How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon‐specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca²⁺‐buffer EGTA, suggesting that synaptic ribbons mediate nano‐domain coupling of Ca²⁺ channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano‐domains that position release‐ready synaptic vesicles adjacent to Ca²⁺ channels.     

The P2-receptor-mediated Ca2+ signalosome of the human pulmonary endothelium - implications for pulmonary arterial hypertension

Purinergic Signalling - Dysfunction of the pulmonary endothelium is associated with most lung diseases. Extracellular nucleotides modulate a plethora of endothelial functions in the lung such as...     

A short LysM protein with high molecular diversity from an arbuscular mycorrhizal fungus,Rhizophagus irregularis

Arbuscular mycorrhizal (AM) fungi form an intimate symbiosis with roots of more than 80% of land plants without eliciting a significant defense response, and how they do so is yet to be determined. Typically, plants mount a defense response upon sensing chitin in fungal walls, and to counteract this response, plant-pathogenic fungi secrete small effector proteins with chitin-binding LysM domains. In the AM fungus, Rhizophagus irregularis, a small, putatively-secreted LysM protein, which we refer to as RiSLM, is among the most highly expressed effector-like proteins during symbiosis. Here, we show that RiSLM expression is reduced during non-functional symbiosis with Medicago mutants, mtpt4-2 and vapyrin. We demonstrate that RiSLM can bind to both chitin and chitosan, and we model the protein-ligand interaction to identify possible binding sites. Finally, we have identified RiSLM homologs in five published R. irregularis isolate genomes and demonstrate that the gene is subject to a high rate of evolution and is experiencing positive selection, while still conserving putative function. Our results present important clues for elucidating a role for a LysM effector, RiSLM, in AM symbiosis.     

Roles of the ClpX IGF loops in ClpP association,dissociation, and protein degradation

IGF‐motif loops project from the hexameric ring of ClpX and are required for docking with the self‐compartmentalized ClpP peptidase, which consists of heptameric rings stacked back‐to‐back. Here, we show that ATP or ATPγS support assembly by changing the conformation of the ClpX ring, bringing the IGF loops closer to each other and allowing efficient multivalent contacts with docking clefts on ClpP. In single‐chain ClpX pseudohexamers, deletion of one or two IGF loops modestly slows association with ClpP but strongly accelerates dissociation of ClpXP complexes. We probe how changes in the sequence and length of the IGF loops affect ClpX–ClpP interactions and show that deletion of one or two IGF loops slows ATP‐dependent proteolysis by ClpXP. We also find that ClpXP degradation is less processive when two IGF loops are deleted.     

The Role of VIN3-LIKE Genes in Environmentally Induced Epigenetic Regulation of Flowering

Given their sessile nature, it is critical for the survival of plants to adapt to their environment. Accordingly, plants have evolved the ability to sense seasonal changes to govern developmental fates such as the floral transition. Temperature and day length are among the seasonal cues that plants sense. We recently reported that VIN3-LIKE 1 (VIL1) is involved in mediating the flowering response to both cold and day length via regulation of two related genes, FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M (FLM), respectively.Key Words: flowering, vernalization, photoperiod, chromatin, histone, gene expressionVernalization renders plants competent to flower after exposure to the prolonged cold of winter.^1,2 Arabidopsis exhibits facultative responses to both vernalization and photoperiod to initiate the floral transition. The facultative nature of the responses makes Arabidopsis a tractable genetic system to study these aspects of flowering time control.In Arabidopsis, vernalization creates competence to flower via silencing of the potent floral repressor, FLC, in a mitotically stable manner.^3,4 Thus, the vernalization response is an environmentally induced epigenetic switch in that exposure to cold permanently affects the plants'' developmental program. This epigenetic switch is associated with increased levels of FLC chromatin methylation on Histone H3 Lys 9 and Lys 27.^5,6 VERNALIZATION INSENSITIVE 3 (VIN3) plays an essential role in this switch since no modifications to FLC chromatin occur in vin3 mutants.⁵ Furthermore, the levels of expression of VIN3 mRNA are tightly correlated with the degree of the vernalization response.⁵ VIN3 encodes Plant HomeoDomain (PHD) finger-containing protein. PHD finger-containing proteins are often associated with protein complexes that are involved in chromatin remodeling.⁷We performed a yeast two-hybrid screen to identify potential protein partners of VIN3. VIN3-LIKE 1 (VIL1) was identified by this screen.⁸ VIL1 encodes a PHD finger-containing protein that is related to VIN3. As expected for proteins that are associated with VIN3, plants containing loss-of-function alleles of VIL1 do not respond to vernalization. Furthermore, no vernalization-mediated histone modifications occur at FLC in vil1 mutants similar to the situation in vin3 mutants. Thus, by yeast two hybrid and genetic analysis, VIL1 is a bona fide VIN3 partner that is required for vernalization-mediated histone modifications at FLC chromatin. Unlike VIN3, the expression of VIL1 does not change over the course of cold exposure. Rather, VIL1 mRNA levels are affected by photoperiod. VIL1 expression is significantly increased in non-inductive photoperiods (short days; SD). Consistent with this expression pattern, vil1 mutants in the Columbia accession exhibit a SD-specific late-flowering phenotype. Furthermore, VIL1 is required for attenuating expression of FLOWERING LOCUS M, a FLC-related gene, in a SD-specific manner. It is possible that the attenuation of FLM by VIL1 has a role in creating the facultative nature of photoperiod response in Arabidopsis since vil1 mutants tend towards an obligate photoperiod response (i.e., vil1 mutants often fail to flower in SD).In Arabidopsis, there are four VIN3-related genes, which we named as VIL1 ∼ VIL4,⁸ and which have also been called VRN5 and VEL1 ∼ VEL3.⁹ The C-terminal domain is highly conserved among these genes and was named the VIN3-Interacting Domain (VID) since it is required for protein-protein interaction between VIN3 and VIL1. The effect of cold on the expression patterns of VIN3-related genes varies. For example, VIL2 and VIL3 are induced specifically by vernalizing cold exposures whereas others such as VIL1 are, for the most part, constitutively expressed. It will be interesting to determine the functions of the remaining VIL genes.FLC is the main target for vernalization in Arabidopsis. Interestingly, FLC orthologs have not been found in vernalization-responsive varieties of cereals. However, in wheat, VRN2 appears to have a role equivalent to that of FLC in Arabidopsis.¹⁰ VRN2 encodes a ZCCT type zinc-finger protein that does not have a homolog in the Arabidopsis genome. In diploid wheat, down regulation of VRN2 is correlated with the vernalization response.¹¹ Interestingly, wheat contains three VIN3-LIKE (VIL) genes, TmVIL1, TmVIL2 and TmVIL3.¹² Furthermore, TmVIL1 is up-regulated by vernalization.¹² However, whether TmVIL1 has a direct role in the vernalization-mediated repression of VRN2 in wheat has not yet been addressed. Similar to VIL1, TmVIL3 shows elevated level of expression in SD. Furthermore, VRN2 is downregulated in SD;^13,14 thus there is a correlation between the induction of TmVIL genes and the downregulation of the floral repressor VRN2 similar to the VIN3/FLC and VIL1/FLM relationships (Fig. 1). Perhaps VIN3-related genes have similar roles both in Arabidopsis and in temperate wheat, but act on different target genes, possibly as a result of convergent evolution. Interestingly, the wheat gene TmVRN3 is homologous to FLOWERING LOCUS T (FT) of Arabidopsis, and TmVRN3 is repressed by TmVRN2 as FT is repressed by FLC,¹⁵ suggesting another similarity in the regulation of flowering time between Arabidopsis and temperate wheat (Fig. 1).Open in a separate windowFigure 1Proposed relationship of VIN3 family genes to the regulatory network controlling flowering time in response to environmental cues in Arabidopsis and diploid wheat (adapted from ref. ¹⁶).Although the PHD finger can be found in various eukaryotes, the VID is unique to plants. It is also noteworthy that VIN3-related genes can be found in various plant species, including rice, which does not have a vernalization response. It will be interesting to address whether the VIN3-related genes from various plant species are more broadly involved in relaying environmental signals to developmental programs.     

Monocyte cholesterol homeostasis correlates with the presence of detergent resistant membrane microdomains.

BACKGROUND: Lipid membrane microdomains are involved in the regulation of biological functions of monocyte membrane proteins. These microdomains show a relative resistance to non-ionic detergents providing an easy analytical tool to study them. METHODS: Here, we applied a rapid detergent-based flow cytometric assay to investigate microdomain association of proteins on monocytes from whole blood samples. The association of known surface antigens with detergent resistant fraction of membranes (DRMs) was compared using monocytes from healthy blood donors, patients with genetic disorders affecting cellular cholesterol traffic and patients with systemic inflammatory response. RESULTS: All investigated surface antigens of Niemann-Pick type C (NPC)-mutant monocytes with impaired cholesterol influx and defective late endosome cholesterol trafficking, presented a strongly increased DRM-association. Though, membrane antigens of ATP binding cassette transporter A1 (ABCA1)-mutant monocytes with impaired cholesterol efflux did not show alterations in DRM-association. Differential CD14-dependent receptor clustering within microdomains was also investigated in response to in vivo lipopolysaccharide (LPS) and/or atherogenic lipoprotein activation. Increased DRM-association of the GPI-anchored proteins CD14, CD55, the Fcgamma receptor CD64, the scavenger receptors CD36, CD91 and CD163, the integrin CD11a, and complement receptor 3 complex CD11b/CD18 were observed from patients with systemic inflammatory response syndrome (SIRS)/sepsis or coronary artery disease (CAD)/myocardial infarction. Interestingly, the tetraspanin CD81 presented increased DRM-association in SIRS/sepsis patients, but not in CAD patients. Moreover, the pentaspanin CD47 and the Fcgamma RIII CD16 showed an increased DRM partition in CAD patients but disassembled from DRMs in SIRS/sepsis patients. CONCLUSIONS: Our results demonstrate that flow cytometric analysis of short time in situ detergent extraction provides a powerful tool for rapid screening of blood monocyte DRMs to preselect patients with potential raft/microdomain abnormalities for more detailed analysis.