability to monitor which specific mechanism is at play at any one time in vivo presents a particular challenge. The activation state of AURKA is often measured by the use of a phospho-specific antibody targeting the phosphorylated Thr288 epitope. However, this has limited effectiveness to detect AURKA activated by Tpx2 at spindle microtubules because of the transient nature of the phospho-Thr288 epitope at this location. Furthermore, in the case of cytoplasmic AURKA activation by Bora, phosphorylation at Thr288 is not required for kinase activation. A live fluorescence energy transfer sensor has been reported for the phosphorylation status of AURKA on Thr288 that detects conformational changes induced by Thr288 phosphorylation rather than the phosphorylation motif itself (Bertolin et al., 2016). If the binding of Tpx2 to phosphorylated AURKA and the binding of phospho-Bora to dephosphorylated AURKA induces similar conformational changes to those induced by autophosphorylation, then this could represent a more generally applicable assay for monitoring the activation state of AURKA.Why is the activation of AURKA so complex? We speculate that the major reason for this complexity is related to kinase action at distinct times and in spatially distinct locations (Fig. 1). Bora acts in the cytoplasm before mitotic entry, and Cep192 acts at the centrosome before and likely after mitotic entry, whereas Tpx2 acts on spindle microtubules after mitotic entry. Thus, the allosteric regulators and their own upstream controllers (e.g., Cyclin A/Cdk1 for Bora) direct AURKA activity to execute distinct functions. In some ways, this complexity of activation represents a different solution than the one adopted by Plk1 or Protein Phosphatase 1 (PP1), which also acts at distinct time points and subcellular locations. Both Plk1 and PP1 employ docking motifs that are post-translationally controlled to dictate their time and sites of action, as well as their substrate specificity. As deeper insights are gained into the control of mitotic kinases and PPases, it will be intriguing to see what additional solutions have evolved to address the challenge of temporally and spatially restricted actions.We end by noting that the different mechanisms described above for AURKA activation are associated with specific pathologies. Doubly activated AURKA, with bound Tpx2 and Thr288 phosphorylation, is not detected on microtubules in normal cells due to the action of PP6. However, this form of AURKA is readily detected in melanoma cells bearing PP6 mutations, which gives rise to pathological chromosome instability and DNA damage (Hammond et al., 2013). Bora is overexpressed in multiple cancer types, including ovarian cancer, where it plays a pro-oncogenic role (Parrilla et al., 2020), and there is significant evidence for ROS signaling, which is involved in centrosomal AURKA activation by Cep192, contributing to a number of disease states. Finally, AURKA itself is overexpressed in numerous cancers associated with drug resistance and poor patient outcome. However, the clinical utility of AURKA inhibitors to date has been limited, likely because of essential roles of AURKA in multiple events in the cell cycle. We posit that the discovery that AURKA is activated through a variety of mechanisms to execute distinct events may afford opportunities to develop drugs targeting a subset of the biological functions of AURKA and hence enable more precise tuning of the therapeutic window.     

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

An immunocytochemical method was used to determine the proportion of cells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyledonary origin, the stably cytokinin-dependent tissue used in the cytokinin bioassay devised by Carlos O. Miller. A standard cell synchronization protocol involving hydroxyurea was used to demonstrate the applicability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell division cycle was restarted by the addition of cytokinin. We have followed the pattern of resumption of S phase after the readdition of cytokinin. This pattern reveals the existence of three subpopulations of cells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, one in S phase, and one in the G₁ phase.Abbreviations BrdU 5-bromo-2-deoxyuridine - DI deionized water - FITC fluorescein isothiocyanate - HU hydroxyurea - l-AOPP l--aminooxy--phenylpropionic acid - LI labeling index - PA polyamine - PI propidium iodide     

Studies on Fertilization in Glycine max

This work studies the whole process of fertilization in Glycine max. The results are summarized as follows: 1. The type of ripened pollen grain of soybean was two-cell type. The generative cell was divided mitotically into two spermatids within the pollen tube. 2. In the 6th hour after self-pollination, the pollen tube entered into the embryo sac and released two sperms. Before the fusion of the male and female nuclei, the cytoplasmic sheath of the spermatids falled off. The cytoplasm of the male gamete did not fuse with that of the egg cell. 3. In the 6th hour after self-pollination, one spermatid nucleus come in contact with the egg nucleus and the other with the secondary nucleus, The contact of the sperimatid nucleus with the egg nucleus was a little earlier than that with the secondary nucleus. 4. The spermatid nucleus entered into the egg nucleus; the chromatic granules of the spermatid despiralized. After the complete fusion of the spermatid nucleus with the egg nucleus, the egg nucleus was darkly stained and the chromonemata increased, afterward the male nucleus appeared. 5. In the 28th hour after self-pollination, the zygote begun the first mitotic division. It took about 20 hours to fuse the male and female gametes, and to form the zygote untile before first mitotic division. It means that the zygote dormancy stage of soybean was about twenty hours. 6. In the 7th hour after self-pollination, the spermatid nucleus fused with the secondary nucleus. The process was similar to that of the spermatid nucleus with the egg nucleus. The chromatic granules gradually despiralized within the secondary nucleus, and the male nucleoli appeared. The velocity of the fusion of the spermatid nucleus with the secondary nucleus was faster than that of the other spermatid nucleus with the egg nucleus. 7. In the 10th hour after self-pollination, the secondary nucleus begun the first mitotic division. 8. The fertilization of soybean was the premitotic type.     

Mitotic disrupter herbicides act by a single mechanism but vary in efficacy

Summary Although there are numerous herbicides that disrupt mitosis as a mechanism of action, to date not one has compared the effects of these disrupters on a single species and over a range of concentrations. Oat seedlings, treated with a range of concentrations of nine different mitotic disrupter herbicides, were examined by immunofluorescence microscopy of tubulin in methacrylate sections. All herbicides caused the same kinds of microtubule disruption, although the concentrations required to cause the effects differed markedly between the herbicides. Effects on spindle and phragmoplast mitotic microtubule arrays were seen at the lowest concentrations and manifested as multipolar spindles and bifurcated phragmoplasts (which subsequently resulted in abnormal cell plate formation). At increasing concentrations, effects on mitotic microtubule arrays manifested as microtubule tufts at kinetochores and reduction of cortical microtubules resulting in arrested prometaphase figures and isodiametric cells. These data indicate that all mitotic disrupter herbicides have a common primary mechanism of action, inhibition of microtubule polymerization, and that marginal effects observed in the past were the result of incomplete inhibition and/or differential sensitivity of the microtubule arrays.Abbreviations DCPA 2,3,5,6-tetrachloroterephthalic acid dimethyl ester - APM amiprophosmethyl - DAPI 4,6-diamidino-2-phenyl indole - MTOC microtubule organizing center     

Seasonal and circadian fluctuations in blood biochemical indicators in mice in natural conditions and exposed to constant light]

The fluctuations of activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and those of the levels of protein, glucose, cholesterol, bilirubin, creatinine, blood urea nitrogen, K+, Cl-, Na+ in blood plasma of mice in natural conditions (NC) and exposed to constant light (CL) were studied in different seasons of the year (in January, April, July, October) on days 18, 24, 6 (at 12 o'clock). Most indices both in NC and CL animals had seasonal rhythm similar for each of them. This proves a primary effect of environmental geoclimatic factors of formation of circadian periodicals as compared to desynchronization in constant light revealed by Kosinor analysis in winter (acrophase from 14.16 till 16.32 o'clock) and autumn (acrophase from 23.03 til 4.40 o'clock). During the same seasons one can observe the maximum desynchronization influences of constant light, which leads to abrupt falling (to the 10-fold and more) of the fluctuations amplitude and in some cases to stabilization of circadian rhythm.     

Mitotic peaks and mitotic time in the desmidiaceae

Summary In a day having 12 hours each of light and darkness mitotic periodicity has been found in Closterium siliqua and Cosmarium botrytis, there being a peak in the dark period. C. botrytis also has a low back-ground mitotic level at other times. The mitotic time was calculated in Closterium siliqua as follows:Prophase=c. 36 min Metaphase=c. 8 min anaphase=c. 6 min Telophase=c. 48 min.This paper forms part of a Ph. D. thesis submitted to the University of London by the first-mentioned author.     

Overexpressing AtPAP15 Enhances Phosphorus Efficiency in Soybean

Low phosphorus (P) availability is a major constraint to crop growth and production, including soybean (Glycine max), on a global scale. However, 50% to 80% of the total P in agricultural soils exists as organic phosphate, which is unavailable to plants unless hydrolyzed to release inorganic phosphate. One strategy for improving crop P nutrition is the enhanced activity of acid phosphatases (APases) to obtain or remobilize inorganic phosphate from organic P sources. In this study, we overexpressed an Arabidopsis (Arabidopsis thaliana) purple APase gene (AtPAP15) containing a carrot (Daucus carota) extracellular targeting peptide in soybean hairy roots and found that the APase activity was increased by 1.5-fold in transgenic hairy roots. We subsequently transformed soybean plants with AtPAP15 and studied three homozygous overexpression lines of AtPAP15. The three transgenic lines exhibited significantly improved P efficiency with 117.8%, 56.5%, and 57.8% increases in plant dry weight, and 90.1%, 18.2%, and 62.6% increases in plant P content, respectively, as compared with wild-type plants grown on sand culture containing phytate as the sole P source. The transgenic soybean lines also exhibited a significant level of APase and phytase activity in leaves and root exudates, respectively. Furthermore, the transgenic lines exhibited improved yields when grown on acid soils, with 35.9%, 41.0%, and 59.0% increases in pod number per plant, and 46.0%, 48.3%, and 66.7% increases in seed number per plant. Taken together, to our knowledge, our study is the first report on the improvement of P efficiency in soybean through constitutive expression of a plant APase gene. These findings could have significant implications for improving crop yield on soils low in available P, which is a serious agricultural limitation worldwide.Phosphorus (P) is a critical macronutrient for plant growth and development. Terrestrial plants generally take up soil P in its inorganic form (Pi; Marschner, 1995). However, 50% to 80% of the total P in agricultural soils exists as organic phosphate, in which, up to 60% to 80% is myoinositol hexakisphosphate (phytate; Iyamuremye et al., 1996; Turner et al., 2002; George and Richardson, 2008). Since phytate-P is not directly available to plants, low P availability becomes one of the limiting factors to plant growth.Plants have developed a number of adaptive mechanisms for better growth on low-P soils, including changes in root morphology and architecture, activation of high-affinity Pi transporters, improvement of internal phosphatase activity, and secretion of organic acids and phosphatases (Raghothama, 1999; Vance et al., 2003). Acid phosphatases (APases) are hydrolytic enzymes with acidic pH optima that catalyze the breakdown of P monoesters to release Pi from organic P compounds, and therefore may play an important role in P nutrition (Vincent et al., 1992; Li et al., 2002). APase activity, including extracellular and intracellular APase activity, is generally increased by Pi starvation in higher plants (Duff et al., 1994). Intracellular APases might play a role in internal Pi homeostasis through remobilization of Pi from older leaves and vacuole stores, whereas extracellular APases are believed to be involved in external P acquisition by mobilizing Pi from organic P compounds (Duff et al., 1994). In the last few years, secreted APases have been purified and characterized in some model plants, such as Arabidopsis (Arabidopsis thaliana; Coello, 2002) and tobacco (Nicotiana tabacum; Lung et al., 2008). Furthermore, an Arabidopsis pup3 mutation that underproduced secreted APases in root tissues accumulated 17% less P in shoots when organic P was supplied as the major P source (Tomscha et al., 2004), indicating the possible role of APases during plant growth in response to Pi starvation.Phytase is a special type of APases with the capability to hydrolyze phytate and its derivatives, which are the predominant inositol phosphates present in seeds and soils. It is generally believed that phytase activation in seeds or resynthesis in plants plays important roles in Pi remobilization through hydrolyzing the phytate into Pi during seed germination (Loewus and Murthy, 2000). Furthermore, phytase in roots and/or root exudation has been demonstrated to be important for utilizing Pi from phytate in the growth medium (Asmar, 1997; Li et al., 1997; Hayes et al., 1999; Richardson et al., 2000).AtPAP15, a purple APase with confirmed phytase activity from Arabidopsis, can hydrolyze myoinositol hexakisphosphate, yielding myoinositol and Pi (Zhang et al., 2008). Overexpression of AtPAP15 in Arabidopsis significantly decreased phytate content in leaves (Zhang et al., 2008). Sequence analysis indicates that AtPAP15 exhibits 74% similarity to the soybean (Glycine max) phytase gene, GmPhy (Hegeman and Grabau, 2001). It seems likely that the possible involvement of phytase in plant P nutrition might be conserved among different plant species. But it is still unclear whether AtPAP15 or other phytases can be used to directly help crops, including the major agronomic crop, soybean, to acquire P under low-P conditions.Soybean is one of the most important food crops, accounting for a large segment of the world market in oil crops and also serving as an important protein source for both human consumption and animal feed (Kereszt et al., 2007). Soybean is mainly cultivated in tropic, subtropic, and temperate areas, where the soils are low in P due to intensive erosion, weathering, and strong P fixation by free iron and aluminum oxides (Sample et al., 1980; Stevenson, 1986). Low P availability is especially problematic for soybean, since root nodules responsible for nitrogen fixation have a high P requirement (Robson, 1983; Vance, 2001).In this study, the Arabidopsis PAP15 gene directed by an extracellular targeting sequence from a carrot (Daucus carota) extensin gene was successfully transformed into both soybean hairy roots and whole soybean plants. Overexpression of AtPAP15 not only increased the secretion of APase from transgenic soybean hairy roots and roots of whole transgenic soybean plants, but also significantly improved APase activity in leaves, as well as P efficiency and yield in the transgenic soybean lines. To the best of our knowledge, this is the first report on the improvement of P efficiency in crop plants through constitutive expression of a plant APase gene. This study could have significant implications for improving crop production on low-P soils, which is a serious agronomic limitation worldwide.     

Regulation of the final stage of mitosis by components of the pre-replicative complex and a polo kinase

The accurate division of duplicated DNA is essential for maintenance of genomic stability in proliferating eukaryotic cells. Errors in DNA replication and chromosomal segregation may lead to cell death or genomic mutations that lead to oncogenic properties. Thus, tight regulation of DNA replication and mitosis is essential for maintaining genomic integrity. Cell division cycle 6 (Cdc6) is an essential factor for initiating DNA replication. Recent work shows that phosphorylation of Cdc6 by pololike kinase 1 (Plk1), one of the essential mitotic kinases, regulates mitotic exit mediated by Cdk1 and separase. Here we discuss how pre-replicative complex factors are connected with Plk1 and affect mitotic exit.Key words: Plk1, Cdc6, DNA replication, mitotic exit, chromosomal segregation