The Drosophila L1CAM homolog Neuroglian signals through distinct pathways to control different aspects of mushroom body axon development.

The spatiotemporal integration of adhesion and signaling during neuritogenesis is an important prerequisite for the establishment of neuronal networks in the developing brain. In this study, we describe the role of the L1-type CAM Neuroglian protein (NRG) in different steps of Drosophila mushroom body (MB) neuron axonogenesis. Selective axon bundling in the peduncle requires both the extracellular and the intracellular domain of NRG. We uncover a novel role for the ZO-1 homolog Polychaetoid (PYD) in axon branching and in sister branch outgrowth and guidance downstream of the neuron-specific isoform NRG-180. Furthermore, genetic analyses show that the role of NRG in different aspects of MB axonal development not only involves PYD, but also TRIO, SEMA-1A and RAC1.

Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin.

PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P(2)-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P(2) binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P(2) 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P(2) binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.

The postsynaptic density 95/disc-large/zona occludens protein syntenin directly interacts with frizzled 7 and supports noncanonical Wnt signaling.

Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)alpha and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCalpha/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.

Nuclear speckles and nucleoli targeting by PIP2-PDZ domain interactions.

PDZ (Postsynaptic density protein, Disc large, Zona occludens) domains are protein-protein interaction modules that predominate in submembranous scaffolding proteins. Recently, we showed that the PDZ domains of syntenin-1 also interact with phosphatidylinositol 4,5-bisphosphate (PIP2) and that this interaction controls the recruitment of the protein to the plasma membrane. Here we evaluate the general importance of PIP2-PDZ domain interactions. We report that most PDZ proteins bind weakly to PIP2, but that syntenin-2, the closest homolog of syntenin-1, binds with high affinity to PIP2 via its PDZ domains. Surprisingly, these domains target syntenin-2 to nuclear PIP2 pools, in nuclear speckles and nucleoli. Targeting to these sites is abolished by treatments known to affect these PIP2 pools. Mutational and domain-swapping experiments indicate that high-affinity binding to PIP2 requires both PDZ domains of syntenin-2, but that its first PDZ domain contains the nuclear PIP2 targeting determinants. Depletion of syntenin-2 disrupts the nuclear speckles-PIP2 pattern and affects cell survival and cell division. These findings show that PIP2-PDZ domain interactions can directly contribute to subnuclear assembly processes.

Interaction between Smad-interacting protein-1 and the corepressor C-terminal binding protein is dispensable for transcriptional repression of E-cadherin.

deltaEF1 and SIP1 (or Zfhx1a and Zfhx1b, respectively) are the only known members of the vertebrate Zfh1 family of homeodomain/zinc finger-containing proteins. Similar to other transcription factors, both Smad-interacting protein-1 (SIP1) and deltaEF1 are capable of repressing E-cadherin transcription through binding to the E2 boxes located in its promoter. In the case of deltaEF1, this repression has been proposed to occur via interaction with the corepressor C-terminal binding protein (CtBP). In this study, we show by coimmunoprecipitation that SIP1 and CtBP interact in vivo and that an isolated CtBP-binding SIP1 fragment depends on CtBP for transcriptional repression. However, and most importantly, full-length SIP1 and deltaEF1 proteins do not depend on their interaction with CtBP to repress transcription from the E-cadherin promoter. Furthermore, in E-cadherin-positive kidney epithelial cells, the conditional synthesis of mutant SIP1 that cannot bind to CtBP abrogates endogenous E-cadherin expression in a similar way as wild-type SIP1. Our results indicate that full-length SIP1 can repress E-cadherin in a CtBP-independent manner.