The dishevelled associated activator of morphogenesis protein 2 (Daam2) regulates neural tube closure.

BACKGROUND: The Wnt signaling pathway is highly conserved in metazoans and regulates a large array of cellular processes including motility, polarity and fate determination, and stem cell homeostasis. Modulation of the actin cytoskeleton via the non-canonical Wnt pathway regulate cell polarity and cell migration that are required for proper vertebrate gastrulation and subsequent neurulation. However, the mechanism(s) of how the non-canonical pathway mediates actin cytoskeleton modulation is not fully understood. RESULTS: Herein, we characterize the role of the Formin-homology protein; dishevelled associated activator of morphogenesis 2 (Daam2) protein in the Wnt signaling pathway. Co-immunoprecipitation assays confirm the binding of Daam2 to dishevelled2 (Dvl2) as well as the domains within these proteins required for interaction; additionally, the interaction between Daam2 and Dvl2 was Wnt-regulated. Sub-cellular localization studies reveal Daam2 is cytoplasmic and regulates the cellular actin cytoskeleton by modulating actin filament formation. During Xenopus development, a knockdown or loss of Daam2 specifically produces neural tube closure defects indicative of a role in non-canonical signaling. Additionally, our studies did not identify any role for Daam2 in canonical Wnt signaling in mammalian culture cells or the Xenopus embryo. CONCLUSIONS: Our studies together identify Daam2 as a component of the non-canonical Wnt pathway and Daam2 is a regulator of neural tube morphogenesis during vertebrate development.

ConFERMing the role of talin in integrin activation and mechanosignaling.

Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell-matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane-cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin-talin-F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein-protein and protein-lipid interfaces, including the membrane-binding and integrin affinity-regulating F1 loop, as well as the interaction with lipid-anchored Rap1 (Rap1a and Rap1b in mammals) GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell-matrix attachment sites.

Phosphorylation of RIAM Activates Its Adaptor Function in Mediating Integrin Signaling.

Integrins are cellular receptors that regulate cell adhesion and many other cellular functions. Integrins can be activated via an "inside-out pathway" that is promoted by RAP1 GTPase. RAP1-GTP-Interacting Adaptor Molecular (RIAM) mediates integrin activation by linking RAP1 GTPase to talin, an integrin activator. RIAM's function in integrin signaling is tightly regulated. In this commentary, we review recent studies of the molecular mechanisms underlying RIAM autoinhibition via both intramolecular interaction and oligomer assembly, and the phosphorylation-dependent activation of RIAM.

A peptide probe for detection of various beta-amyloid oligomers.

Aggregation of ß-amyloid (Aß) is implicated in the pathology of Alzheimer's disease (AD). A considerable amount of data has identified soluble Aß oligomers as potentially significant toxic agents. Rapid, specific and quantitative detection is preferred for accurate profiling of structurally unstable Aß oligomers as well as for implementation of high-throughput assays for pharmaceutical applications. PG46 is an engineered Aß variant, constructed by integrating Aß self-recognition sequences with the conformation-sensitive biarsenical fluorescent dye, FlAsH. PG46 was found to be an effective peptide probe, which detected Aß oligomers specifically and quantitatively within one hour. However, PG46 was highly aggregation-prone and displayed a limited repertoire of detectable Aß oligomers. Here, we report the creation of a novel molecular probe, PG44, by C-terminal truncation of PG46. PG44 exhibited a reduced self-aggregation propensity and a different conformation when compared to PG46, and generated specific FlAsH fluorescence signals as a result of binding to various Aß oligomers, including those not readily detectable by PG46. We also show that sensitivity of PG44 for detection of certain Aß oligomers may be increased by lowering PG44 concentration and thus decreasing the extent of self-aggregation of PG44. Our results suggest that PG44 can serve as an important molecular probe with a broadened repertoire of detectable Aß oligomeric aggregates. We believe that detection of Aß oligomers using our peptide probe would potentially contribute toward a better understanding of the molecular basis of Aß oligomerization and the development of Aß oligomer-based early diagnostics as well as therapeutic drugs targeting Aß oligomers.

Modulation of beta-amyloid aggregation by engineering the sequence connecting beta-strand forming domains.

Aggregation of beta-amyloid (Aß) into oligomers and fibrils is associated with the pathology of Alzheimer's disease. The major structural characteristics of Aß fibrils include the presence of ß sheet-loop-ß sheet conformations. Several lines of study suggested a potentially important role of the Aß loop forming sequence (referred to as the Aß linker region) in Aß aggregation. Effects of mutations in several charged residues within the Aß linker region on aggregation have been extensively studied. However, little is known about oligomerization effects of sequence variation in other residues within the Aß linker region. Moreover, modulation effects of the Aß linker mutants on Aß aggregation have yet to be characterized. Here, we created and characterized Aß linker variants containing sequences preferentially found in specific ß turn conformations. Our results indicate that a propensity to form oligomers may be changed by local sequence variation in the Aß linker region without mutating the charged residues. Strikingly, one Aß linker variant rapidly formed protofibrillar oligomers, which did not convert to fibrillar aggregates in contrast to Aß aggregating to fibrils under similar incubation conditions. Moreover, our results suggest that molecular forces critical in oligomerization and fibrillization may differ at least for those involved in the linker region. When co-incubated with Aß, some Aß linker variants were found to induce accumulation of Aß oligomers. Our results suggest that engineering of the Aß linker region as described in this paper may represent a novel approach to control Aß oligomerization and create Aß oligomerization modulators.

A strategy for designing a peptide probe for detection of ß-amyloid oligomers.

Aggregation of ß-amyloid (Aß) is implicated in the pathology of Alzheimer's disease. Development of a robust strategy to detect Aß oligomeric intermediates, which have been identified as significant toxic agents, would be highly beneficial in the screening of drug candidates as well as enhancing our understanding of Aß oligomerization. Rapid, specific and quantitative detection, currently unavailable, would be highly preferred for accurate and reliable probing of transient Aß oligomers. Here, we report the development of a novel peptide probe, PG46, based on the nature of Aß self-assembly and the conformation-sensitive fluorescence of the biarsenical dye, FlAsH. PG46 was found to bind to Aß oligomers and displayed an increase in FlAsH fluorescence upon binding. No such event was observed when PG46 was co-incubated with Aß low-molecular-weight species or Aß fibrils. Aß oligomer detection was fast, and occurred within one hour without any additional sample incubation or preparation. We anticipate that the development of a strategy for detection of amyloid oligomers described in this study will be directly relevant to a host of other amyloidogenic proteins.