Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119- dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119- dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior–Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse

The synaptic ribbon is critical for sound encoding at high rates and with temporal precision.

We studied the role of the synaptic ribbon for sound encoding at the synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in mice lacking RIBEYE (RBEKO/KO). Electron and immunofluorescence microscopy revealed a lack of synaptic ribbons and an assembly of several small active zones (AZs) at each synaptic contact. Spontaneous and sound-evoked firing rates of SGNs and their compound action potential were reduced, indicating impaired transmission at ribbonless IHC-SGN synapses. The temporal precision of sound encoding was impaired and the recovery of SGN-firing from adaptation indicated slowed synaptic vesicle (SV) replenishment. Activation of Ca2+-channels was shifted to more depolarized potentials and exocytosis was reduced for weak depolarizations. Presynaptic Ca2+-signals showed a broader spread, compatible with the altered Ca2+-channel clustering observed by super-resolution immunofluorescence microscopy. We postulate that RIBEYE disruption is partially compensated by multi-AZ organization. The remaining synaptic deficit indicates ribbon function in SV-replenishment and Ca2+-channel regulation

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin (Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/µg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates eight Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation

The Political De-Determination of Legal Rules and the Contested Meaning of the ‘No Bailout’ Clause

Traditional debates on legal theory have devoted a great deal of attention to the question of the determinacy of legal rules. With the aid of social sciences and linguistics, this article suggests a way out of the ‘determinate-indeterminate’ dichotomy that has dominated the academic debate on the topic so far. Instead, a dynamic approach is proposed, in which rules are deemed to undergo processes of political ‘de-determination’ and ‘re-determination’. To illustrate this, the article uses the example of Art. 125 of the Treaty on the Functioning of the European Union, the ‘no bailout’ provision, which played a major role in the management of the Euro-crisis. As will be shown, with the start of the crisis, this provision, whose meaning was once scarcely controversial, became the object of intense interpretative disagreement. As it became politically relevant, the rule also became the site of interpretative competitions, until the intervention of the European Court of Justice disambiguated and redefined its meaning

Approach to diagnosis and pathological examination in bronchial Dieulafoy disease: a case series

<p>Abstract</p> <p>Background</p> <p>There are limited series concerning Dieulafoy disease of the bronchus. We describe the clinical presentation of a series of 7 patients diagnosed with Dieulafoy disease of the bronchus and provide information about the pathological diagnosis approach.</p> <p>Patients and methods</p> <p>A retrospective review of patients who underwent surgery for massive and unexplained recurrent hemoptysis in a referral center during a 11-year period.</p> <p>Results</p> <p>Seven heavy smoker (49 pack years) patients (5 males) mean aged 54 years experienced a massive hemoptysis (350–1000 ml) unrelated to a known lung disease and frequently recurrent. Bronchial contrast extravasation was observed in 3 patients, combining both CT scan and bronchial arteriography. Efficacy of bronchial artery embolization was achieved in 40% of cases before surgery. Pathological examination demonstrated a minute defect in 3 cases and a large and dysplasic superficial bronchial artery in the submucosa in all cases.</p> <p>Conclusion</p> <p>Dieulafoy disease should be suspected in patients with massive and unexplained episodes of recurrent hemoptysis, in order to avoid hazardous endoscopic biopsies and to alert the pathologist if surgery is performed.</p