The exocyst complex is required for developmental and regenerative neurite growth in vivo.

The exocyst complex is an important regulator of intracellular trafficking and tethers secretory vesicles to the plasma membrane. Understanding of its role in neuron outgrowth remains incomplete, and previous studies have come to different conclusions about its importance for axon and dendrite growth, particularly in vivo. To investigate exocyst function in vivo we used Drosophila sensory neurons as a model system. To bypass early developmental requirements in other cell types, we used neuron-specific RNAi to target seven exocyst subunits. Initial neuronal development proceeded normally in these backgrounds, however, we considered this could be due to residual exocyst function. To probe neuronal growth capacity at later times after RNAi initiation, we used laser microsurgery to remove axons or dendrites and prompt regrowth. Exocyst subunit RNAi reduced axon regeneration, although new axons could be specified. In control neurons, a vesicle trafficking marker often concentrated in the new axon, but this pattern was disrupted in Sec6 RNAi neurons. Dendrite regeneration was also severely reduced by exocyst RNAi, even though the trafficking marker did not accumulate in a strongly polarized manner during normal dendrite regeneration. The requirement for the exocyst was not limited to injury contexts as exocyst subunit RNAi eliminated dendrite regrowth after developmental pruning. We conclude that the exocyst is required for injury-induced and developmental neurite outgrowth, but that residual protein function can easily mask this requirement.

Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system.

Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutants for genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.

RalGPS2 is involved in tunneling nanotubes formation in 5637 bladder cancer cells.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.

InsP3R-SEC5 interaction on phagosomes modulates innate immunity to Candida albicans by promoting cytosolic Ca2+ elevation and TBK1 activity.

BACKGROUND: Candida albicans (C. albicans) invasion triggers antifungal innate immunity, and the elevation of cytoplasmic Ca2+ levels via the inositol 1,4,5-trisphosphate receptor (InsP3R) plays a critical role in this process. However, the molecular pathways linking the InsP3R-mediated increase in Ca2+ and immune responses remain elusive. RESULTS: In the present study, we find that during C. albicans phagocytosis in macrophages, exocyst complex component 2 (SEC5) promotes InsP3R channel activity by binding to its C-terminal α-helix (H1), increasing cytosolic Ca2+ concentrations ([Ca2+]c). Immunofluorescence reveals enriched InsP3R-SEC5 complex formation on phagosomes, while disruption of the InsP3R-SEC5 interaction by recombinant H1 peptides attenuates the InsP3R-mediated Ca2+ elevation, leading to impaired phagocytosis. Furthermore, we show that C. albicans infection promotes the recruitment of Tank-binding kinase 1 (TBK1) by the InsP3R-SEC5 interacting complex, leading to the activation of TBK1. Subsequently, activated TBK1 phosphorylates interferon regulatory factor 3 (IRF-3) and mediates type I interferon responses, suggesting that the InsP3R-SEC5 interaction may regulate antifungal innate immune responses not only by elevating cytoplasmic Ca2+ but also by activating the TBK1-IRF-3 pathway. CONCLUSIONS: Our data have revealed an important role of the InsP3R-SEC5 interaction in innate immune responses against C. albicans.

Dexamethasone-induced cellular tension requires a SGK1-stimulated Sec5-GEF-H1 interaction.

Dexamethasone, a synthetic glucocorticoid, is often used to induce osteoblast commitment of mesenchymal stem cells (MSCs), and this process requires RhoA-dependent cellular tension. The underlying mechanism is unclear. In this study, we show that dexamethasone stimulates expression of fibronectin and integrin α5 (ITGA5), accompanied by an increase in the interaction of GEF-H1 (also known as ARHGEF2) with Sec5 (also known as EXOC2), a microtubule (MT)-regulated RhoA activator and a component of the exocyst, respectively. Disruption of this interaction abolishes dexamethasone-induced cellular tension and GEF-H1 targeting to focal adhesion sites at the cell periphery without affecting dexamethasone-induced levels of ITGA5 and fibronectin, and the extracellular deposition of fibronectin at adhesion sites is specifically inhibited. We demonstrate that dexamethasone stimulates the expression of serum-glucocorticoid-induced protein kinase 1 (SGK1), which is necessary and sufficient for the induction of the Sec5-GEF-H1 interaction. Given the function of SGK1 in suppressing MT growth, our data suggest that the induction of SGK1 through treatment with dexamethasone alters MT dynamics to increase Sec5-GEF-H1 interactions, which promote GEF-H1 targeting to adhesion sites. This mechanism is essential for the formation of fibronectin fibrils and their attachment to integrins at adhesion sites in order to generate cellular tension.

Decreased proteinase A excretion by strengthening its vacuolar sorting and weakening its constitutive secretion in Saccharomyces cerevisiae.

Proteinase A (PrA), encoded by PEP4 gene, is detrimental to beer foam stability. There are two transport pathways for the new synthesized PrA in yeast, sorting to the vacuole normally, or excreting out of the cells under stress conditions. They were designated as the Golgi-to-vacuole pathway and the constitutive secretory pathway, respectively. To reduce PrA excretion in some new way instead of its coding gene deletion, which had a negative effect on cell metabolism and beer fermentation, we modified the PrA transport based on these above two pathways. In the Golgi-to-vacuole pathway, after the verification that Vps10p is the dominant sorting receptor for PrA Golgi-to-vacuolar transportation by VPS10 deletion, VPS10 was then overexpressed. Furthermore, SEC5, encoding exocyst complexes' central subunit (Sec5p) in the constitutive secretory pathway, was deleted. The results show that PrA activity in the broth fermented with WGV10 (VPS10 overexpressing strain) and W∆SEC5 (SEC5 deletion strain) was lowered by 76.96 and 32.39%, compared with the parental strain W303-1A, at the end of main fermentation. There are negligible changes in fermentation performance between W∆SEC5 and W303-1A, whereas, surprisingly, WGV10 had a significantly improved fermentation performance compared with W303-1A. WGV10 has an increased growth rate, resulting in higher biomass and faster fermentation speed; finally, wort fermentation is performed thoroughly. The results show that the biomass production of WGV10 is always higher than that of W∆SEC5 and W303-1A at all stages of fermentation, and that ethanol production of WGV10 is 1.41-fold higher than that of W303-1A. Obviously, VPS10 overexpression is beneficial for yeast and is a more promising method for reduction of PrA excretion.

A signalling cascade for Ral.

Ras is the most mutated oncoprotein in cancer. Among the three oncogenic effectors of Ras - Raf, PI3 Kinase and RalGEF>Ral - signalling through RalGEF>Ral (Ras-like) is by far the least well understood. A variety of signals and binding partners have been defined for Ral, yet we know little of how Ral functions in vivo. This review focuses on previous research in Drosophila that defined a function for Ral in apoptosis and established indirect relationships among Ral, the CNH-domain MAP4 Kinase misshapen, and the JNK MAP kinase basket. Most of the described signalling components are not essential in C. elegans, facilitating subsequent analysis using developmental patterning of the C. elegans vulval precursor cells (VPCs). The functions of two paralogous CNH-domain MAP4 Kinases were defined relative to Ras>Raf, Notch and Ras>RalGEF>Ral signalling in VPCs. MIG-15, the nematode ortholog of misshapen, antagonizes both the Ral-dependent and Ras>Raf-dependent developmental outcomes. In contrast, paralogous GCK-2, the C. elegans ortholog of Drosophila happyhour, propagates the 2°-promoting signal of Ral. Manipulations via CRISPR of Ral signalling through GCK-2 coupled with genetic epistasis delineated a Ras>RalGEF>Ral>Exo84>GCK-2>MAP3KMLK-1> p38PMK-1 cascade. Thus, genetic analysis using invertebrate experimental organisms defined a cascade from Ras to p38 MAP kinase.

SEC5 is involved in M2 polarization of macrophages via the STAT6 pathway, and its dysfunction in decidual macrophages is associated with recurrent spontaneous abortion.

Decidual macrophages (dMϕs) play critical roles in the establishment of microhomeostasis at the maternal-fetal interface during pregnancy. Impaired macrophage polarization during early pregnancy is associated with recurrent spontaneous abortion (RSA). In the present study, the SEC5 expression level was found to be significantly decreased in primary dMϕs of patients with RSA, and downregulation of SEC5 expression inhibited M2 polarization and STAT6 phosphorylation, whereas SEC5 overexpression in the Mϕs promoted M2 polarization and STAT6 phosphorylation in vitro. We subsequently found that SEC5 interacted with STAT6 in THP-1-derived Mϕs. The abundance of phosphorylated STAT6 (pSTAT6) protein was obviously increased, with a predominant distribution in the nucleus, after M2 polarization of Mϕs, and SEC5 protein was colocalized with pSTAT6. Moreover, a significantly reduced pSTAT6 expression level was observed in the dMϕs of patients with RSA. M2 polarization of Mϕs showed a stimulatory effect on the proliferation and invasion of human extravillous trophoblasts (EVTs) in vitro, and downregulation of SEC5 expression in Mϕs effectively reversed this effect. In a mouse model of LPS-induced early pregnancy loss, the uterine SEC5 expression level and the number of M2-Mϕs at the maternal-fetal interface were significantly reduced. More interestingly, heterozygous SEC5-deficient (SEC5-/+) pregnant mice were more sensitive to LPS-induced pregnancy loss. Taken together, these data indicate that SEC5 participates in the regulation of M2 polarization of Mϕs by interacting with STAT6 and that decreased SEC5 expression inhibits the M2 polarization of dMϕs and results in early pregnancy loss by interfering with the physical activities of EVTs and immunotolerance at the maternal-fetal interface.

Hedgehog Signaling Regulates Epithelial Morphogenesis to Position the Ventral Embryonic Midline.

Despite much progress toward understanding how epithelial morphogenesis is shaped by intra-epithelial processes including contractility, polarity, and adhesion, much less is known regarding how such cellular processes are coordinated by extra-epithelial signaling. During embryogenesis, the coelomic epithelia on the two sides of the chick embryo undergo symmetrical lengthening and thinning, converging medially to generate and position the dorsal mesentery (DM) in the embryonic midline. We find that Hedgehog signaling, acting through downstream effectors Sec5 (ExoC2), an exocyst complex component, and RhoU (Wrch-1), a small GTPase, regulates coelomic epithelium morphogenesis to guide DM midline positioning. These effects are accompanied by changes in epithelial cell-cell alignment and N-cadherin and laminin distribution, suggesting Hedgehog regulation of cell organization within the coelomic epithelium. These results indicate a role for Hedgehog signaling in regulating epithelial morphology and provide an example of how transcellular signaling can modulate specific cellular processes to shape tissue morphogenesis.

Ral Signals through a MAP4 Kinase-p38 MAP Kinase Cascade in C. elegans Cell Fate Patterning.

C. elegans vulval precursor cell (VPC) fates are patterned by an epidermal growth factor (EGF) gradient. High-dose EGF induces 1° VPC fate, and lower dose EGF contributes to 2° fate in support of LIN-12/Notch. We previously showed that the EGF 2°-promoting signal is mediated by LET-60/Ras switching effectors, from the canonical Raf-MEK-ERK mitogen-activated protein (MAP) kinase cascade that promotes 1° fate to the non-canonical RalGEF-Ral that promotes 2° fate. Of oncogenic Ras effectors, RalGEF-Ral is by far the least well understood. We use genetic analysis to identify an effector cascade downstream of C. elegans RAL-1/Ral, starting with an established Ral binding partner, Exo84 of the exocyst complex. Additionally, RAL-1 signals through GCK-2, a citron-N-terminal-homology-domain-containing MAP4 kinase, and PMK-1/p38 MAP kinase cascade to promote 2° fate. Our study delineates a Ral-dependent developmental signaling cascade in vivo, thus providing the mechanism by which lower EGF dose is transduced.

The RalGEF/Ral pathway: evaluating an intervention opportunity for Ras cancers.

Recognition that Ral guanine nucleotide exchange factors (RalGEFs) are direct Ras effectors and that Ral G-protein activation is a direct consequence of Ras activation has spurred focused efforts to establish the contribution of RalGEF/Ral signaling to oncogenic transformation. Here, we provide a broad-strokes overview of the mechanistic organization of the RalGEF/Ral signaling network, evaluate the evidence for participation of this network in tumorigenic regulatory milieus, consider targeting strategies, and discuss the challenges to and opportunities for clinical development of these targeting strategies.