Wiechers, Marianne F.
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Profiling of Human Neural Crest Chemoattractant Activity as a Replacement of Fetal Bovine Serum for In Vitro Chemotaxis Assays
Fetal bovine serum (FBS) is the only known stimulus for the migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis as-says to be incorporated in a test battery for reproductive and developmental toxicity. We con-firmed here in an optimized transwell assay that FBS triggers directed migration along a con-centration gradient. The responsible factor was found to be a protein in the 30-100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chem-otactic for NCCs in animal models (e.g., VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined, whether human cells would produce any fac-tor able to trigger NCC migration in a broad screening approach. We found that HepG2 hepa-toma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as a direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease sensitivity suggested also a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants.
Reggies/flotillins regulate retinal axon regeneration in the zebrafish optic nerve and differentiation of hippocampal and N2a neurons
The reggies/flotillins proteins upregulated during axon regeneration in retinal ganglion cells (RGCs) are scaffolding proteins of microdomains and involved in neuronal differentiation. Here, we show that reggies regulate axon regeneration in zebrafish (ZF) after optic nerve section (ONS) in vivo as well as axon/neurite extension in hippocampal and N2a neurons in vitro through signal transduction molecules modulating actin dynamics. ZF reggie-1a, -2a, and -2b downregulation by reggie-specific morpholino (Mo) antisense oligonucleotides directly after ONS significantly reduced ZF RGC axon regeneration: RGC axons from reggie Mo retinas were markedly reduced. Moreover, the number of axon-regenerating RGCs, identified by insertion of A488-coupled dextran, decreased by 69% in retinas 7 d after Mo application. At 10 and 14 d, RGCs decreased by 53 and 33%, respectively, in correlation with the gradual inactivation of the Mos. siRNA-mediated knockdown of reggie-1 and -2 inhibited the differentiation and axon/neurite extension in hippocampal and N2a neurons. N2a cells had significantly shorter filopodia, more cells had lamellipodia and fewer neurites, defects which were rescued by a reggie-1 construct without siRNA-binding sites. Furthermore, reggie knockdown strongly perturbed the balanced activation of the Rho family GTPases Rac1, RhoA, and cdc42, influenced the phosphorylation of cortactin and cofilin, the formation of the N-WASP, cortactin and Arp3 complex, and affected p38, Ras, ERK1/2 (extracellular signal-regulated kinases 1 and 2), and focal adhesion kinase activation. Thus, as suggested by their prominent re-expression after lesion, the reggies represent neuron-intrinsic factors for axon outgrowth and regeneration, being crucial for the coordinated assembly of signaling complexes regulating cytoskeletal remodeling.
Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2
Using confocal laser scanning and double immunogold electron microscopy, we demonstrate that reggie-1 and -2 are colocalized in
Substrate properties of zebrafish Rtn4b/Nogo and axon regeneration in the zebrafish optic nerve
This study explored why lesioned retinal ganglion cell (RGC) axons regenerate successfully in the zebrafish optic nerve despite the presence of Rtn4b, the homologue of the rat neurite growth inhibitor RTN4-A/Nogo-A. Rat Nogo-A and zebrafish Rtn4b possess characteristic motifs (M1-4) in the Nogo-A-specific region, which contains delta20, the most inhibitory region of rat Nogo-A. To determine whether zebrafish M1-4 is inhibitory as rat M1-4 and Nogo-A delta20, proteins were recombinantly expressed and used as substrates for zebrafish single cell RGCs, mouse hippocampal neurons and goldfish, zebrafish and chick retinal explants. When offered as homogenous substrates, neurites of hippocampal neurons and of zebrafish single cell RGCs were inhibited by zebrafish M1-4, rat M1-4, and Nogo-A delta20. Neurite length increased when zebrafish single cell RGCs were treated with receptor-type-specific antagonists and, respectively, with morpholinos (MO) against S1PR2 and S1PR5a-which represent candidate zebrafish Nogo-A receptors. In a stripe assay, however, where M1-4 lanes alternate with polylysine-(Plys)-only lanes, RGC axons from goldfish, zebrafish, and chick retinal explants avoided rat M1-4 but freely crossed zebrafish M1-4 lanes-suggesting that zebrafish M1-4 is growth permissive and less inhibitory than rat M1-4. Moreover, immunostainings and dot blots of optic nerve and myelin showed that expression of Rtn4b is very low in tissue and myelin at 3-5 days after lesion when axons regenerate. Thus, Rtn4b seems to represent no major obstacle for axon regeneration in vivo because it is less inhibitory for RGC axons from retina explants, and because of its low abundance.
PrPc capping in T cells promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads to signal transduction : [Langfassung]
The cellular prion protein (PrPc) resides in lipid rafts, yet the type of raft and the physiological function of PrPc are unclear. We show here that cross-linking of PrPc with specific antibodies leads to 1) PrPc capping in Jurkat and human peripheral blood T cells; 2) to cocapping with the intracellular lipid raft proteins reggie-1 and reggie-2; 3) to signal transduction as seen by MAP kinase phosphorylation and an elevation of the intracellular Ca2+ concentration; 4) to the recruitment of Thy-1, TCR/CD3, fyn, lck and LAT into the cap along with local tyrosine phosphorylation and F-actin polymerization, and later, internalization of PrPc together with the reggies into limp-2 positive lysosomes. Thus, PrPc association with reggie rafts triggers distinct transmembrane signal transduction events in T cells that promote the focal concentration of PrPc itself by guiding activated PrPc into preformed reggie caps and then to the recruitment of important interacting signaling molecules.
FAST TRACK : Identification of Reggie-1 and Reggie-2 as Plasmamembrane-Associated Proteins Which Cocluster with Activated GPI-Anchored Cell Adhesion Molecules in Non-Caveolar Micropatches in Neurons
Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosylphosphatidylinositol (GPI)-anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie-1 and -2 (80% identical to goldfish reggie proteins) shows that reggie-2 is practically identical to mouse flotillin-1. Flotillin-1 and epidermal surface antigen (ESA) (flotillin-2) are suggested to represent possible membrane proteins in caveolae. Rat reggie-1 is 99% homologous to ESA in overlapping sequences but has a 49-amino-acid N-terminus not present in ESA. Antibodies (ABs) which recognize reggie-1 or -2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons
[dorsal root ganglia (DRGs), retinal ganglion, and PC-12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti-caveolin negative) neurons and show anti-reggie-1 immunogoldlabeled clusters at the plasmamembrane of DRGs. When ABs against the GPI-anchored cell adhesion molecules (CAMs) F3 and Thy-1 are applied to live DRGs, the GPI-linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti-reggie antibodies. Thus, reggie-1 and reggie-2 identify sites where activated GPIlinked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains).
Reggies/Flotillins regulate E-cadherin-mediated cell contact formation by affecting EGFR trafficking
The reggie/flotillin proteins were implicated in membrane trafficking and together with the cellular prion protein (PrP) in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies as well as PrP downregulation in epithelial A431 cells caused overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion resulted from the reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduced Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl, and led to increased surface-exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhanced cell motility and macropinocytosis through which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restored normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling, regulate AJ formation and dynamics and thereby cell adhesion.
Isolation and identification of the plasma membrane-associated intracellular protein reggie-2 from goldfish brain by chromatography and Fourier-transform ion cyclotron resonance mass spectrometry
The neuronal protein reggie-2 is localized at the cytoplasmic face of the plasma membrane and participates, together with reggie-1, in the formation of plasma membrane microdomains. Reggie-2 exhibits several potential phosphorylation sites but whether the relevant sites are modified accordingly is not yet known. In order to obtain a detailed, molecular characterization of the primary structure of the native protein, an effective procedure for the isolation of the different reggie proteins from animal tissue is required. The specific properties of the proteins, particularly their membrane association and low abundance, make approaches for isolation such as affinity chromatography and 2D gel electrophoresis unfeasible. This study describes a rapid and efficient procedure for the isolation of reggie-2 by use of two consecutive HPLC steps and subsequent SDS-PAGE. The protein fractions were characterized by SDS-PAGE and Western blot analysis as well as by mass spectrometry. In the primary structure analysis by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS), the efficiency of high-resolution Fourier-transform ion cyclotron resonance-MALDI-MS was demonstrated, enabling the direct, unequivocal, and sensitive characterization of posttranslationally and/or chemically modified proteins.