A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei

Tissue-specific patterns of methylated deoxycytidine residues in the mammalian genome are preserved by postreplicative methylation of newly synthesized DNA. DNA methyltransferase (MTase) is here shown to associate with replication foci during S phase but to display a diffuse nucleoplasmic distribution in non-S phase cells. Analysis of DNA MTase-β-galactosidase fusion proteins has shown that association with replication foci is mediated by a novel targeting sequence located near the N-terminus of DNA MTase. This sequence has the properties expected of a targeting sequence in that it is not required for enzymatic activity, prevents proper targeting when deleted, and, when fused to β-galactosidase, causes the fusion protein to associate with replication foci in a cell cycle-dependent manner

Up-regulation of flotillins, new marker of metastatic development, induces cell invasion and metastatic development

Flotillin 1 and 2 are two ubiquitous, highly conserved and related proteins present in man

Upregulated flotillins and sphingosine kinase 2 derail AXL vesicular traffic to promote epithelial-mesenchymal transition

Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called upregulated flotillin-induced trafficking (UFIT). Here, we found that in non-tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane domains and endosomes. Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation.This work was supported by the Fondation pour la Recherche Médicale (DEQ20161136700) and the Fondation ARC pour la recherche sur le cancer (ARCPJA32020060002078). Collection of breast tumor samples was supported by the Russian Science Foundation (19-75-30016). C.G.-R. was supported by the Institut National de la Santé et de la Recherche Médicale.Peer reviewe

Mitotic progression, arrest, exit or death relies on centromere structural integrity, rather than de novo transcription

Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain a robust spindle assembly checkpoint (SAC) response. Here, we used live-cell imaging of human cells, RNA-seq and qPCR to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA- intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC signaling and cell fate. However, we were unable to detect significant changes in mitotic transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on Aurora B centromeric localization, SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, mitotic progression, arrest, exit or death is determined by centromere structural integrity, rather than de novo transcription

Activation of AMP-activated Protein Kinase Is Essential for Lysophosphatidic Acid-induced Cell Migration in Ovarian Cancer Cells

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-beta 3 (PLC-beta 3) and calcium/calmodulin-dependent protein kinase kinase beta (CaMKK beta) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPK beta 1, PLC-beta 3, or (CaMKK beta) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPK beta 1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.close161

RhoA leads to up-regulation and relocalization of utrophin in muscle fibers.

International audienceUp-regulation of utrophin, a homolog of dystrophin, is known to ameliorate the dystrophic phenotype in animal models of Duchenne muscular dystrophy. We have previously demonstrated that the active form of RhoA (RhoAV14) increases the expression of utrophin and its localization at the plasma membrane in cultured myoblasts. In this paper, we ask whether RhoAV14 can up-regulate utrophin also in mice. A plasmid encoding for RhoAV14 was injected into skeletal muscles followed by electroporation. Muscles expressing RhoAV14 were analyzed by Western-immunoblotting, real time PCR amplification and immunohistochemistry. We found that RhoAV14 increased utrophin protein expression and distribution specifically at the plasma membrane in muscle fibers without any effect on utrophin transcription. Utrophin up-regulation, uncoupled from that of its mRNA, has been previously observed in pathological processes and in normal regenerating conditions

P-cadherin-mediated Rho GTPase regulation during collective cell migration

International audienc