Molecular characterisation of the small GTPase CDC42 in the ectomycorrhizal fungus Tuber borchii Vittad.

Summary. The small GTPase CDC42 is ubiquitously expressed in eukaryotes, where it participates in the regulation of the cytoskeleton and a wide range of cellular processes, including cytokinesis, gene expression, cell cycle progression, apoptosis, and tumorigenesis. As very little is known on the molecular level about mycorrhizal morphogenesis and development and these events depend on a tightly regulated reorganisation of the cytoskeleton network in filamentous fungi, we focused on the molecular characterisation of the cdc42 gene in Tuber borchii Vittad., an ascomycetous hypogeous fungus forming ectomycorrhizae. The entire gene was isolated from a T. borchii cDNA library and Southern blot analyses showed that only one copy of cdc42 is present in the T. borchii genome. The predicted amino acid sequence is very similar to those of other known small GTPases and the similar domain structures suggest a similar function. Real-time PCR analyses revealed an increased expression of Tbcdc42 during the phase preparative to the instauration of symbiosis, in particular after stimulation with root exudate extracts. Immunolocalisation experiments revealed an accumulation of CDC42 in the apical tips of the growing hyphae. When a constitutively active Tbcdc42 mutant was expressed in Saccharomyces cerevisiae, morphological changes typical of pseudohyphal growth were observed. Our results suggest a fundamental role of CDC42 in cell polarity development in T. borchii. Correspondence and reprints: Istituto di Chimica Biologica “G. Fornaini”, Università degli Studi di Urbino, Via Saffi 2, 61029 Urbino, Italy.     

Cdc42和球形肌动蛋白在卵母细胞胞质分裂中的定位分析

研究活性Cdc42与球形肌动蛋白(G-actin)在爪蟾卵母细胞胞质分裂中的定位关系。分别用GFP-wGBDmRNA与罗丹明-594-微管蛋白、Alexa-488-球形肌动蛋白与罗丹明-594-微管蛋白、GFP-wGBDmRNA与Alexa-594-球形肌动蛋白共同显微注射爪蟾卵母细胞。利用共聚焦显微镜,时间延迟摄影方法,分别观察活体卵母细胞中活性Cdc42、球形肌动蛋白在胞质分裂过程中的定位,以及活性Cdc42与球形肌动蛋白在胞质分裂中的定位关系。在卵母细胞胞质分裂中,活性Cdc42与球形肌动蛋白存在空间上共定位现象,并且在时相上具有一致性。结果提示活性Cdc42和球形肌动蛋白在卵母细胞胞质分裂过程中密切相关。     

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.     

Identification of the functional expression of adenosine A<Subscript>3</Subscript> receptor in pancreas using transgenic mice expressing jellyfish apoaequorin

To investigate the functional expression of adenosine A₃ receptor (A3AR) in mammalian living tissues, we generated an apoaequorin-transgenic mouse that expresses jellyfish apoaequorin throughout its body. The expression of apoaequorin under the control of a strong CAG promoter was detected in various tissues, including the abdominal skin, adipose, ear, brain, esophagus, heart, inferior vena cava vessel, kidney, lens, liver, lung, pancreas, skeletal muscle, spleen, tail, testis, and thymus. The transgene was mapped to the C1–2 region of chromosome 16 by Fluorescence in situ hybridization analysis. Among these transgenic mouse tissues, we succeeded in detecting elevated responses of intracellular Ca²⁺ as a light emission of aequorin induced by the A3AR agonist in the pancreas, brain, and testis, the last two of which are known to be main tissues abundantly expressing A3AR. The A3AR agonist led to the phosphorylation of both extracellular signal-regulated kinase 1/2 and protein kinase B in mouse pancreas, and all the intracellular responses via A3AR were antagonized by the A3AR-specific antagonist. In addition, the mRNA expression of A3AR and the A3AR-induced intracellular responses were also found in the rat pancreatic acinar cell line AR42J. These results suggest that pancreas is one of the main tissues functionally expressing A3AR in mammalians in vivo, and that the present approach using transgenic mice that express apoaequorin throughout their bodies will facilitate the functional analysis of proteins of interest. Kazuya Yamano and Katsuhiro Mori contributed equally to this work     

StarD13 negatively regulates invadopodia formation and invasion in high-grade serous (HGS) ovarian adenocarcinoma cells by inhibiting Cdc42

Metastasis remains the main challenge to overcome for treating ovarian cancers. In this study, we investigate the potential role of the Cdc42 GAP StarD13 in the modulation of cell motility, invasion in ovarian cancer cells. StarD13 depletion does not affect the 2D motility of ovarian cancer cells. More importantly, StarD13 inhibits matrix degradation, invadopodia formation and cell invasion through the inhibition of Cdc42. StarD13 does not localize to mature TKS4-labeled invadopodia that possess matrix degradation ability, while a Cdc42 FRET biosensor, detects Cdc42 activation in these invadopodia. In fact, StarD13 localization and Cdc42 activation appear mutually exclusive in invadopodial structures. Finally, for the first time we uncover a potential role of Cdc42 in the direct recruitment of TKS4 to invadopodia. This study emphasizes the specific role of StarD13 as a narrow spatial regulator of Cdc42, inhibiting invasion, suggesting the suitability of StarD13 for targeted therapy.     

Signaling networks regulating leukocyte podosome dynamics and function

Podosomes are ventral adhesion structures prominent in cells of the myeloid lineage. A common aspect of these cells is that they are highly motile and must to traverse multiple tissue barriers in order to perform their functions. Recently podosomes have gathered attention from researchers as important cellular structures that can influence cell adhesion, motility and matrix remodeling. Adhesive and soluble ligands act via transmembrane receptors and propagate signals to the leukocyte cytoskeleton via small G proteins of the Rho family, tyrosine kinases and scaffold proteins and are able to induce podosome formation and rearrangements. Manipulation of the signals that regulate podosome formation and dynamics can therefore be a strategy to interfere with leukocyte functions in a multitude of pathological settings, such as infections, atherosclerosis and arthritis. Here, we review the major signaling molecules that act in the formation and regulation of podosomes.     

What's Nu(SAP) in mitosis and cancer?

Unperturbed mitosis is a prerequisite for the generation of two genetically identical daughter cells. Nucleolar-spindle associated protein (NuSAP) is an important mitotic regulator. The activity of NuSAP is essential for a variety of cellular events that occur during mitosis starting from spindle assembly to cytokinesis. In addition to playing crucial roles during mitosis, NuSAP has been in the spotlight recently due to different studies exhibiting its importance in embryogenesis and cancer. In this review, we have extensively mined the current literature and made connections between different studies involving NuSAP. Importantly, we have assembled data pertaining to NuSAP from several proteomic studies and analyzed it thoroughly. Our review focuses on the role of NuSAP in mitosis and cancer, and brings to light several unanswered questions regarding the regulation of NuSAP in mitosis and its role in carcinogenesis.     

Quinazolinones as γ-secretase modulators

Synthesis, SAR and evaluation of styrenyl quinazolinones as novel gamma secretase modulators are presented in this communication. Starting from literature and in-house leads we evaluated a range of quinazolinones which showed good modulation of γ-secretase activity.     

海南坡鹿CDC42 cDNA的克隆、原核表达及纯化

应用RT-PCR方法对海南坡鹿细胞分裂周期蛋白42(Cell division cycle 42,CDC42)编码区进行扩增,将扩增产物与PET-42a载体连接,重组质粒鉴定正确后,进行生物信息学分析;构建pET42a-hdCDc42表达载体,经IPTG诱导表达,将表达产物进行SDS-PAGE、可溶性分析、纯化及Wes...