The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion

Background: Cell adhesion molecules are plasma membrane proteins specialized in cell-cell recognition and adhesion. Two related adhesion molecules, Necl-1 and Necl-2/SynCAM, were recently described and shown to fulfill important functions in the central nervous system. The purpose of the work was to investigate the distribution, and the properties of Necl-3/SynCAM-2, a previously uncharacterized member of the Necl family with which it shares a conserved modular organization and extensive sequence homology. Results: We show that Necl-3/SynCAM-2 is a plasma membrane protein that accumulates in several tissues, including those of the central and peripheral nervous system. There, Necl-3/SynCAM-2 is expressed in ependymal cells and in myelinated axons, and sits at the interface between the axon shaft and the myelin sheath. Several independent assays demonstrate that Necl-3/SynCAM-2 functionally and selectively interacts with oligodendrocytes. We finally prove that Necl-3/SynCAM-2 is a bona fide adhesion molecule that engages in homo- and heterophilic interactions with the other Necl family members, leading to cell aggregation. Conclusion: Collectively, our manuscripts and the works onNecl-1 and SynCAM/Necl-2 reveal a complex set of interactions engaged in by the Necl proteins in the nervous system. Our work also support the notion that the family of Necl proteins fulfils key adhesion and recognition functions in the nervous system, in particular between different cell types

An RNA Polymerase II Complex Containing All Essential Initiation Factors Binds to the Activation Domain of PAR Leucine Zipper Transcription Factor Thyroid Embryonic Factor

Transcription initiation of protein-encoding genes involves the assembly of RNA polymerase II and a number of general transcription factors at the promoter. A mammalian RNA polymerase II complex containing all of the components required for promoter-specific transcription initiation can be isolated by immunopurification with a monoclonal antibody directed against the cyclin-dependent kinase CDK7, a subunit of the general transcription factor TFIIH. In vitro transcription by this immunopurified RNA polymerase II complex is effectively stimulated by thyroid embryonic factor (TEF), a basic leucine zipper transcription factor. Thus, the RNA polymerase II complex must also contain components required for activated transcription that interact with the transactivation domain of TEF. This conjecture was verified by affinity selection experiments in which the TEF transcription activation domain was used as a bait. Indeed, an RNA polymerase II complex containing all of the accessory proteins required for transcription initiation can be enriched by its affinity to recombinant proteins containing the TEF transactivation domain. These results are compatible with a mechanism by which TEF can recruit an RNA polymerase II holoenzyme to the promoter in a single step

The two PAR leucine zipper proteins, TEF and DBP, display similar circadian and tissue-specific expression, but have different target promoter preferences

The two highly related PAR basic region leucine zipper proteins TEF and DBP accumulate according to a robust circadian rhythm in liver and kidney. In liver nuclei, the amplitude of daily oscillation has been estimated to be 50‐fold and 160‐fold for TEF and DBP, respectively. While DBP mRNA expression is the principal determinant of circadian DBP accumulation, the amplitude of TEF mRNA cycling is insufficient to explain circadian TEF fluctuation. Conceivably, daily variations in TEF degradation or nuclear translocation efficiency may explain the discrepancy between mRNA and protein accumulation. In vitro, TEF and DBP bind the same DNA sequences. Yet, in co‐transfection experiments, these two proteins exhibit different activation potentials for two reporter genes examined. While TEF stimulates transcription from the albumin promoter more potently than DBP, only DBP is capable of activating transcription efficiently from the cholesterol 7 alpha hydroxylase (C7alphaH) promoter. However, a TEF‐DBP fusion protein, carrying N‐terminal TEF sequences and the DNA binding/dimerization domain of DBP, enhances expression of the C7alphaH‐CAT reporter gene as strongly as wild‐type DBP. Our results suggest that the promoter environment, rather than the affinity with which PAR proteins recognize their cognate DNA sequences in vitro, determines the promoter preferences of TEF and DBP

The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion

Abstract Background Cell adhesion molecules are plasma membrane proteins specialized in cell-cell recognition and adhesion. Two related adhesion molecules, Necl-1 and Necl-2/SynCAM, were recently described and shown to fulfill important functions in the central nervous system. The purpose of the work was to investigate the distribution, and the properties of Necl-3/SynCAM-2, a previously uncharacterized member of the Necl family with which it shares a conserved modular organization and extensive sequence homology. Results We show that Necl-3/SynCAM-2 is a plasma membrane protein that accumulates in several tissues, including those of the central and peripheral nervous system. There, Necl-3/SynCAM-2 is expressed in ependymal cells and in myelinated axons, and sits at the interface between the axon shaft and the myelin sheath. Several independent assays demonstrate that Necl-3/SynCAM-2 functionally and selectively interacts with oligodendrocytes. We finally prove that Necl-3/SynCAM-2 is a bona fide adhesion molecule that engages in homo- and heterophilic interactions with the other Necl family members, leading to cell aggregation. Conclusion Collectively, our manuscripts and the works on Necl-1 and SynCAM/Necl-2 reveal a complex set of interactions engaged in by the Necl proteins in the nervous system. Our work also support the notion that the family of Necl proteins fulfils key adhesion and recognition functions in the nervous system, in particular between different cell types.</p

(A) The image in the red-pass channel shows Fc-Necl-3 revealed by a rhodamine-coupled secondary antibody

<p><b>Copyright information:</b></p><p>Taken from "The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion"</p><p>http://www.biomedcentral.com/1471-2202/8/90</p><p>BMC Neuroscience 2007;8():90-90.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2176061.</p><p></p> (B) The green-pass channel shows that Fc-Necl-3 binds to oligodendrocytes, which are identified by the specific mAb RIP revealed with an Alexa 594 secondary antibody. Note that Rip-negative cell-types are not bound by Fc-Necl-3 (arrows in A and B). The green and red images are merged in (C). Nuclei were visualized with DAPI in (A), (B). The field shown in the figure is representative of more than 100 rhodamine-positive cells, all of which were also Rip-positive

12 μm sagittal sections of the caudate putamen in a two-month-old rat were processed for immunohistochemistry

<p><b>Copyright information:</b></p><p>Taken from "The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion"</p><p>http://www.biomedcentral.com/1471-2202/8/90</p><p>BMC Neuroscience 2007;8():90-90.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2176061.</p><p></p> The sections were incubated with anti-Necl-3 and anti Rip antibodies, which were revealed with secondary antibodies as in Fig. 5. (A), Rip immunoreactivity in the green-pass channel; (B), Necl-3 immunoreactivity in the red-pass channel. The green and red images are merged in (C). Nuclei were visualized with DAPI in (A) and (B). All images represent the average projection of 9 z-stacks of 0.12 micron each. Note that although present in the same areas, the Necl-3 and oligodendrocytic immunoreactivities are not overlapping

12 μm trigeminal nerve sections of a four-month-old rat were processed for immunohistochemistry

<p><b>Copyright information:</b></p><p>Taken from "The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion"</p><p>http://www.biomedcentral.com/1471-2202/8/90</p><p>BMC Neuroscience 2007;8():90-90.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2176061.</p><p></p> The sections were incubated with anti-Necl-3 and anti neurofilament antibodies (A, B, C, 10 z-stacks of 0.24 micron each) or with anti-Necl-3 and anti MBP antibodies (D, E, F, 10 z-stacks of 0.12 micron each), which were revealed with secondary antibodies as in Fig. 5. (A), neurofilament immunoreactivity in the green-pass channel; (B), Necl-3 immunoreactivity in the red-pass channel; panel (D), Rip immunoreactivity in the green-pass channel; (E), Necl-3 immunoreactivity in the red-pass channel. The green and red images are merged in (C) and (F). Nuclei were visualized with DAPI in (A), (B), (D), and (E)

12 μm sagittal brain sections of a two-month-old rat were processed for immunohistochemistry

<p><b>Copyright information:</b></p><p>Taken from "The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion"</p><p>http://www.biomedcentral.com/1471-2202/8/90</p><p>BMC Neuroscience 2007;8():90-90.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2176061.</p><p></p> The vestibular nucleus is visualized in the top sections (A, B, C) as the average projection of 20 z-stacks of 0.24 micron each, and in the bottom sections the pons (D, E, F) as the average projection of 26 z-stacks of 0.12 micron each. The sections were incubated with anti-Necl-3 and anti Rip antibodies (A, B, C) or with anti-Necl-3 and anti MBP antibodies (D, E, F), which were revealed with secondary antibodies as in Fig. 5. (A), Rip immunoreactivity in the green-pass channel; (B), Necl-3 immunoreactivity in the red-pass channel; (D), MBP immunoreactivity in the green-pass channel; (E), Necl-3 immunoreactivity in the red-pass channel. The green and red images are merged in (C) and (F). Nuclei were visualized with DAPI in (A), (B), (D), and (E)