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

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

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

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

Ett nytt cellskelett i kampen mot cancer

Forskare vid Lunds universitet har upptäckt ett cellskelett som ger struktur åt mito-kondrierna, cellens energifabriker. Skelettet är nödvändigt för mitokondriernas funktion, men forskarna fann även att cancerceller trots defekter utnyttjar skelettet för att kunna bibehålla sin energiproduktion – och därigenom överleva. Cellskelettet i mitokondrierna fungerar ungefär som ett armeringsnät och hjälper till att bibehålla cellens uppbyggnad och bidrar till stabiliteten i cellens olika rum. Det gör att olika molekyler lättare kan ta sig runt i cellen, skriver Maria Alvarado Kristensson, docent och forskare i molekylär patologi vid Lunds universitet

γ-tubulin as a signal-transducing molecule and meshwork with therapeutic potential

Cancer: Cytoskeleton regulator identified as promising drug target The association of γ-tubulin with tumor growth suggests that γ-tubulin inhibitors could be used as anticancer therapeutics. γ-tubulin is a member of the tubulin family that is best known for regulating the assembly of microtubules, part of the cell’s cytoskeleton. Maria Alvarado-Kristensson at Lund University, Sweden, reviews recent studies on γ-tubulin-rich structures that form a meshwork in both the cell cytoplasm and nucleus. These structures not only ensure the proper positioning of organelles but also that key signaling proteins are in the right place at the right time for cell cycle progression. High levels of γ-tubulin are found in various tumors and compounds that target γ-tubulin activity reduce tumor growth in animal models. Further work will determine whether drugs that target γ-tubulin will be more effective and have fewer side effects than those developed to target α- and β-tubulin

Choreography of the centrosome

More than a century ago, the centrosome was discovered and described as “the true division organ of the cell”. Electron microscopy revealed that a centrosome is an amorphous structure or pericentriolar protein matrix that surrounds a pair of well-organized centrioles. Today, the importance of the centrosome as a microtubule-organizing center and coordinator of the mitotic spindle is questioned, because centrioles are absent in up to half of all known eukaryotic species, and various mechanisms for acentrosomal microtubule nucleation have been described. This review recapitulates the known functions of centrosome movements in cellular homeostasis and discusses knowledge gaps in this field

Regulation of neutrophil apoptosis

The human neutrophil is the most abundant granulocyte and the major type of cell involved in an acute inflammatory response. Neutrophils are armed with various systems of enzymes, that can find and kill pathogens, but unfortunately, these "weapons" cannot distinguish between the host tissues and the "invaders." Therefore, an extensive neutrophil reaction leads to continuous release of toxic metabolites, which causes successive self-destruction of host tissues and possibly also organ failure. Such a series of destructive events has been implicated in diseases such as rheumatoid arthritis, myocardial infarction/reperfusion injury, atherogenesis, asthma, cystic fibrosis, emphysema, and vasculitis. Resolution of an acute inflammatory process depends on termination of neutrophil emigration from blood vessels and clearance of extravasated neutrophils and their metabolic products. Outside the blood vessels, neutrophils spontaneously undergo apoptosis, and are therefore removed by phagocytic cells at the site of inflammation. Neutrophil apoptosis can be modulated by several factors in the local environment, such as the Fas ligand (FasL), but the molecular mechanisms involved are poorly understood. In this dissertation thesis, I describe and elucidate intracellular signalling mechanisms that are involved in regulation of spontaneous and Fas-induced apoptosis in human neutrophils. Using two different methods it was possible to detect constitutive activity of p38 mitogen-activated protein kinase (p38) in newly isolated neutrophils. The p38 survival signal was transiently lost during both spontaneous and Fas-induced apoptosis, favoured induction of the apoptotic process. During the transient loss of p38 activity there was a temporary Fas-induced increase in phosphatidylinositol 3-kinase (PI3K) activity, which also had a pro-apoptotic impact on the neutrophils. In addition, my experiments showed that the active form of p38 associates with caspase 8 and caspase 3, which is necessary for p38-induced phosphorylation of serine-362 and serine-150 on these caspases. These biochemical modifications impair the activities, and possibly also the stability, of caspase 8 and 3 and thereby weaken the capacity of these enzymes to induce apoptosis. The results in this dissertation also demonstrate that the protein phosphatase type 2A (PP2A) can directly and independently decrease the phosphorylation levels of both p38 and caspase 3. Consequently, PP2A can increase the activity of caspase 3 by dual mechanisms and thereby promote the apoptotic response in human neutrophils