Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven,auxin-dependent patterning

Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP₂) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus.     

Tissue Mechanics Regulate Mitotic Nuclear Dynamics during Epithelial Development

1. Download : Download high-res image (217KB)
2. Download : Download full-size image

     

The tyrosine kinase v-Src modifies cytotoxicities of anticancer drugs targeting cell division

v-Src oncogene causes cell transformation through its strong tyrosine kinase activity. We have revealed that v-Src-mediated cell transformation occurs at a low frequency and it is attributed to mitotic abnormalities-mediated chromosome instability. v-Src directly phosphorylates Tyr-15 of cyclin-dependent kinase 1 (CDK1), thereby causing mitotic slippage and reduction in Eg5 inhibitor cytotoxicity. However, it is not clear whether v-Src modifies cytotoxicities of the other anticancer drugs targeting cell division. In this study, we found that v-Src restores cancer cell viability reduced by various microtubule-targeting agents (MTAs), although v-Src does not alter cytotoxicity of DNA-damaging anticancer drugs. v-Src causes mitotic slippage of MTAs-treated cells, consequently generating proliferating tetraploid cells. We further demonstrate that v-Src also restores cell viability reduced by a polo-like kinase 1 (PLK1) inhibitor. Interestingly, treatment with Aurora kinase inhibitor strongly induces cell death when cells express v-Src. These results suggest that the v-Src modifies cytotoxicities of anticancer drugs targeting cell division. Highly activated Src-induced resistance to MTAs through mitotic slippage might have a risk to enhance the malignancy of cancer cells through the increase in chromosome instability upon chemotherapy using MTAs.     

The usefulness of the chlorophyll fluorescence parameters in harvest prediction in 10 genotypes of winter triticale under optimal growth conditions

The aim of the experiment was to detect differences in the activity of the photosynthetic apparatus in 10 genotypes of winter triticale. Measurements of gas exchange and chlorophyll fluorescence were performed. Among the tested genotypes, the photosynthetic apparatus of Timbo and Piano was the most active, while the photosynthesis of both Babor and Boreas was highly reduced. Additionally, we have found significant correlations between the yield and some parameters of chlorophyll fluorescence and leaf gas exchange. Parameters of chlorophyll fluorescence are useful for estimation of the functional state of the photosynthetic apparatus and could become selection criteria in plant breeding. Moreover, such parameters permit definition of the effectiveness of the utilization of chlorophyll a excitation energy and co‐operation of the light phase reactions, with reactions occurring during the dark phase of photosynthesis.     

Comparative morphology of selected characters of the Pentatomidae foreleg (Hemiptera,Heteroptera)

Heteropteran legs are very diverse within and among taxa, and such variation is frequently correlated with life habits. Structural modifications are commonly present in the legs of the Pentatomoidea but are poorly studied. Using scanning electron microscopy, the tibia and pretarsal microstructure of 82 species of Pentatomidae (Heteroptera), three species of Scutelleridae, and ten species of Thyreocoridae were described, focusing on the pretarsal structure, the foretibial apparatus, and the foretibial comb. The Pentatomidae, the Scutelleridae, and the Thyreocoridae have uniform pretarsal structures. Variation can be found in the length of the parempodial setae and in the shape of the parempodial projections. The foretibial combs of the Pentatomidae, the Thyreocoridae, and the Scutelleridae are described for the first time, and we have demonstrated that there is low structural variation in the foretibial comb complex of the studied species. The setae organization and distribution on the foretibial apparatus is uniform in the families studied. However, the Asopinae (Pentatomidae) bear a foretibial apparatus that is uniquely organized. The taxonomic and phylogenetic relevance of the pretarsal traits, the foretibial apparatus, and the foretibial comb are discussed.     

Cyclin B1 is an efficacy-predicting biomarker for Chk1 inhibitors

Chk1 is the major mediator of cell-cycle checkpoints in response to various forms of genotoxic stress. Although it was previously speculated that checkpoint abrogation due to Chk1 inhibition may potentiate the efficacy of DNA-damaging agents through induction of mitotic catastrophe, there has not been direct evidence proving this process. Here, through both molecular marker and morphological analysis, we directly demonstrate that specific downregulation of Chk1 expression by Chk1 siRNA potentiates the cytotoxicities of topoisomerase inhibitors through the induction of premature chromosomal condensation and mitotic catastrophe. More importantly, we discovered that the cellular cyclin B1 level is the major determinant of the potentiation. We show that downregulation of cyclin B1 leads to impairment of the induction of mitotic catastrophe and correspondingly a reduction of the potentiation ability of either Chk1 siRNA or a small molecule Chk1 inhibitor. More significantly, we have extended the study by examining a panel of 10 cancer cell-lines with different tissue origins for their endogenous levels of cyclin B1 and the ability of a Chk1 inhibitor to sensitize the cells to DNA-damaging agents. The cellular levels of cyclin B1 positively correlate with the degrees of potentiation achieved. Of additional interest, we observed that the various colon cancer cell lines in general appear to express higher levels of cyclin B1 and also display higher sensitivity to Chk1 inhibitors, implying that Chk1 inhibitor may be more efficacious in treating colon cancers. In summary, we propose that cyclin B1 is a biomarker predictive of the efficacy of Chk1 inhibitors across different types of cancers. Unlike previously established efficacy-predictive biomarkers that are usually the direct targets of the therapeutic agents, cyclin B1 represents a non-drug-target biomarker that is based on the mechanism of action of the target inhibitor. This finding may be potentially very useful for the stratification of patients for Chk1 inhibitor clinical trials and hence, maximize its chance of success.     

CaMKKβ-AMPKα2 signaling contributes to mitotic Golgi fragmentation and the G2/M transition in mammalian cells

Before a cell enters mitosis, the Golgi apparatus undergoes extensive fragmentation. This is required for the correct partitioning of the Golgi apparatus into daughter cells, and inhibition of this process leads to cell cycle arrest in G2 phase. AMP-activated protein kinase (AMPK) plays critical roles in regulating growth and reprogramming metabolism. Recent studies have suggested that AMPK promotes mitotic progression and Golgi disassembly, and that this seems independent of the cellular energy status. However, the molecular mechanism underlying these events is not well understood. Here, we show that both treatment with compound C and depletion of AMPKα2 (but not AMPKα1) delays the G2/M transition in synchronized HeLa cells, as evidenced by flow cytometry and mitotic index analysis. Furthermore, knockdown of AMPKα2 specifically delays further fragmentation of isolated Golgi stacks. Interestingly, pAMPKα^Thr172 signals transiently appear in the perinuclear region of late G2/early prophase cells, partially co-localizing with the Golgi matrix protein, GM-130. These Golgi pAMPKα^Thr172 signals were also specifically abolished by AMPKα2 knockdown, indicating specific spatio-temporal activation of AMPKα2 at Golgi complex during late G2/early prophases. We also found that the specific CaMKKβ inhibitor, STO-609, reduces the pAMPKα ^Thr172 signals in the perinuclear region of G2 phase cells and delays mitotic Golgi fragmentation. Taken together, these data suggest that AMPKα2 is the major catalytic subunit of AMPKα which regulates Golgi fragmentation and G2/M transition, and that the CaMKKβ activates AMPKα2 during late G2 phase.     

Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.     

Impact of cranberry juice on initial adhesion of the EPS producing bacterium Burkholderia cepacia

The impact of cranberry juice was investigated with respect to the initial adhesion of three isogenic strains of the bacterium Burkholderia cepacia with different extracellular polymeric substance (EPS) producing capacities, viz. a wild-type cepacian EPS producer PC184 and its mutant strains PC184rml with reduced EPS production and PC184bceK with a deficiency in EPS production. Adhesion experiments conducted in a parallel-plate flow chamber demonstrated that, in the absence of cranberry juice, strain PC184 had a significantly higher adhesive capacity compared to the mutant strains. In the presence of cranberry juice, the adhesive capacity of the EPS-producing strain PC184 was largely reduced, while cranberry juice had little impact on the adhesion behavior of either mutant strain. Thermodynamic modeling supported the results from adhesion experiments. Surface force apparatus (SFA) and scanning electron microscope (SEM) studies demonstrated a strong association between cranberry juice components and bacterial EPS. It was concluded that cranberry juice components could impact bacterial initial adhesion by adhering to the EPS and impairing the adhesive capacity of the cells, which provides an insight into the development of novel treatment strategies to block the biofilm formation associated with bacterial infection.     

Phylogenetic relationships of green algae assigned to the genus Planophila (Chlorophyta): evidence from 18S rDNA sequence data and ultrastructure

Phylogenetic analyses, based upon nuclear small-subunit ribosomal RNA gene sequences, of four ‘chlorosarcinoid’ species referred to Planophila Gerneck show that the genus is polyphyletic. The type species, P. laetevirens Gerneck, is closely related to species in the Ulotrichales, Ulvophyceae. The monotypic sarcinoid genus Pseudendocloniopsis is the closest relative of Planophila; the two genera represent the addition of a new morphological type to the Ulotrichales. Planophila microcystis (Dangeard) Kornmann & Sahling forms a clade at the base of the Ulvophyceae with Oltmannsiellopsis, and thus belongs to the Oltmannsiellopsidales. This result is also supported by the Oltmannsiellopsis-like ultrastructure of P. microcystis zoospores. Planophila sp. B from Antarctica, which has Trebouxia-like pyrenoid structure, is a trebouxiophyte closely related to Chlorella-like unicellular coccoids, Stichococcus bacillaris and Prasiola species. This is the first robustly supported molecular phylogenetic analysis that places Prasiola in the Trebouxiophyceae. As shown previously, P. terrestris Groover & Hofstetter belongs to the Chaetopeltidales, Chlorophyceae. Dangemannia gen. nov. (type species : D. microcystis (Dangeard) comb. nov.), Floydiella gen. nov. (type species : F. terrestris (Groover & Hofstetter) comb. nov.) and Pabia gen. nov. (type: P. signiensis sp. nov.) are proposed.