Lipid Binding Defects and Perturbed Synaptogenic Activity of a Collybistin R290H Mutant That Causes Epilepsy and Intellectual Disability

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects—or synaptopathies—are at the basis of many neurological and psychiatric disorders. In key areas of the mammalian brain, such as the hippocampus or the basolateral amygdala, the clustering of the scaffolding protein Gephyrin and of γ-aminobutyric acid type A receptors at inhibitory neuronal synapses is critically dependent upon the brain-specific guanine nucleotide exchange factor Collybistin (Cb). Accordingly, it was discovered recently that an R290H missense mutation in the diffuse B-cell lymphoma homology domain of Cb, which carries the guanine nucleotide exchange factor activity, leads to epilepsy and intellectual disability in human patients. In the present study, we determined the mechanism by which the Cb^R290H mutation perturbs inhibitory synapse formation and causes brain dysfunction. Based on a combination of biochemical, cell biological, and molecular dynamics simulation approaches, we demonstrate that the R290H mutation alters the strength of intramolecular interactions between the diffuse B-cell lymphoma homology domain and the pleckstrin homology domain of Cb. This defect reduces the phosphatidylinositol 3-phosphate binding affinity of Cb, which limits its normal synaptogenic activity. Our data indicate that impairment of the membrane lipid binding activity of Cb and a consequent defect in inhibitory synapse maturation represent a likely molecular pathomechanism of epilepsy and mental retardation in humans.     

Nitrogen fertigation of greenhouse-grown cucumber

Summary This greenhouse study investigated the response of trickle-irrigated cucumber (Cucumis sativa cv. ‘Petita’) to three N levels applied with every irrigation via the irrigation stream. The plants were grown in pots filled with 12 kg of soil. Water containing 5.8, 11.8, or 17.8 mmol N/l, and uniformly supplied with 2.0 and 3.9 mmol/l of P and K, respectively, was applied two to three times daily. In all treatments of 0.3 leaching fraction was allowed. The resulting total N applications were 15.7, 31., and 47.2 g N/plant. The total amount of water applied was 1851/plant. Total N and NO₃-N, in lajinae and petioles, increased with increasing N level whereas P and K in generated decreased. Although different NO₃/NH₄ ratios in the treatments may have influeced the response to N, it could be concluded that the highest yield was obtained with 11.8 mmol N/1 due to increased number of fruit. In the root volume of this treatment the NO₃-N concentration in the soil solution was aroun 7 mmol/1 for most of the growing season. The dry matter concentration of fruits was not affected by the N levels. It was concluded that 11.8 mmol N/1 applied with every irrigation via the irrigation stream is adequate to cover the needs of greenhous-grown cucumber for higher yield (9.42 kg/plant over a harvesting period of 93 days).     

Effects of seminiferous tubule secreted factor(s) on Leydig cell cyclic AMP production in mature rat

The effects of spent media from seminiferous tubules (STM) on Percoll-purified rat Leydig cells were investigated. Intracellular and extracellular cyclic AMP (cAMP) accumulation and testosterone production were measured. After a 5 h incubation period, STM reduces both the basal and LH-dependent cAMP levels (38 and 20%, respectively for intra- and extracellular cAMP) while, simultaneously, a stimulation of testosterone production is observed (47 to 50%, respectively in the absence or presence of LH). The reduction of cAMP levels observed after 5 h is likely to be due to the potentiating effect of the STM factor on the LH-dependent initial rise of the cAMP level which, in turn, induces a desensitization of the Leydig cell adenylate cyclase. This substance is a thermolabile protein (Mr greater than 50 000) produced by the Sertoli cell, independent of FSH and testosterone controls, and different from the LHRH-like substance.     

From trickle to flood: the large-scale,cryptic invasion of California by tropical fruit flies

Since 1954, when the first tropical tephritid fruit fly was detected in California, a total of 17 species in four genera and 11 386 individuals (adults/larvae) have been detected in the state at more than 3348 locations in 330 cities. We conclude from spatial mapping analyses of historical capture patterns and modelling that, despite the 250+ emergency eradication projects that have been directed against these pests by state and federal agencies, a minimum of five and as many as nine or more tephritid species are established and widespread, including the Mediterranean, Mexican and oriental fruit flies, and possibly the peach, guava and melon fruit flies. We outline and discuss the evidence for our conclusions, with particular attention to the incremental, chronic and insidious nature of the invasion, which involves ultra-small, barely detectable populations. We finish by considering the implications of our results for invasion biology and for science-based invasion policy.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.     

Enterobacter sp. AA26 gut symbiont as a protein source for Mediterranean fruit fly mass-rearing and sterile insect technique applications

Background

The interaction between gut bacterial symbionts and Tephritidae became the focus of several studies that showed that bacteria contributed to the nutritional status and the reproductive potential of its fruit fly hosts. Anastrepha fraterculus is an economically important fruit pest in South America. This pest is currently controlled by insecticides, which prompt the development of environmentally friendly methods such as the sterile insect technique (SIT). For SIT to be effective, a deep understanding of the biology and sexual behavior of the target species is needed. Although many studies have contributed in this direction, little is known about the composition and role of A. fraterculus symbiotic bacteria. In this study we tested the hypothesis that gut bacteria contribute to nutritional status and reproductive success of A. fraterculus males.

Results

AB affected the bacterial community of the digestive tract of A. fraterculus, in particular bacteria belonging to the Enterobacteriaceae family, which was the dominant bacterial group in the control flies (i.e., non-treated with AB). AB negatively affected parameters directly related to the mating success of laboratory males and their nutritional status. AB also affected males’ survival under starvation conditions. The effect of AB on the behaviour and nutritional status of the males depended on two additional factors: the origin of the males and the presence of a proteinaceous source in the diet.

Conclusions

Our results suggest that A. fraterculus males gut contain symbiotic organisms that are able to exert a positive contribution on A. fraterculus males’ fitness, although the physiological mechanisms still need further studies.

     

RUN and FYVE domain–containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain–containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17–positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4–treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.     

Rem uncouples excitation–contraction coupling in adult skeletal muscle fibers

In skeletal muscle, excitation–contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca²⁺ channel (Ca_V1.1) to be communicated to the type 1 ryanodine-sensitive Ca²⁺ release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein–protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca_V1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α_1S subunit of Ca_V1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary β_1a subunit of Ca_V1.1 is a conduit for this intermolecular communication. However, a direct role for β_1a has been difficult to test because β_1a serves two other functions that are prerequisite for conformational coupling between Ca_V1.1 and RYR1. Specifically, β_1a promotes efficient membrane expression of Ca_V1.1 and facilitates the tetradic ultrastructural arrangement of Ca_V1.1 channels within plasma membrane–SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established β subunit–interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca²⁺ transients without greatly affecting Ca_V1.1 targeting, intramembrane gating charge movement, or releasable SR Ca²⁺ store content. In contrast, a β_1a-binding–deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca²⁺ release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca_V1.1 from RYR1-mediated SR Ca²⁺ release via its ability to interact with β_1a. Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the β_1a subunit in skeletal-type EC coupling.