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1.
J Comp Neurol ; 525(14): 2991-3009, 2017 Oct 01.
Article in English | MEDLINE | ID: mdl-28560734

ABSTRACT

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.


Subject(s)
Axons/physiology , Myelin Proteins/metabolism , Nerve Regeneration , Nogo Proteins/metabolism , Optic Nerve Injuries/physiopathology , Optic Nerve/physiology , Retinal Ganglion Cells/physiology , Zebrafish Proteins/metabolism , Amino Acid Motifs , Animals , Cells, Cultured , Chick Embryo , Goldfish , Hippocampus/pathology , Hippocampus/physiopathology , Mice, Inbred C57BL , Myelin Proteins/chemistry , Myelin Sheath/metabolism , Neuronal Outgrowth/physiology , Nogo Proteins/chemistry , Nogo Receptors/antagonists & inhibitors , Nogo Receptors/metabolism , Optic Nerve/pathology , Optic Nerve Injuries/pathology , Rats , Retina/pathology , Retina/physiopathology , Tissue Culture Techniques , Tissue Scaffolds , Zebrafish , Zebrafish Proteins/chemistry
3.
Exp Neurol ; 289: 31-45, 2017 03.
Article in English | MEDLINE | ID: mdl-27993509

ABSTRACT

Reggie-1 and -2 (flotillins) reside at recycling vesicles and promote jointly with Rab11a the targeted delivery of cargo. Recycling is essential for synapse formation suggesting that reggies and Rab11a may regulate the development of spine synapses. Recycling vesicles provide cargo for dendritic growth and recycle surface glutamate receptors (AMPAR, GluA) for long-term potentiation (LTP) induced surface exposure. Here, we show reduced number of spine synapses and impairment of an in vitro correlate of LTP in hippocampal neurons from reggie-1 k.o. (Flot2-/-) mice maturating in culture. These defects apparently result from reduced trafficking of PSD-95 revealed by live imaging of 10 div reggie-1 k.o. (Flot2-/-) neurons and likely impairs co-transport of cargo destined for spines: N-cadherin and the glutamate receptors GluA1 and GluN1. Impaired cargo trafficking and fewer synapses also emerged in reggie-1 siRNA, reggie-2 siRNA, and reggie-1 and -2 siRNA-treated neurons and was in siRNA and k.o. neurons rescued by reggie-1-EGFP and CA-Rab11a-EGFP. While correlative expressional changes of specific synapse proteins were observed in reggie-1 k.o. (Flot2-/-) brains in vivo, this did not occur in neurons maturating in vitro. Our work suggests that reggie-1 and reggie-2 function at Rab11a recycling containers in the transport of PSD-95, N-cadherin, GluA1 and GluN1, and promote (together with significant signaling molecules) spine-directed trafficking, spine synapse formation and the in vitro correlate of LTP.


Subject(s)
Hippocampus/cytology , Long-Term Potentiation/physiology , Membrane Proteins/metabolism , Neurons/physiology , Receptors, AMPA/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Animals, Newborn , Cadherins/metabolism , Cells, Cultured , Disks Large Homolog 4 Protein , Endocytosis/drug effects , Endocytosis/genetics , Female , Guanylate Kinases/genetics , Guanylate Kinases/metabolism , Long-Term Potentiation/drug effects , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/metabolism , Protein Transport/genetics , Pyridinium Compounds/pharmacokinetics , Quaternary Ammonium Compounds/pharmacokinetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Time Factors , rab GTP-Binding Proteins/genetics
4.
Dev Dyn ; 246(1): 41-49, 2017 01.
Article in English | MEDLINE | ID: mdl-27666728

ABSTRACT

BACKGROUND: The conditional Cre/lox system has recently emerged as a valuable tool for studies on both embryonic and adult Zebrafish. Temporal control and site-specific recombination are achieved by using the ligand-inducible CreERT2 and administration of the drug tamoxifen (TAM) or its active metabolite, 4-Hydroxytamoxifen (4-OHT). RESULTS: Here we report the generation of a transgenic Zebrafish line, which expresses an mCherry-tagged variant of CreERT2 under the control of the myelin basic protein a (mbpa) promoter. Our analysis shows that larval and adult expression of the transgene recapitulates the endogenous mbpa expression pattern in oligodendrocytes. Furthermore, combination with a Cre-dependent EGFP reporter results in EGFP-expressing oligodendrocytes in the spinal cord, brain, and optic nerve in TAM- or 4-OHT-treated larvae and 4-month-old fish, but not in untreated controls. CONCLUSIONS: The transgenic Zebrafish line Tg(mbpa:mCherry-T2A-CreERT2 ) elicits CreERT2 expression specifically in myelinating glia cells. Cre-inducible targeted recombination of genes in oligodendrocytes will be useful to elucidate cellular and molecular mechanisms of myelination in vivo during development (myelination) and regeneration (remyelination) after injury to the central nervous system (CNS). It will also allow targeted expression and overexpression of genes of interest (transgenes) in oligodendrocytes at defined developmental and adult stages. Developmental Dynamics 246:41-49, 2017. © 2016 Wiley Periodicals, Inc.


Subject(s)
Integrases/metabolism , Myelin Sheath/metabolism , Oligodendroglia/metabolism , Zebrafish/embryology , Animals , Animals, Genetically Modified , Demyelinating Diseases , Gene Expression Regulation, Developmental , Genes, Reporter , Myelin Basic Protein/genetics , Oligodendroglia/ultrastructure , Promoter Regions, Genetic , Recombination, Genetic , Transgenes , Zebrafish/metabolism
5.
PLoS Genet ; 12(6): e1006116, 2016 06.
Article in English | MEDLINE | ID: mdl-27362352

ABSTRACT

Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro-and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds.


Subject(s)
Cell Membrane/metabolism , Detergents/metabolism , Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Bacillus subtilis/metabolism , Microscopy, Fluorescence/methods , Protein Transport/physiology
6.
Eur J Cell Biol ; 94(11): 531-45, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26299802

ABSTRACT

Reggies/flotillins are implicated in trafficking of membrane proteins to their target sites and in the regulation of the Rab11a-dependent targeted recycling of E-cadherin to adherens junctions (AJs). Here we demonstrate a function of reggies in focal adhesion (FA) formation and α5- and ß1-integrin recycling to FAs. Downregulation of reggie-1 in HeLa and A431 cells by siRNA and shRNA increased the number of FAs, impaired their distribution and modified FA turnover. This was coupled to enhanced focal adhesion kinase (FAK) and Rac1 signaling and gain in plasma membrane motility. Wild type and constitutively-active (CA) Rab11a rescued the phenotype (normal number of FAs) whereas dominant-negative (DN) Rab11a mimicked the loss-of-reggie phenotype in control cells. That reggie-1 affects integrin trafficking emerged from the faster loss of internalized antibody-labeled ß1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and α5- or ß1-integrin, trafficking close to the substrate-near membrane and making kiss-and-run contacts with FAs. Thus, reggie-1 in interaction with Rab11a controls Rac1 and FAK activation and coordinates the targeted recycling of α5- and ß1-integrins to FAs to regulate FA formation and membrane dynamics.


Subject(s)
Focal Adhesions/metabolism , Integrin alpha5/metabolism , Integrin beta1/metabolism , Membrane Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , HeLa Cells , Humans , Membrane Proteins/genetics , Protein Transport , rac1 GTP-Binding Protein/metabolism
7.
Neural Dev ; 10: 6, 2015 Mar 20.
Article in English | MEDLINE | ID: mdl-25888884

ABSTRACT

BACKGROUND: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals. RESULTS: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 µM MO1) and 74% (140 µM MO1), respectively, with MO2: 59% (70 µM MO2) and 73% (140 µM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO. CONCLUSIONS: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.


Subject(s)
Myelin Proteins/physiology , Nerve Regeneration/physiology , Optic Nerve Injuries/genetics , Optic Nerve/physiology , Retinal Ganglion Cells/metabolism , Zebrafish Proteins/physiology , Animals , Axonal Transport , Axotomy , Endoplasmic Reticulum/metabolism , Morpholinos/pharmacology , Myelin Proteins/antagonists & inhibitors , Myelin Proteins/biosynthesis , Myelin Proteins/genetics , Optic Nerve Injuries/metabolism , Up-Regulation , Zebrafish , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/biosynthesis , Zebrafish Proteins/genetics
8.
PLoS One ; 8(7): e70327, 2013.
Article in English | MEDLINE | ID: mdl-23936187

ABSTRACT

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.


Subject(s)
Intracellular Space/metabolism , Prions/chemistry , Prions/metabolism , Protein Structure, Tertiary , Actin Cytoskeleton/metabolism , Animals , Animals, Genetically Modified , Cadherins/metabolism , Cell Adhesion/genetics , Cell Communication/genetics , Cell Line , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Glycosylation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , MCF-7 Cells , Mice , Mice, Transgenic , Microscopy, Confocal , Mutation , Prions/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
9.
Mol Biol Cell ; 24(17): 2689-702, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23825023

ABSTRACT

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1-decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca(2+) switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca(2+) repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.


Subject(s)
Cadherins/metabolism , Membrane Proteins/metabolism , Receptors, Transferrin/metabolism , Sorting Nexins/metabolism , rab GTP-Binding Proteins/metabolism , Cadherins/genetics , Cell Line, Tumor , Cell Movement/physiology , Down-Regulation , HeLa Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/immunology , Phylogeny , Protein Transport
10.
Eukaryot Cell ; 12(4): 529-44, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23376944

ABSTRACT

The SPFH protein superfamily is assumed to occur universally in eukaryotes, but information from protozoa is scarce. In the Paramecium genome, we found only Stomatins, 20 paralogs grouped in 8 families, STO1 to STO8. According to cDNA analysis, all are expressed, and molecular modeling shows the typical SPFH domain structure for all subgroups. For further analysis we used family-specific sequences for fluorescence and immunogold labeling, gene silencing, and functional tests. With all family members tested, we found a patchy localization at/near the cell surface and on vesicles. The Sto1p and Sto4p families are also associated with the contractile vacuole complex. Sto4p also makes puncta on some food vacuoles and is abundant on vesicles recycling from the release site of spent food vacuoles to the site of nascent food vacuole formation. Silencing of the STO1 family reduces mechanosensitivity (ciliary reversal upon touching an obstacle), thus suggesting relevance for positioning of mechanosensitive channels in the plasmalemma. Silencing of STO4 members increases pulsation frequency of the contractile vacuole complex and reduces phagocytotic activity of Paramecium cells. In summary, Sto1p and Sto4p members seem to be involved in positioning specific superficial and intracellular microdomain-based membrane components whose functions may depend on mechanosensation (extracellular stimuli and internal osmotic pressure).


Subject(s)
Cell Membrane/physiology , Genome, Protozoan , Membrane Microdomains/physiology , Membrane Proteins/metabolism , Paramecium tetraurelia/physiology , Transport Vesicles/physiology , Cell Membrane/chemistry , Gene Expression Regulation , Gene Silencing , Mechanotransduction, Cellular/physiology , Membrane Microdomains/chemistry , Membrane Proteins/genetics , Multigene Family , Paramecium tetraurelia/chemistry , Phagocytosis/physiology , Phagosomes/chemistry , Phagosomes/physiology , Protein Structure, Tertiary , Transport Vesicles/chemistry , Vacuoles/chemistry , Vacuoles/physiology
11.
Neurobiol Dis ; 51: 168-76, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23174179

ABSTRACT

The ability of fish retinal ganglion cells (RGCs) to regenerate their axons was shown to require the re-expression and function of the two proteins reggie-1 and -2. RGCs in mammals fail to upregulate reggie expression and to regenerate axons after lesion suggesting the possibility that induced upregulation might promote regeneration. In the present study, RGCs in adult rats were induced to express reggie-1 by intravitreal injection of adeno-associated viral vectors (AAV2/1) expressing reggie-1 (AAV.R1-EGFP) 14d prior to optic nerve crush. Four weeks later, GAP-43-positive regenerating axons had crossed the lesion and grown into the nerve at significantly higher numbers and length (up to 5mm) than the control transduced with AAV.EGFP. Consistently, after transduction with AAV.R1-EGFP as opposed to AAV.EGFP, primary RGCs in vitro grew long axons on chondroitin sulfate proteoglycan (CSPG) and Nogo-A, both glial cell-derived inhibitors of neurite growth, suggesting that reggie-1 can provide neurons with the ability to override inhibitors of neurite growth. This reggie-1-mediated enhancement of growth was reproduced in mouse hippocampal and N2a neurons which generated axons 40-60% longer than their control counterparts. This correlates with the reggie-1-dependent activation of Src and PI3 kinase (PI3K), of the Rho family GTPase Rac1 and downstream effectors such as cofilin. This increased growth also depends on TC10, the GTPase involved in cargo delivery to the growth cone. Thus, the upregulation of reggie-1 in mammalian neurons provides nerve cells with neuron-intrinsic properties required for axon growth and successful regeneration in the adult mammalian CNS.


Subject(s)
Axons/metabolism , Membrane Proteins/biosynthesis , Nerve Regeneration/physiology , Neurites/metabolism , Optic Nerve/metabolism , Animals , Blotting, Western , Mice , Rats , Rats, Wistar , Signal Transduction/physiology , Transduction, Genetic , Up-Regulation
12.
Mol Biol Cell ; 23(10): 1812-25, 2012 May.
Article in English | MEDLINE | ID: mdl-22438585

ABSTRACT

The reggie/flotillin proteins are 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 down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.


Subject(s)
Adherens Junctions/metabolism , Cadherins/metabolism , ErbB Receptors/metabolism , Membrane Proteins/metabolism , Adherens Junctions/ultrastructure , Cell Adhesion , Cell Line, Tumor , Cell Movement , Endocytosis , Gene Knockdown Techniques , Humans , Membrane Proteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Prions/genetics , Prions/metabolism , Protein Processing, Post-Translational , Protein Transport , RNA Interference , Signal Transduction , beta Catenin/metabolism
13.
Cell Tissue Res ; 349(1): 71-7, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22350847

ABSTRACT

The microdomain-forming proteins reggie-1 and reggie-2 (alias flotillins) were found to be upregulated in axon-regenerating fish retinal ganglion cells (RGCs). They were subsequently shown to be indispensible for axon regeneration and neurite extension in fish and mammals. Our current concept proposes that reggies--often together with the cellular Prion protein (PrP)--regulate the turnover of membrane and specific membrane proteins at the growth cone, which is the prerequisite for neurite elongation and guidance.


Subject(s)
Axons/physiology , Membrane Proteins/metabolism , Nerve Regeneration/physiology , Animals , Humans , Lysosomes/metabolism , Retinal Ganglion Cells/metabolism , Transport Vesicles/metabolism
14.
J Neurosci ; 31(49): 18013-25, 2011 Dec 07.
Article in English | MEDLINE | ID: mdl-22159115

ABSTRACT

The role of prion protein (PrP) is insufficiently understood partially because PrP-deficient (-/-) neurons from C57BL/6J mice seem to differentiate normally and are functionally mildly impaired. Here, we reassessed this notion and, unexpectedly, discovered that PrP(-/-) hippocampal growth cones were abnormally small and poor in filopodia and cargo-containing vesicles. Based on our findings that PrP-PrP trans-interaction recruits E-cadherin to cell contact sites and reggie microdomains, and that reggies are essential for growth by regulating membrane trafficking, we reasoned that PrP and reggie might promote cargo (N-cadherin) delivery via PrP-reggie-connected signaling upon PrP activation (by PrP-Fc-induced trans-interaction). In wild-type but not PrP(-/-) neurons, PrP activation led to (1) enhanced PrP-reggie cocluster formation, (2) reggie-associated fyn and MAP kinase activation, (3) Exo70 and N-cadherin (cargo) recruitment to reggie, (4) the preference of the growth cone for PrP-Fc as substrate, and (5) longer neurites. Conversely, PrP-reggie-induced N-cadherin recruitment was blocked by mutant TC10, the GTPase downstream of reggie, triggering exocyst-assisted cargo delivery. This implies that PrP functions in reggie-mediated signaling and cargo trafficking, thus promoting growth cone complexity and vitality and thereby growth cone elongation.


Subject(s)
Cadherins/metabolism , Growth Cones/drug effects , Membrane Proteins/metabolism , Neurons/cytology , Prions/pharmacology , Analysis of Variance , Animals , Animals, Newborn , Cells, Cultured , Exocytosis/drug effects , Exocytosis/genetics , Hippocampus/cytology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurites/drug effects , Neurites/physiology , Peptides/pharmacology , Prions/genetics , Protein Transport/drug effects , Protein Transport/genetics , RNA, Small Interfering/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Transfection/methods , Vesicular Transport Proteins/metabolism
15.
J Neurochem ; 116(5): 708-13, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21214550

ABSTRACT

The two proteins reggie-1/flotillin-2 and reggie-2/flotillin-1 form microdomains at the plasma membrane and at intracellular compartments where src tyrosine kinases associate with them. Specific GPI-anchored proteins, in particular prion protein and Thy-1, co-cluster with reggie microdomains at the plasma membrane and elicit signal transduction in association with reggies which regulates the activation of several GTPases involved in the recruitment of specific membrane proteins from intracellular carriers to target sites of the cell membrane in a cell type-specific manner. For example, prion protein and reggie regulate the recruitment and targeted delivery of the T cell receptor complex to the T cell cap, of E-cadherin to cell-cell contact sites in epithelial cells, and of bulk membrane and growth receptors to the growth cone in developing neurons. Evidence is accumulating that reggies are involved in guiding the cell-type-specific membrane proteins from the intracellular compartments to their target sites at the cell membrane, a function required in all cells which explains why reggies are expressed in many or all cells in invertebrates and vertebrates.


Subject(s)
Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Animals , Axons/physiology , Cadherins/metabolism , Humans , Models, Biological , Neurons/cytology , Prions/physiology , Signal Transduction/physiology , Thy-1 Antigens/physiology , src-Family Kinases/metabolism
16.
Biochim Biophys Acta ; 1812(3): 415-22, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21147218

ABSTRACT

The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type specific manner. In this review, reggie microdomains, lipid rafts, related scaffolding proteins and caveolin-which, however, are responsible for their own microdomains and functions-are introduced. Moreover, the function of the reggies in axon growth is demonstrated: neurons fail to extend axons after reggie knockdown. Furthermore, our current concept of the molecular mechanism underlying reggie function is presented: the association of glycosyl-phophatidyl inositol (GPJ)-anchored surface proteins with reggie microdomains elicits signals which activate src tyrosine and mitogen-activated protein kinases, as well as small guanosine 5'-triphosphate-hydrolyzing enzymes. This leads to the mobilization of intracellular vesicles and to the recruitment of bulk membrane and specific cargo proteins, such as cadherin, to specific sites of the plasma membrane such as the growth cone of elongating axons. Thus, reggies regulate the targeted delivery of cargo-a process which is required for process extension and growth. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.


Subject(s)
Axons/physiology , Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Nerve Regeneration , Zebrafish/metabolism , Animals , Cell Differentiation , Zebrafish/embryology
17.
Mol Biol Evol ; 28(4): 1363-70, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21098000

ABSTRACT

Unlike mammals, fish are able to regenerate axons in their central nervous system. This difference has been partly attributed to the loss/acquisition of inhibitory proteins during evolution. Nogo-A--the longest isoform of the reticulon4 (rtn4) gene product--is commonly found in mammalian myelin where it acts as a potent inhibitor of axonal regeneration. Interestingly, fish RTN4 isoforms were previously reported to lack the most inhibitory Nogo-A-specific region (NSR). Nevertheless, fish axons collapse on contact with mammalian NSR, suggesting that fish possess a functional Nogo-A receptor but not its ligand. To reconcile these findings, we revisited the early evolution of rtn4. Mining of current genome databases established the unequivocal presence of NSR-coding sequences in fish rtn4 paralogues. Further comparative analyses indicate that the common ancestor of fish and tetrapods had an NSR-coding rtn4 gene, which underwent duplication and divergent evolution in bony fish. Our genomic survey also revealed that the cephalochordate Branchiostoma floridae contains a single rtn gene lacking the NSR. Hence, Nogo-A most probably arose independently in the rtn4 gene of a gnathostome ancestor before the split of the fish and tetrapod lineages. Close examination of the NSR uncovered clusters of structural and sequential similarities with neurocan (NCAN), an inhibitory proteoglycan of the glial scar. Notably, the shared presence of transposable elements in ncan and rtn4 genes suggests that Nogo-A originated via insertion of an ncan-like sequence into the rtn4 gene of an early jawed vertebrate with myelinated axons.


Subject(s)
Biological Evolution , Jaw , Myelin Proteins/genetics , Protein Isoforms/genetics , Vertebrates/genetics , Amino Acid Sequence , Animals , Axons/physiology , Fishes/genetics , Humans , Molecular Sequence Data , Nogo Proteins , Phylogeny , Sequence Alignment , Vertebrates/classification
18.
Front Biosci (Landmark Ed) ; 15(3): 1075-85, 2010 06 01.
Article in English | MEDLINE | ID: mdl-20515742

ABSTRACT

The prion protein (PrP) has been implicated in many diverse functions, making it difficult to pinpoint its basic physiological role. Our most recent studies in zebrafish, mammalian and invertebrate cells indicate that PrP regulates cell-cell communication, as well cell-matrix interactions at focal adhesions. In addition, we previously have shown that upon antibody-mediated cross-linking, PrP can be induced to cluster in the preformed T-cell cap. Here we review these data and discuss how the spatial link between PrP and the microdomain-forming proteins reggie-1 (flotillin-2) and reggie-2 (flotillin-1) may contribute to PrP signaling, leading to the local assembly of membrane protein complexes at sites involved in cellular communication, such as cell-cell contacts, focal adhesions, the T-cell cap, and synapses.


Subject(s)
Cell Communication , Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Prions/metabolism , Animals , Focal Adhesions , Humans , Models, Biological , Signal Transduction
19.
Trends Cell Biol ; 20(1): 6-13, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19896850

ABSTRACT

The proteins reggie-1 and reggie-2 were originally discovered in neurons during axon regeneration. Subsequently, they were independently identified as markers of lipid rafts in flotation assays and were hence named flotillins. Since then, reggie/flotillin proteins have been found to be evolutionarily conserved and are present in all vertebrate cells - yet their function has remained elusive and controversial. Recent results now show that reggie/flotillin proteins are indeed necessary for axon regeneration and growth: no axons form when reggies/flotillins are downregulated and signaling pathways controlling actin dynamics are perturbed. Their widespread expression and conservation, however, suggest that these proteins regulate basic cellular functions beyond regeneration. It is argued here that the reggie/flotillin proteins regulate processes vital to all cells - the targeted delivery of bulk membrane and specific membrane proteins from internal vesicle pools to strategically important sites including cell contact sites, the T cell cap, regenerating axons and growth cones and other protrusions.


Subject(s)
Membrane Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Animals , Humans , Membrane Proteins/genetics , Nerve Regeneration , Protein Transport , Signal Transduction
20.
J Neurosci ; 29(49): 15489-98, 2009 Dec 09.
Article in English | MEDLINE | ID: mdl-20007473

ABSTRACT

In contrast to mammals, lesioned axons in the zebrafish (ZF) optic nerve regenerate and restore vision. This correlates with the absence of the NogoA-specific N-terminal domains from the ZF nogo/rtn-4 (reticulon-4) gene that inhibits regeneration in mammals. However, mammalian nogo/rtn-4 carries a second inhibitory C-terminal domain, Nogo-66, being 70% identical with ZF-Nogo66. The present study examines, (1) whether ZF-Nogo66 is inhibitory and effecting similar signaling pathways upon Nogo66-binding to the Nogo66 receptor NgR and its coreceptors, and (2) whether Rat-Nogo66 on fish, and ZF-Nogo66 on mouse neurons, cause inhibition via NgR. Our results from "outgrowth, collapse and contact assays" suggest, surprisingly, that ZF-Nogo66 is growth-permissive for ZF and mouse neurons, quite in contrast to its Rat-Nogo66 homolog which inhibits growth. The opposite effects of ZF- and Rat-Nogo66 are, in both fish and mouse, transmitted by GPI (glycosylphosphatidylinositol)-anchored receptors, including NgR. The high degree of sequence homology in the predicted binding site is consistent with the ability of ZF- and mammalian-Nogo66 to bind to NgRs of both species. Yet, Rat-Nogo66 elicits phosphorylation of the downstream effector cofilin whereas ZF-Nogo66 has no influence on cofilin phosphorylation--probably because of significantly different Rat- versus ZF-Nogo66 sequences outside of the receptor-binding region effecting, by speculation, recruitment of a different set of coreceptors or microdomain association of NgR. Thus, not only was the NogoA-specific domain lost in fish, but Nogo66, the second inhibitory domain in mammals, and its signaling upon binding to NgR, was modified so that ZF-Nogo/RTN-4 does not impair axon regeneration.


Subject(s)
Axons/physiology , Myelin Proteins/metabolism , Nerve Regeneration/physiology , Optic Nerve/physiology , Receptors, Cell Surface/metabolism , Zebrafish Proteins/metabolism , Actin Depolymerizing Factors/metabolism , Animals , Glycosylphosphatidylinositols/metabolism , Growth Cones/physiology , HeLa Cells , Hippocampus/physiology , Humans , In Vitro Techniques , Mice , Myelin Proteins/genetics , Neurites/physiology , Neurons/physiology , Nogo Proteins , Rats , Retina/physiology , Retinal Ganglion Cells/physiology , Signal Transduction , Species Specificity , Zebrafish
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