Maria Marx

D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro

Synaptobrevin 2 (Sb2), syntaxin1 (Stx1), and synaptosomal-associated protein of 25 kDa (SNAP-25) are the main components of the soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex involved in fusion of synaptic vesicles with the presynaptic plasma membrane. We report the characterization of D53, a novel SNARE-binding protein preferentially expressed in neural and neuro-endocrine cells. Its two-dimensional organization, established by the hydrophobic cluster analysis, is reminiscent of SNARE proteins. D53 contains two putative helical regions, one of which includes a large coiled-coil domain involved in the interaction with Sb2 in vitro. Following subcellular fractionation, endogenous D53 was specifically detected in the membrane-containing fraction of PC12 cells, where it co-immunoprecipitated with Sb2. Analysis by confocal microscopy showed that, in these cells, endogenous D53 co-localized partially with the transferrin receptor in early endosomes. In vitro assays revealed that binding properties of D53 to Stx1 and Sb2 are comparable with those of SNAP-25. Furthermore, D53 forms Sb2/Stx1/D53 complexes in vitro in a manner similar to SNAP-25. We propose that D53 could be involved in the assembly or disassembly of endosomal SNARE complexes by regulating Sb2/Stx interaction.

Characterization of a Leucine Zipper-containing Protein Identified by Retroviral Insertion in Avian Neuroretina Cells

We reported previously that post-mitotic chicken embryonic neuroretina (NR) cells are induced to proliferate following in vitro infection with RAV-1, a retrovirus that does not carry an oncogene. NR cell multiplication results from the frequent activation and subsequent retroviral transduction of two related serine/threonine protein kinases, the c-mil/c-raf or c-Rmil/B-raf genes. We also showed that a very early event in the activation of these proto-oncogenes is the synthesis of chimeric mRNAs containing viral and cellular sequences joined by a splicing mechanism. In the current study, we have examined the ability of RAV-1 to induce proliferation of quail NR cells. By using the reverse transcription-polymerase chain reaction technique, we identified, in several proliferating quail NR cultures infected with RAV-1, a chimeric mRNA containing cellular sequences joined to the RAV-1 splice donor site. These cellular sequences are derived from a gene designated R10, which is expressed through a 1.9-kilobase (kb) mRNA detected in several embryonic tissues. A second transcript of 2.3 kb is specifically expressed in the NR, where both transcripts are developmentally regulated. The R10 cDNA encodes a 251-amino acid polypeptide that contains a leucine zipper motif. It exhibits significant similarity with the putative D52/N8L protein, encoded by an mRNA reported previously to be overexpressed in human breast and lung carcinomas. By using polyclonal antibodies specific for its amino-terminal and leucine zipper-containing regions, we identified the R10 gene product as a cytoplasmic protein of 23 kDa in cultured avian fibroblasts. A second protein of 30 kDa is detected in post-mitotic NR cells that express the 2.3-kb transcript. We also show, by in vitro transcription/translation and immunoprecipitation, that the R10 protein can readily form homodimers, presumably through its leucine zipper motif.

Expression pattern of the drm/gremlin gene during chicken embryonic development.

The drm gene encodes a cystine knot-containing secreted and cell membrane-associated glycoprotein shown to be an antagonist of BMPs. Drm was recently reported to play a crucial role in limb bud development, by its capacity to bind BMPs. Here, we have studied the expression pattern of drm transcripts during chicken development, by using whole-mount in situ hybridization. We show that, from stage 22HH to stage 26HH, in addition to limb buds, drm is expressed in cephalic neural crest-derived branchial arches I, II and III, in the medio-dorsal lip of the myotome and in the superficial dermatome

Amplification of the provirus region in Rous sarcoma virus-transformed Chinese hamster cells and segregation of the amplified copies in somatic cell hybrids

Four independent clones of RSV-transformed Chinese hamster fibroblasts were isolated. Southern blots and dot hybridization studies showed that in three out of four clones there were four to eight times as many integrated proviruses as in the fourth clone which contained at least one complete provirus. Restriction mapping studies showed that although the integration site varied from clone to clone, all the proviral copies in the same clone shared the same flanking cellular sequences. In one clone there are at least two polymorphic proviral variants A and B, one with and one without a BglI site. Further experiments were performed to see if the variants could be physically separated. RSV-Transformed Chinese hamster cells resistant to thioguanine were fused with mouse cells to give somatic hybrids which preferentially segregate Chinese hamster chromosomes. Ten out of eleven hybrids positive for virus rescue have lost up to 90% of the parental provirus copies. Four of these hybrids were found to contain the provirus variant A alone, one the variant B alone, and the rest contained both variants. All the proviruses retained in somatic hybrids shared the flanking cellular sequences of the parental provirus. Provirus segregation in somatic hybrids confirms that multiple (about ten) copies of the provirus region are present in the karyotype of parental RSV-transformed cells and, furthermore, suggests that the amplified copies of this region are translocated to different chromosomes.

Small deletion in v-src SH3 domain of a transformation defective mutant of rous sarcoma virus restores wild type transforming properties

RSV mutant virus PA101T was obtained while assaying the tumorigenicity of parental PA101 virus in chickens. PA101 is a transformation defective mutant of RSV which has a low src kinase activity. However, PA101 retained a temperature-sensitive ability to induce sustained proliferation of neuroretina cells. PA101T appeared as a wild-type phenotype revertant of PA101. Molecular cloning and sequencing of PA101T showed that this reversion is due to additional mutations in PA101 src gene. These mutations are a deletion eliminating three amino acids in the N-terminal region of SH3 domain and mutation of Ala 426 to Val. Analysis of the properties of chimeric src genes associating either half of PA101T with the complementary regions of PA101 or wild-type virus showed that the N-terminal moiety of PA101T src, which contains the deletion, confers wild-type transforming properties, whereas its C-terminal moiety, which contains single amino acid mutation, confers a partially temperature-sensitive phenotype. These results are consistent with other reports showing that mutations or deletions in this region of SH3 activate the transforming potential of c-src. They support the hypothesis that the N-terminal region of SH3 interacts with a cellular negative regulator of src activity.

Localized expression of drm/gremlin in the central nervous system of the chicken embryo

Drm/Gremlin is a member of the Dan family of bone morphogenetic protein (BMP) antagonists known to function in vertebrate limb outgrowth and lung morphogenesis. Its expression detected in neurons and astrocytes of the adult brain suggested a possible role in brain morphogenesis and/or neuronal versus glial differentiation. To investigate this role, we analysed its expression pattern in the central nervous system of the chicken embryo, by in situ hybridization. In the brain, we found that drm is mainly expressed in the medial pallium in the dorsal telencephalon and in the ventral diencephalon. drm was detected in the meninges of the spinal cord. We also found that drm was expressed in the developing optic nerve and at the optic nerve/pecten junction. In all these territories, distinct bmps are expressed. Taken together, these data suggest that Drm could play a role in the development of the medial pallium and during optic nerve and pecten development by modulating BMP signaling. Developmental Dynamics 229:688–694, 2004.

Notch signaling activation suppresses v-Src-induced transformation of neural cells by restoring TGF-β-mediated differentiation.

Background We have been investigating how interruption of differentiation contributes to the oncogenic process and the possibility to reverse the transformed phenotype by restoring differentiation. In a previous report, we correlated the capacity of intracellular Notch (ICN) to suppress v-Src-mediated transformation of quail neuroretina (QNR/v-srcts) cells with the acquisition by these undifferentiated cells of glial differentiation markers. Methodology/Principal Findings In this work, we have identified autocrine TGF-β3 signaling activation as a major effector of Notch-induced phenotypic changes, sufficient to induce transition in differentiation markers expression, suppress morphological transformation and significantly inhibit anchorage-independent growth. We also show that this signaling is constitutive of and contributes to ex-vivo autonomous QNR cell differentiation and that its down-regulation is essential to achieve v-Src-induced transformation. Conclusions/Significance These results support the possibility that Notch signaling induces differentiation and suppresses transformation by a novel mechanism, involving secreted proteins. They also underline the importance of extracellular signals in controlling the balance between normal and transformed phenotypes.

Role of QN1 protein in cell proliferation arrest and differentiation during the neuroretina development

In this report, we describe the involvement of the quail neuroretina 1 (QN1) protein in retinal development. The Qn1 cDNA was isolated as a gene specifically expressed at the onset of neuronal cell cycle withdrawal (Bidou et al., Mech. Dev. 43 (1993) 159). Qn1 is located in the cytoplasm in proliferating cells during the early stages of the development. Its distribution changes, becoming predominantly nuclear, in neurons during establishment of the quiescent state upon the differentiation. We decreased the amount of QN1 protein by an antisense strategy in vitro or in vivo. This decrease of the amount of QN1 protein results in additional mitosis and in severe abnormalities such as retinal dysplasia. Our results suggest that QN1 plays a key role at the onset of neuronal cell cycle withdrawal.