Gonzalo P. Solis

Regulation of Embryonic Cell Adhesion by the Prion Protein

Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Ca+2-independent homophilic cell adhesion and signaling; and (2) modulates Ca+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.

Reggie-1/flotillin-2 promotes secretion of the long-range signalling forms of Wingless and Hedgehog in Drosophila

The lipid‐modified morphogens Wnt and Hedgehog diffuse poorly in isolation yet can spread over long distances in vivo, predicting existence of two distinct forms of these mophogens. The first is poorly mobile and activates short‐range target genes. The second is specifically packed for efficient spreading to induce long‐range targets. Subcellular mechanisms involved in the discriminative secretion of these two forms remain elusive. Wnt and Hedgehog can associate with membrane microdomains, but the function of this association was unknown. Here we show that a major protein component of membrane microdomains, reggie‐1/flotillin‐2, plays important roles in secretion and spreading of Wnt and Hedgehog in Drosophila. Reggie‐1 loss‐of‐function results in reduced spreading of the morphogens, while its overexpression stimulates secretion of Wnt and Hedgehog and expands their diffusion. The resulting changes in the morphogen gradients differently affect the short‐ and long‐range targets. In its action reggie‐1 appears specific for Wnt and Hedgehog. These data suggest that reggie‐1 is an important component of the Wnt and Hedgehog secretion pathway dedicated to formation of the mobile pool of these morphogens.

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.

Disparate evolution of prion protein domains and the distinct origin of Doppel- and prion-related loci revealed by fish-to-mammal comparisons

Prions result from the misfolding and selective accumulation of the host‐encoded prion protein (PrP) in the brain. Despite intensive research on mammalian models, basic questions about the biological role of PrP and the evolutionary origin of prion disease remain unanswered. Following our previous identification of novel fish PrP homologues, here we generated new fish PrP sequences and performed genomic analysis to demonstrate the existence of two homologous PrP loci in bony fish, which display extensive molecular variation and are highly expressed in adult and developing fish brains. The fish PrP genomic regions contain PrP‐related loci directly downstream of each PrP locus, suggesting an independent origin of prion‐related proteins in fish and mammals. Our structural prediction analysis uncovers a conserved molecular “bauplan” for all vertebrate PrPs. The C‐ and N‐terminal protein domains have evolved independently from one another, the former having retained its basic globular structure despite high sequence divergence and the latter having undergone differential expansion‐degeneration cycles in its repetitive domains. Our evolutionary analysis redefines fundamental concepts on the functional significance of PrP domains and opens up new possibilities for the experimental analysis of prion misfolding and neurodegeneration in a non‐mammalian model like the zebrafish.

Linking membrane microdomains to the cytoskeleton: Regulation of the lateral mobility of reggie-1/flotillin-2 by interaction with actin

The reggies/flotillins are oligomeric scaffolding proteins for membrane microdomains. We show here that reggie‐1/flotillin‐2 microdomains are organized along cortical F‐actin in several cell types. Interaction with F‐actin is mediated by the SPFH domain as shown by in vivo co‐localization and in vitro binding experiments. Reggie‐1/flotillin‐2 microdomains form independent of actin, but disruption or stabilization of the actin cytoskeleton modulate the lateral mobility of reggie‐1/flotillin‐2 as shown by FRAP. Furthermore, reggie/flotillin microdomains can efficiently be immobilized by actin polymerisation, while exchange of reggie‐1/flotillin‐2 molecules between microdomains is enhanced by actin disruption as shown by tracking of individual microdomains using TIRF microscopy.

Reggies/flotillins regulate E-cadherin–mediated cell contact formation by affecting EGFR trafficking

In epithelial cells, the reggie/flotillin proteins regulate—in association with PrP—the formation of E-cadherin adherens junctions (AJs) via the EGFR. Reggies control the EGF-mediated phosphorylation and internalization of EGFR. EGF signaling at the plasma membrane induces the macropinocytosis of E-cadherin and thus the formation of AJs.

Regulation of focal adhesion formation and filopodia extension by the cellular prion protein (PrPC)

While the prion protein (PrP) is clearly involved in neuropathology, its physiological roles remain elusive. Here, we demonstrate PrP functions in cell–substrate interaction in Drosophila S2, N2a and HeLa cells. PrP promotes cell spreading and/or filopodia formation when overexpressed, and lamellipodia when downregulated. Moreover, PrP normally accumulates in focal adhesions (FAs), and its downregulation leads to reduced FA numbers, increased FA length, along with Src and focal adhesion kinase (FAK) activation. Furthermore, its overexpression elicits the formation of novel FA‐like structures, which require intact reggie/flotillin microdomains. Altogether, PrP modulates process formation and FA dynamics, possibly via signal transduction involving FAK and Src.

Prion Protein Promotes Growth Cone Development through Reggie/Flotillin-Dependent N-Cadherin Trafficking

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.

No Nogo66- and NgR-Mediated Inhibition of Regenerating Axons in the Zebrafish Optic Nerve

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.