Spectroscopic investigation of local mechanical impedance of living cells

The mechanical properties of PC12 living cells have been studied at the nanoscale with a Force Feedback Microscope using two experimental approaches. Firstly, the local mechanical impedance of the cell membrane has been mapped simultaneously to the cell morphology at constant force. As the force of the interaction is gradually increased, we observed the appearance of the sub-membrane cytoskeleton. We shall compare the results obtained with this method with the measurement of other existing techniques. Secondly, a spectroscopic investigation has been performed varying the indentation of the tip in the cell membrane and consequently the force applied on it. In contrast with conventional dynamic atomic force microscopy techniques, here the small oscillation amplitude of the tip is not necessarily imposed at the cantilever first eigenmode. This allows the user to arbitrarily choose the excitation frequency in developing spectroscopic AFM techniques. The mechanical response of the PC12 cell membrane is found to be frequency dependent in the 1 kHz - 10 kHz range. The damping coefficient is reproducibly observed to decrease when the excitation frequency is increased.Comment: 8 pages, 8 figure

Myeloproliferative disorder FOP-FGFR1 fusion kinase recruits phosphoinositide-3 kinase and phospholipase Cγ at the centrosome

<p>Abstract</p> <p>Background</p> <p>The t(6;8) translocation found in rare and agressive myeloproliferative disorders results in a chimeric gene encoding the FOP-FGFR1 fusion protein. This protein comprises the N-terminal region of the centrosomal protein FOP and the tyrosine kinase of the FGFR1 receptor. FOP-FGFR1 is localized at the centrosome where it exerts a constitutive kinase activity.</p> <p>Results</p> <p>We show that FOP-FGFR1 interacts with the large centrosomal protein CAP350 and that CAP350 is necessary for FOP-FGFR1 localisation at centrosome. FOP-FGFR1 activates the phosphoinositide-3 kinase (PI3K) pathway. We show that p85 interacts with tyrosine 475 of FOP-FGFR1, which is located in a YXXM consensus binding sequence for an SH2 domain of p85. This interaction is in part responsible for PI3K activation. Ba/F3 cells that express FOP-FGFR1 mutated at tyrosine 475 have reduced proliferative ability. Treatment with PI3K pathway inhibitors induces death of FOP-FGFR1 expressing cells. FOP-FGFR1 also recruits phospholipase Cγ1 (PLCγ1) at the centrosome. We show that this enzyme is recruited by FOP-FGFR1 at the centrosome during interphase.</p> <p>Conclusion</p> <p>These results delineate a particular type of oncogenic mechanism by which an ectopic kinase recruits its substrates at the centrosome whence unappropriate signaling induces continuous cell growth and MPD.</p

The Rho-associated protein kinase p160ROCK is required for centrosome positioning

The p160–Rho-associated coiled-coil–containing protein kinase (ROCK) is identified as a new centrosomal component. Using immunofluorescence with a variety of p160ROCK antibodies, immuno EM, and depletion with RNA interference, p160ROCK is principally bound to the mother centriole (MC) and an intercentriolar linker. Inhibition of p160ROCK provoked centrosome splitting in G1 with the MC, which is normally positioned at the cell center and shows little motion during G1, displaying wide excursions around the cell periphery, similar to its migration toward the midbody during cytokinesis. p160ROCK inhibition late after anaphase in mitosis triggered MC migration to the midbody followed by completion of cell division. Thus, p160ROCK is required for centrosome positioning and centrosome-dependent exit from mitosis

Fifteen years of research on oral–facial–digital syndromes: from 1 to 16 causal genes

Oral–facial–digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype

Optimisation des propriétés photoréfractives du matériau Bi12GeO20: modélisation de la croissance par simulation numérique, dopage au cuivre et élaboration de couches minces

Dans le but d'optimiser et, conjointement, de mieux comprendre l'effet photoréfractif dans le matériau Bi12GeO20 (BGO), nous avons oriente nos recherches dans trois grandes directions. Dans un premier temps, une modélisation par simulation numérique de la croissance cristalline de BGO par la technique Czochralski a été entreprise afin de déterminer les conditions expérimentales les plus appropriées a l'obtention de cristaux de grande qualité optique. Dans un deuxième temps, nous nous sommes intéresses au dopage au cuivre de la matrice BGO. Celui-ci a été caractérise par des mesures physico-chimiques classiques complétées par des études spectroscopiques. Une interprétation de l'effet photochromique du matériau dope ou non a ete proposée. Enfin, nous avons élaboré par ablation laser des films monocristallins de BGO sur du saphir. L'existence d'un effet électrooptique linéaire, de propriétés photoconductrices et de doublage de frequence a été mise en évidence.non disponibl

De-repression of the RAC activator ELMO1 in cancer stem cells drives progression of TGFβ-deficient squamous cell carcinoma from transition zones.

International audienceSquamous cell carcinomas occurring at transition zones are highly malignant tumors with poor prognosis. The identity of the cell population and the signaling pathways involved in the progression of transition zone squamous cell carcinoma are poorly understood, hence representing limited options for targeted therapies. Here, we identify a highly tumorigenic cancer stem cell population in a mouse model of transitional epithelial carcinoma and uncover a novel mechanism by which loss of TGFβ receptor II (Tgfbr2) mediates invasion and metastasis through de-repression of ELMO1, a RAC-activating guanine exchange factor, specifically in cancer stem cells of transition zone tumors. We identify ELMO1 as a novel target of TGFβ signaling and show that restoration of Tgfbr2 results in a complete block of ELMO1 in vivo. Knocking down Elmo1 impairs metastasis of carcinoma cells to the lung, thereby providing insights into the mechanisms of progression of Tgfbr2-deficient invasive transition zone squamous cell carcinoma

Myomegalin is necessary for the formation of centrosomal and Golgi-derived microtubules

Summary The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in γ-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs), mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1), recently described as a nucleation activator, and is related to a family of γ-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits γ-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells

Myeloproliferative disorder FOP-FGFR1 fusion kinase recruits phosphoinositide-3 kinase and phospholipase Cγ at the centrosome-5

with mycFOP-FGFR1. Costaining using anti-CAP350 (green) and anti-myc (red) antibody reveals colocalization at the centrosome of CAP350 and mycFOP-FGFR1. Scale bar 5 μm. (B) FOP-FGFR1 interacts with CAP350 in lysates from Cos-1 cells transiently transfected with vectors expressing CAP350 C-terminus, and either FOP-FGFR1 or FGFR1. Immunoprecipitations are done with anti-FGFR1 antibody and bound CAP350 is revealed by western blotting using anti-CAP350 antibody. Anti-FGFR1 western blotting confirms the efficiency of the transfection. (C) Western blot with anti-CAP350 antibody shows reduced level of CAP350 in HeLa cell lysates after siRNA treatment for 48 h or 72 h with siCAP duplex as compared to sicontrol duplex. γ-tubulin is shown as a loading control. (D) Immunofluorescence of CAP350 depleted with siRNA CAP350 (bottom) and control (top) in FOP-FGFR1 GFP HeLa cells (a, b) and anti-CAP350 antibody (b, f) and γ-tubulin antibody (c, g). CAP350 depleted cells lack FOP-FGFR1. Scale bar 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Myeloproliferative disorder FOP-FGFR1 fusion kinase recruits phosphoinositide-3 kinase and phospholipase Cγ at the centrosome"</p><p>http://www.molecular-cancer.com/content/7/1/30</p><p>Molecular Cancer 2008;7():30-30.</p><p>Published online 15 Apr 2008</p><p>PMCID:PMC2373309.</p><p></p