ABSTRACT
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.
Subject(s)
Cell Differentiation/physiology , Hippocampus/cytology , Membrane Proteins/metabolism , Nerve Regeneration/physiology , Neurons/physiology , Optic Nerve Injuries/physiopathology , Retina/pathology , Animals , Animals, Newborn , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Dextrans , Down-Regulation/drug effects , Green Fluorescent Proteins/genetics , Immunoprecipitation , Insulin-Like Growth Factor I/pharmacology , Membrane Proteins/genetics , Mice , Nerve Regeneration/drug effects , Nerve Regeneration/genetics , Neuroblastoma , Neurons/drug effects , Oligodeoxyribonucleotides, Antisense/pharmacology , Optic Nerve Injuries/metabolism , Optic Nerve Injuries/pathology , Organ Preservation Solutions , RNA, Small Interfering/metabolism , Retina/physiology , Signal Transduction/drug effects , Signal Transduction/genetics , Transfection/methods , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Zebrafish , cdc42 GTP-Binding Protein/metabolism , rac GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/metabolismABSTRACT
The reggies/flotillins were discovered as proteins upregulated during axon regeneration. Here, we show that expression of a trans-negative reggie-1/flotillin-2 deletion mutant, R1EA, which interferes with oligomerization of the reggies/flotillins, inhibited insulin-like growth factor (IGF)-induced neurite outgrowth in N2a neuroblastoma cells and impaired in vitro differentiation of primary rat hippocampal neurons. Cells expressing R1EA formed only short and broad membrane protrusions often with abnormally large growth cones. R1EA expression strongly perturbed the balanced activation of the Rho-family GTPases Rac1 and cdc42. Furthermore, focal adhesion kinase (FAK) activity was also enhanced by R1EA expression, while other signaling pathways like ERK1/2, PKC or PKB signaling were unaffected. These severe signaling defects were caused by an impaired recruitment of the reggie/flotillin-associated adaptor molecule CAP/ponsin to focal contacts at the plasma membrane. Thus, the reggies/flotillins are crucial for coordinated assembly of signaling complexes regulating cytoskeletal remodeling.
Subject(s)
Cytoskeleton/physiology , Membrane Proteins/physiology , Microfilament Proteins/metabolism , Neurons/cytology , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , 3-Phosphoinositide-Dependent Protein Kinases , Animals , Blotting, Western , Cell Differentiation , Cell Membrane/metabolism , Cells, Cultured , Fluorescent Antibody Technique, Indirect , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gene Deletion , Green Fluorescent Proteins/metabolism , Hippocampus/cytology , Hippocampus/metabolism , Humans , Insulin-Like Growth Factor I/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mutation/genetics , Neurites/physiology , Neuroblastoma/metabolism , Neuroblastoma/pathology , Neurons/metabolism , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, WistarABSTRACT
The reggie/flotillin proteins oligomerize and associate into clusters which form scaffolds for membrane microdomains. Besides their localization at the plasma membrane, the reggies/flotillins reside at various intracellular compartments; however, the trafficking pathways used by reggie-1/flotillin-2 remain unclear. Here, we show that trafficking of reggie-1/flotillin-2 is BFA sensitive and that deletion mutants of reggie-1/flotillin-2 accumulate in the Golgi complex in HeLa, Jurkat and PC12 cells, suggesting Golgi-dependent trafficking of reggie-1/flotillin-2. Using total internal reflection fluorescence microscopy, we observed fast cycling of reggie-1/flotillin-2-positive vesicles at the plasma membrane, which engaged in transient interactions with the plasma membrane only. Reggie-1/flotillin-2 cycling was independent of clathrin, but was inhibited by cholesterol depletion and microtubule disruption. Cycling of reggie-1/flotillin-2 was negatively correlated with cell-cell contact formation but was stimulated by serum, epidermal growth factor and by cholesterol loading mediated by low density lipoproteins. However, reggie-1/flotillin-2 was neither involved in endocytosis of the epidermal growth factor itself nor in endocytosis of GPI-GFPs or the GPI-anchored cellular prion protein (PrP(c)). Reggie-2/flotillin-1 and stomatin-1 also exhibited cycling at the plasma membrane similar to reggie-1/flotillin-2, but these vesicles and microdomains only partially co-localized with reggie-2/flotillin-1. Thus, regulated vesicular cycling might be a general feature of SPFH protein-dependent trafficking.
