Exomer Is Part of a Hub Where Polarized Secretion and Ionic Stress Connect.

Plasma membrane and membranous organelles contribute to the physiology of the Eukaryotic cell by participating in vesicle trafficking and the maintenance of ion homeostasis. Exomer is a protein complex that facilitates vesicle transport from the trans-Golgi network to the plasma membrane, and its absence leads to the retention of a set of selected cargoes in this organelle. However, this retention does not explain all phenotypes observed in exomer mutants. The Schizosaccharomyces pombe exomer is composed of Cfr1 and Bch1, and cfr1Δ and bch1Δ were sensitive to high concentrations of potassium salts but not sorbitol, which showed sensitivity to ionic but not osmotic stress. Additionally, the activity of the plasma membrane ATPase was higher in exomer mutants than in the wild-type, pointing to membrane hyperpolarization, which caused an increase in intracellular K+ content and mild sensitivity to Na+, Ca2+, and the aminoglycoside antibiotic hygromycin B. Moreover, in response to K+ shock, the intracellular Ca2+ level of cfr1Δ cells increased significantly more than in the wild-type, likely due to the larger Ca2+ spikes in the mutant. Microscopy analyses showed a defective endosomal morphology in the mutants. This was accompanied by an increase in the intracellular pools of the K+ exporting P-type ATPase Cta3 and the plasma membrane Transient Receptor Potential (TRP)-like Ca2+ channel Pkd2, which were partially diverted from the trans-Golgi network to the prevacuolar endosome. Despite this, most Cta3 and Pkd2 were delivered to the plasma membrane at the cell growing sites, showing that their transport from the trans-Golgi network to the cell surface occurred in the absence of exomer. Nevertheless, shortly after gene expression in the presence of KCl, the polarized distribution of Cta3 and Pkd2 in the plasma membrane was disturbed in the mutants. Finally, the use of fluorescent probes suggested that the distribution and dynamics of association of some lipids to the plasma membrane in the presence of KCl were altered in the mutants. Thus, exomer participation in the response to K+ stress was multifaceted. These results supported the notion that exomer plays a general role in protein sorting at the trans-Golgi network and in polarized secretion, which is not always related to a function as a selective cargo adaptor.

The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain.

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8-10 aa)C signature motif. Structure-function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.

Traffic Through the Trans-Golgi Network and the Endosomal System Requires Collaboration Between Exomer and Clathrin Adaptors in Fission Yeast.

Despite its biological and medical relevance, traffic from the Golgi to the plasma membrane (PM) is one of the least understood steps of secretion. Exomer is a protein complex that mediates the trafficking of certain cargoes from the trans-Golgi network/early endosomes to the PM in budding yeast. Here, we show that in Schizosaccharomyces pombe the Cfr1 and Bch1 proteins constitute the simplest form of an exomer. Cfr1 co-immunoprecipitates with Assembly Polypeptide adaptor 1 (AP-1), AP-2, and Golgi-localized, gamma-adaptin ear domain homology, ARF-binding (GGA) subunits, and cfr1+ interacts genetically with AP-1 and GGA genes. Exomer-defective cells exhibit multiple mild defects, including alterations in the morphology of Golgi stacks and the distribution of the synaptobrevin-like Syb1 protein, carboxypeptidase missorting, and stress sensitivity. S. pombe apm1Δ cells exhibit a defect in trafficking through the early endosomes that is severely aggravated in the absence of exomer. apm1Δ cfr1Δ cells exhibit a dramatic disorganization of intracellular compartments, including massive accumulation of electron-dense tubulovesicular structures. While the trans-Golgi network/early endosomes are severely disorganized in the apm1Δ cfr1Δ strain, gga21Δ gga22Δ cfr1Δ cells exhibit a significant disturbance of the prevacuolar/vacuolar compartments. Our findings show that exomer collaborates with clathrin adaptors in trafficking through diverse cellular compartments, and that this collaboration is important to maintain their integrity. These results indicate that the effect of eliminating exomer is more pervasive than that described to date, and suggest that exomer complexes might participate in diverse steps of vesicle transport in other organisms.

The AP-2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast.

In metazoans the AP-2 complex has a well-defined role in clathrin-mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co- immunoprecipitation between the fission yeast AP-2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4-64 uptake. In an otherwise wild-type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP-2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP-2 in endocytosis has been conserved throughout evolution.

Acute in vitro effects of dronedarone, an iodine-free derivative, and amiodarone, on the rabbit sinoatrial node automaticity: a comparative study.

Amiodarone is a potent antiarrhythmic drug commonly used in the treatment of supraventricular and ventricular arrhythmias. Dronedarone is a recently developed iodine-free compound (Sanofi Recherche), structurally related to amiodarone. Amiodarone and dronedarone have shown similar long-term effects on sinoatrial node automaticity in vivo and in vitro in the rabbit heart. In the present study, we used a microelectrode technique to compare the acute in vitro electrophysiologic effects of amiodarone (100 microM) and dronedarone (100 microM) on the rabbit sinus node. Like amiodarone, dronedarone induces a marked reduction in sinus node automaticity, evidenced by decreases in spontaneous beating rate, action potential amplitude, and slope of phase 4 depolarization. Isoproterenol dose-dependently increases sinus node automaticity in the presence of either amiodarone or dronedarone. The data suggest that dronedarone may be a useful antiarrhythmic alternative to amiodarone in the treatment of supraventricular arrhythmias.

In vitro effects of acute amiodarone and dronedarone on epicardial, endocardial, and M cells of the canine ventricle.

Amiodarone (AM) is an antiarrhythmic agent widely used in the treatment of ventricular and supraventricular arrhythmias. Dronedarone (DR) is a new compound with a pharmacological profile similar to that of AM, but iodine free. We previously demonstrated that chronic AM treatment reduces transmural dispersion of repolarization (TDR) in the canine heart. We used standard microelectrode technique to evaluate the effects of acute AM (100 microM) and DR (30 microM) on epicardial (EPI), endocardial (ENDO), and M region tissues obtained from the left ventricular wall of the canine heart. Amiodarone (100 microM, 120 min of exposure) produced little change in the action potential duration of ENDO and EPI tissues, but it shortened the action potential of M cells, especially at slow rates, leading to a decrease in TDR. Similar results were observed with DR. Acute AM (100 microM) and DR (30 microM) eliminated d-sotalol-induced early afterdepolarizations (EADs) and triggered activity in 3 of 3 and 2 of 6 M cell preparations, respectively. The reduction of TDR and the elimination of EAD-induced triggered activity differentiates AM and DR from other class III agents. These effects may explain the efficacy and low arrhythmogenicity of acute AM and suggest a potential safe use of DR as an antiarrhythmic agent.

Estudio comparativo de los efectos de la dronedarona y la amiodarona sobre el automatismo del nódulo sinusal del conejo / Comparative study of the effects of dronedarone and amiodarone on automaticity of the sinus node of the rabbit

La amiodarona es un agente antiarrítmico de gran eficacia en el tratamiento de las arritmias supraventriculares y ventriculares. La dronedarona es un nuevo derivado de la amiodarona libre de yodo con un perfil farmacológico similar. En este estudio se comparan los efectos electrofisiológicos in vitro de la amiodarona y la dronedarona sobre preparados de nódulo sinusal del corazón de conejo. La amiodarona (30 µM) y la dronedarona (10µM) prolongaron la longitud del ciclo espontánea y disminuyeron la amplitud del potencial de acción y la pendiente de la fase 4. En este modelo, la presencia de dronedarona no modificó la intensidad de la respuesta automática ante la exposición a isoproterenol. La dronedarona, como la amiodarona, deprime el automatismo sinusal, debido probablemente a un mecanismo de bloqueo cálcico

Efectos agudos de la amiodarona y de la dronedarona en células epicárdicas, M y endocárdicas del ventrículo canino / Acute effects of amiodarone and dronedarone on epicardial, M and endocardial cells of the canine ventricle

La amiodarona y la dronedarona, in vitro, desarrollan efectos electrofisiológicos similares entre sí y con el tratamiento crónico de amiodarona. Disminuyen la dispersión de la refractariedad ventricular, acortando principalmente el potencial de acción de las células M prolongándolo levemente en el endocardio y el epicardio e inhibiendo y revirtiendo el aumento desproporcionado de la duración del potencial de las células M y las posdespolarizaciones precoces inducidas por el d-sotalol. No se observó un efecto depresor de la Vmáx en estas células con ninguna de las dos drogas. La dronedarona parece ser más potente, ya que con concentraciones menores se alcanzaron resultados similares a la amiodarona

Estudio comparativo de los efectos de la dronedarona y la amiodarona sobre el automatismo del nódulo sinusal del conejo / Comparative study of the effects of dronedarone and amiodarone on automaticity of the sinus node of the rabbit

La amiodarona es un agente antiarrítmico de gran eficacia en el tratamiento de las arritmias supraventriculares y ventriculares. La dronedarona es un nuevo derivado de la amiodarona libre de yodo con un perfil farmacológico similar. En este estudio se comparan los efectos electrofisiológicos in vitro de la amiodarona y la dronedarona sobre preparados de nódulo sinusal del corazón de conejo. La amiodarona (30 AM) y la dronedarona (10AM) prolongaron la longitud del ciclo espontánea y disminuyeron la amplitud del potencial de acción y la pendiente de la fase 4. En este modelo, la presencia de dronedarona no modificó la intensidad de la respuesta automática ante la exposición a isoproterenol. La dronedarona, como la amiodarona, deprime el automatismo sinusal, debido probablemente a un mecanismo de bloqueo cálcico (AU)

Efectos agudos de la amiodarona y de la dronedarona en células epicárdicas, M y endocárdicas del ventrículo canino / Acute effects of amiodarone and dronedarone on epicardial, M and endocardial cells of the canine ventricle

La amiodarona y la dronedarona, in vitro, desarrollan efectos electrofisiológicos similares entre sí y con el tratamiento crónico de amiodarona. Disminuyen la dispersión de la refractariedad ventricular, acortando principalmente el potencial de acción de las células M prolongándolo levemente en el endocardio y el epicardio e inhibiendo y revirtiendo el aumento desproporcionado de la duración del potencial de las células M y las posdespolarizaciones precoces inducidas por el d-sotalol. No se observó un efecto depresor de la Vmáx en estas células con ninguna de las dos drogas. La dronedarona parece ser más potente, ya que con concentraciones menores se alcanzaron resultados similares a la amiodarona (AU)