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Membrane microdomains or “lipid rafts” have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the dichotomy between caveolin-1 and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2 potentiates while caveolin-1 inhibits nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated protein kinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequestering the cognate receptors, TrkA and p75NTR, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75NTR that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75NTR) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis. [ABSTRACT FROM AUTHOR]

The development of gene transfection technologies has greatly advanced our understanding of life sciences. While use of viral vectors has clear efficacy, it requires specific expertise and biological containment conditions. Electroporation has become an effective and commonly used method for introducing DNA into neurons and in intact brain tissue. The present study describes the use of the Neon® electroporation system to transfect genes into dorsal root ganglia neurons isolated from embryonic mouse Day 13.5-16. This cell type has been particularly recalcitrant and refractory to physical or chemical methods for introduction of DNA. By optimizing the culture condition and parameters including voltage and duration for this specific electroporation system, high efficiency (60-80%) and low toxicity (>60% survival) were achieved with robust differentiation in response to Nerve growth factor (NGF). Moreover, 3-50 times fewer cells are needed (6 × 104) compared with other traditional electroporation methods. This approach underlines the efficacy of this type of electroporation, particularly when only limited amount of cells can be obtained, and is expected to greatly facilitate the study of gene function in dorsal root ganglia neuron cultures. [ABSTRACT FROM AUTHOR]

Robo-Slit and Plexin-Semaphorin signaling participate in various developmental and pathogenic processes. During commissural axon guidance in the spinal cord, chemorepulsion by Semaphorin3B and Slits controls midline crossing. Slit processing generates an N-terminal fragment (SlitN) that binds to Robo1 and Robo2 receptors and mediates Slit repulsive activity, as well as a C-terminal fragment (SlitC) with an unknown receptor and bioactivity. We identified PlexinA1 as a Slit receptor and found that it binds the C-terminal Slit fragment specifically and transduces a SlitC signal independently of the Robos and the Neuropilins. PlexinA1-SlitC complexes are detected in spinal cord extracts, and ex vivo, SlitC binding to PlexinA1 elicits a repulsive commissural response. Analysis of various ligand and receptor knockout mice shows that PlexinA1-Slit and Robo-Slit signaling have complementary roles during commissural axon guidance. Thus, PlexinA1 mediates both Semaphorin and Slit signaling, and Slit processing generates two active fragments, each exerting distinct effects through specific receptors. [ABSTRACT FROM AUTHOR]

Selenium-Binding Proteini (SBP1) gene expression was studied in Arabidopsis (Arabidopsis thaliana) seedlings challenged with several stresses, including cadmium (Cd), selenium (selenate [Se(VI)] and selenite [Se(IV)]), copper (Cu), zinc (Zn), and hydrogen peroxide (H2O2) using transgenic lines expressing the luciferase (LUC) reporter gene under the control of the SBP1 promoter. In roots and shoots of SBP1::LUC lines, LUC activity increased in response to Cd, Se(VI), Cu, and H2O2 but not in response to Se(IV) or Zn. The pattern of expression of SBP1 was similar to that of PRH43, which encodes the 5' -Adenylylphosphosulfate Reductase2, a marker for the induction of the sulfur assimilation pathway, suggesting that an enhanced sulfur demand triggers SBP1 up-regulation. Correlated to these results, SBP1 promoter showed enhanced activity in response to sulfur starvation. The sulfur starvation induction of SBP1 was abolished by feeding the plants with glutathione (GSH) and was enhanced when seedlings were treated simultaneously with buthionine sulfoxide, which inhibits GSH synthesis, indicating that GSH level participates in the regulation of SBP1 expression. Changes in total GSH level were observed in seedlings challenged with Cd, Se(VI), and H2O2. Accordingly, cad2-.1 seedlings, affected iii GSH synthesis, were more sensitive than wild-type plants to these three stresses. Moreover, wild-type and cad2-1 seedlings overexpressing SBP1 showed a significant enhanced tolerance to Se(VI) and H2O2 in addition to the previously described resistance to Cd, highlighting that SBP1 expression decreases sensitivity to stress requiring GSH for tolerance. These results are discussed with regard to the potential regulation and function of SBP1 in plants. [ABSTRACT FROM AUTHOR]

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration. [Display omitted] • Uncoordinated-5 receptor D (Unc5D) and glypican-3 (GPC3) form an octameric complex • Anti-GPC3 nanobodies (Nanoglue, Nanobreak) enhance or weaken Unc5-GPC3 binding • Unc5/GPC3 signaling causes cell contact-repulsion in vitro • In vivo , Unc5/GPC3 modulate the migration of cortical neurons and neuroblastoma cells Detailed structural analysis and modulation of binding between uncoordinated-5 receptor D (Unc5D) and morphogen receptor glypican-3 (GPC3) reveal how finely balanced Unc5-GPC3 interactions regulate the migration of neuronal and cancer cells. [ABSTRACT FROM AUTHOR]