Centrosome Movements Are TUBG1-Dependent

The centrosome of mammalian cells is in constant movement and its motion plays a part in cell differentiation and cell division. The purpose of this study was to establish the involvement of the TUBG meshwork in centrosomal motility. In live cells, we used a monomeric red-fluorescenceprotein-tagged centrin 2 gene and a green-fluorescence-protein-tagged TUBG1 gene for labeling the centrosome and the TUBG1 meshwork, respectively. We found that centrosome movements occurredin cellular sites rich in GTPase TUBG1 and single-guide RNA mediated a reduction in the expression of TUBG1, altering the motility pattern of centrosomes. We propose that the TUBG1 meshwork enables the centrosomes to move by providing them with an interacting platform that mediates positional changes. These findings uncover a novel regulatory mechanism that controls the behavior of centrosomes

p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38–mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response

Expression of the RNA-binding protein RBM3 is associated with a favourable prognosis and cisplatin sensitivity in epithelial ovarian cancer

<p>Abstract</p> <p>Background</p> <p>We recently demonstrated that increased expression of the RNA-binding protein RBM3 is associated with a favourable prognosis in breast cancer. The aim of this study was to examine the prognostic value of RBM3 mRNA and protein expression in epithelial ovarian cancer (EOC) and the cisplatin response upon RBM3 depletion in a cisplatin-sensitive ovarian cancer cell line.</p> <p>Methods</p> <p>RBM3 mRNA expression was analysed in tumors from a cohort of 267 EOC cases (Cohort I) and RBM3 protein expression was analysed using immunohistochemistry (IHC) in an independent cohort of 154 prospectively collected EOC cases (Cohort II). Kaplan Meier analysis and Cox proportional hazards modelling were applied to assess the relationship between RBM3 and recurrence free survival (RFS) and overall survival (OS). Immunoblotting and IHC were used to examine the expression of RBM3 in a cisplatin-resistant ovarian cancer cell line A2780-Cp70 and its cisplatin-responsive parental cell line A2780. The impact of RBM3 on cisplatin response in EOC was assessed using siRNA-mediated silencing of RBM3 in A2780 cells followed by cell viability assay and cell cycle analysis.</p> <p>Results</p> <p>Increased RBM3 mRNA expression was associated with a prolonged RFS (HR = 0.64, 95% CI = 0.47-0.86, <it>p = 0.003</it>) and OS (HR = 0.64, 95% CI = 0.44-0.95, <it>p = 0.024</it>) in Cohort I. Multivariate analysis confirmed that RBM3 mRNA expression was an independent predictor of a prolonged RFS, (HR = 0.61, 95% CI = 0.44-0.84, <it>p = 0.003</it>) and OS (HR = 0.62, 95% CI = 0.41-0.95; <it>p = 0.028</it>) in Cohort I. In Cohort II, RBM3 protein expression was associated with a prolonged OS (HR = 0.53, 95% CI = 0.35-0.79, <it>p = 0.002</it>) confirmed by multivariate analysis (HR = 0.61, 95% CI = 0.40-0.92, <it>p = 0.017</it>). RBM3 mRNA and protein expression levels were significantly higher in the cisplatin sensitive A2780 cell line compared to the cisplatin resistant A2780-Cp70 derivative. siRNA-mediated silencing of RBM3 expression in the A2780 cells resulted in a decreased sensitivity to cisplatin as demonstrated by increased cell viability and reduced proportion of cells arrested in the G2/M-phase.</p> <p>Conclusions</p> <p>These data demonstrate that RBM3 expression is associated with cisplatin sensitivity <it>in vitro </it>and with a good prognosis in EOC. Taken together these findings suggest that RBM3 may be a useful prognostic and treatment predictive marker in EOC.</p

PLK1 Interacts and Phosphorylates Axin That Is Essential for Proper Centrosome Formation

10.1371/journal.pone.0049184PLoS ONE711

Small molecule sensitization to TRAIL is mediated via nuclear localization, phosphorylation and inhibition of chaperone activity of Hsp27

10.1038/cddis.2013.413Cell Death and Disease410

Diffusion is capable of translating anisotropic apoptosis initiation into a homogeneous execution of cell death

<p>Abstract</p> <p>Background</p> <p>Apoptosis is an essential cell death process throughout the entire life span of all metazoans and its deregulation in humans has been implicated in many proliferative and degenerative diseases. Mitochondrial outer membrane permeabilisation (MOMP) and activation of effector caspases are key processes during apoptosis signalling. MOMP can be subject to spatial coordination in human cancer cells, resulting in intracellular waves of cytochrome-c release. To investigate the consequences of these spatial anisotropies in mitochondrial permeabilisation on subsequent effector caspase activation, we devised a mathematical reaction-diffusion model building on a set of partial differential equations.</p> <p>Results</p> <p>Reaction-diffusion modelling suggested that even if strong spatial anisotropies existed during mitochondrial cytochrome c release, these would be eliminated by free diffusion of the cytosolic proteins that instantiate the apoptosis execution network. Experimentally, rapid sampling of mitochondrial permeabilisation and effector caspase activity in individual HeLa cervical cancer cells confirmed predictions of the reaction-diffusion model and demonstrated that the signalling network of apoptosis execution could efficiently translate spatial anisotropies in mitochondrial permeabilisation into a homogeneous effector caspase response throughout the cytosol. Further systems modelling suggested that a more than 10,000-fold impaired diffusivity would be required to maintain spatial anisotropies as observed during mitochondrial permeabilisation until the time effector caspases become activated.</p> <p>Conclusions</p> <p>Multi-protein diffusion efficiently contributes to eliminating spatial asynchronies which are present during the initiation of apoptosis execution and thereby ensures homogeneous apoptosis execution throughout the entire cell body. For previously reported biological scenarios in which effector caspase activity was shown to be targeted selectively to specific subcellular regions additional mechanisms must exist that limit or spatially coordinate caspase activation and/or protect diffusing soluble caspase substrates from unwanted proteolysis.</p

The Game of Tubulins

Members of the tubulin superfamily are GTPases; the activities of GTPases are necessary for life. The members of the tubulin superfamily are the constituents of the microtubules and the γ-tubulin meshwork. Mutations in members of the tubulin superfamily are involved in developmental brain disorders, and tubulin activities are the target for various chemotherapies. The intricate functions (game) of tubulins depend on the activities of the GTP-binding domain of α-, β-, and γ-tubulin. This review compares the GTP-binding domains of γ-tubulin, α-tubulin, and β-tubulin and, based on their similarities, recapitulates the known functions and the impact of the γ-tubulin GTP-binding domain in the regulation of the γ-tubulin meshwork and cellular homeostasis

Gamma-tubules — novel filaments in the fight against cancer

The word skeleton sounds very significant, and indeed humans could not exist without a skeleton! Similarly, a cellular skeleton or cytoskeleton is crucial for the functions of a cell. Several different types of cytoskeleton have been identified that are necessary, for among other things, muscle movement and the activities of the brain, and consequently humans could not function at all without cytoskeletons. This also means that a number of diseases are connected with defects in cytoskeletal elements, and various chemotherapies that are presently used to treat a variety of tumors aim to impair the functions of cytoskeletons. Inasmuch as cytoskeletons play a crucial role in the actions of cells and of the human body, and considering that modulation of the activities of cytoskeletons is essential for the treatment of patients, the finding of a novel cytoskeletal element should open up a new area of research—a new scientific world

A simple and fast method for fixation of cultured cell lines that preserves cellular structures containing gamma-tubulin

When using fluorescence microscope techniques to study cells, it is essential that the cell structure and contents are preserved after preparation of the samples, and that the preparation method employed does not create artefacts that can be perceived as cellular structure/components. γ-Tubulin forms filaments that in some cases are immunostained with an anti-γ-tubulin antibody, but this immunostaining is not reproducible [1,2]. In addition, the C terminal region of γ-tubulin (green fluorescence protein tagged [GFP]-γ-tubulin334––449) forms cytosolic GFP-labeled structures, which can easily be imaged in live cells but are not preserved in fixed cells [1,3]. The purpose of this study was to identify a fixation technique that preserves cellular constituents containing γ-tubulin. • This protocol describes a method that preserves γ-tubulin-containing structures in fixed cells.• The technique entails two-step fixation. A pre-fixation step using paraformaldehyde is followed by a final fixation and permeabilization step performed at −80 °C.• In comparison with other methodology for fixation [4–6], the technique presented here uses a short pre-fixation step with a mixture of paraformaldehyde and sucrose followed by a short fixation/permeabilization step with a mixture of methanol and acetone at −80 °C