Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

DNA-Binding Magnetic Nanoreactor Beads for Digital PCR Analysis.

Digital PCR (dPCR) is based on the separation of target amplification reactions into many compartments with randomly distributed template molecules. Here, we present a novel digital PCR format based on DNA binding magnetic nanoreactor beads (mNRBs). Our approach relies on the binding of all nucleic acids present in a sample to the mNRBs, which both provide a high-capacity binding matrix for capturing nucleic acids from a sample and define the space available for PCR amplification by the internal volume of their hydrogel core. Unlike conventional dPCR, this approach does not require a precise determination of the volume of the compartments used but only their number to calculate the number of amplified targets. We present a procedure in which genomic DNA is bound, the nanoreactors are loaded with PCR reagents in an aqueous medium, and amplification and detection are performed in the space provided by the nanoreactor suspended in fluorocarbon oil. mNRBs exhibit a high DNA binding capacity of 1.1 ng DNA/mNRB (95% CI 1.0-1.2) and fast binding kinetics with ka = 0.21 s-1 (95% CI 0.20-0.23). The dissociation constant KD was determined to be 0.0011 µg/µL (95% CI 0.0007-0.0015). A simple disposable chamber plate is used to accommodate the nanoreactor beads in a monolayer formation for rapid thermocycling and fluorescence detection. The performance of the new method was compared with conventional digital droplet PCR and found to be equivalent in terms of the precision and linearity of quantification. In addition, we demonstrated that mNRBs provide quantitative capture and loss-free analysis of nucleic acids contained in samples in different volumes.

The Na+/H+ Exchanger Nhe1 Modulates Network Excitability via GABA Release.

Brain functions are extremely sensitive to pH changes because of the pH-dependence of proteins involved in neuronal excitability and synaptic transmission. Here, we show that the Na+/H+ exchanger Nhe1, which uses the Na+ gradient to extrude H+, is expressed at both inhibitory and excitatory presynapses. We disrupted Nhe1 specifically in mice either in Emx1-positive glutamatergic neurons or in parvalbumin-positive cells, mainly GABAergic interneurons. While Nhe1 disruption in excitatory neurons had no effect on overall network excitability, mice with disruption of Nhe1 in parvalbumin-positive neurons displayed epileptic activity. From our electrophysiological analyses in the CA1 of the hippocampus, we conclude that the disruption in parvalbumin-positive neurons impairs the release of GABA-loaded vesicles, but increases the size of GABA quanta. The latter is most likely an indirect pH-dependent effect, as Nhe1 was not expressed in purified synaptic vesicles itself. Conclusively, our data provide first evidence that Nhe1 affects network excitability via modulation of inhibitory interneurons.

Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247.

Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide-cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis.