Identifying RNA splicing factors using IFT genes in Chlamydomonas reinhardtii

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 ( fla9 ) and IFT121 ( ift121-2 ), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii . The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3′ splice site mutation in IFT81 , and a frameshift mutant of FRA10 , which suppresses the 5′ splice site mutation in IFT121 . Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1 , which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening. </jats:p

Primary cilia of human endothelial cells disassemble under laminar shear stress

We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-α-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells

Síndrome de Bayés, accidente cerebrovascular y demencia

Bayés’s síndrome is a clinical entity based on the association between advanced interatrial block and the development of supraventricular tachyarrhythmia, being atrial fibrillation (AF), most frequent. This association was discovered by Prof. Antoni Bayés de Luna in the’80s. Further studies by other groups found a strong relationship between Bayés’s syndrome and thromboembolic phenomena, being stroke the most serious. Moreover, patients with this syndrome has an increased incidence of cognitive impairment and dementia. This observation triggered the question about whether the use of an anticoagulation therapy prior to the documentation of AF could prevent A-IAB associated thromboembolic events. There are ongoing studies in different phases of development aiming to compare the efficacy of anticoagulation in patients with A-IAB with no prior documentation of AF. The outcomes of these studies will allow determining the efficacy of this early therapeutic intervention, and help deciding the role of anticoagulation in patients with A-IAB and no demonstrated AF.El síndrome de Bayés es una entidad clínica que se define como la asociación entre la presencia de bloqueo interauricular avanzado (BIA-A), y el desarrollo de taquiarritmias supraventriculares (TSV), siendo la fibrilación auricular (FA), la más frecuente. Esta asociación salió a la luz con los estudios del Prof. Antoni Bayés de Luna en los años ‘80. Estudios realizados posteriormente por otros grupos encontraron una fuerte asociación entre este síndrome y la ocurrencia de fenómenos tromboembólicos, siendo el accidente cerebrovascular (ACV) el más grave.&nbsp; Del mismo modo, se documentó una asociación con deterioro cognitivo y demencia. Esto generó la pregunta acerca de si la terapia de anticoagulación previa a la documentación de FA podría prevenir la ocurrencia de tromboembolismos asociados al BIA-A y de esta manera preservar el estado cognitivo. Esta pregunta permanece, aún hoy, sin respuesta definitiva. Existen estudios en diferentes estadios de su desarrollo cuyo objetivo es comparar la efectividad de la anticoagulación en pacientes con BIA-A y sin FA documentada. Los resultados de los mismos permitirán establecer la efectividad de esta opción terapéutica temprana y, de manera conclusiva, definir si, para decidir anticoagular un paciente, la FA tiene que ser previamente documentada o no

The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking

Two intraflagellar transport (IFT) complexes, IFT-A and IFT-B, build and maintain primary cilia and are required for activity of the Sonic hedgehog (Shh) pathway. A weak allele of the IFT-A gene, Ift144, caused subtle defects in cilia structure and ectopic activation of the Shh pathway. In contrast, strong loss of IFT-A, caused by either absence of Ift144 or mutations in two IFT-A genes, blocked normal ciliogenesis and decreased Shh signaling. In strong IFT-A mutants, the Shh pathway proteins Gli2, Sufu, and Kif7 localized correctly to cilia tips, suggesting that these pathway components were trafficked by IFT-B. In contrast, the membrane proteins Arl13b, ACIII, and Smo failed to localize to primary cilia in the absence of IFT-A. We propose that the increased Shh activity seen in partial loss-of-function IFT-A mutants may be a result of decreased ciliary ACIII and that the loss of Shh activity in the absence of IFT-A is a result of severe disruptions of cilia structure and membrane protein trafficking

Primary cilia signaling mediates intraocular pressure sensation

Lowe syndrome is a rare X-linked congenital disease that presents with congenital cataracts and glaucoma, as well as renal and cerebral dysfunction. OCRL, an inositol polyphosphate 5-phosphatase, is mutated in Lowe syndrome. We previously showed that OCRL is involved in vesicular trafficking to the primary cilium. Primary cilia are sensory organelles on the surface of eukaryotic cells that mediate mechanotransduction in the kidney, brain, and bone. However, their potential role in the trabecular meshwork (TM) in the eye, which regulates intraocular pressure, is unknown. Here, we show that TM cells, which are defective in glaucoma, have primary cilia that are critical for response to pressure changes. Primary cilia in TM cells shorten in response to fluid flow and elevated hydrostatic pressure, and promote increased transcription of TNF-α, TGF-β, and GLI1 genes. Furthermore, OCRL is found to be required for primary cilia to respond to pressure stimulation. The interaction of OCRL with transient receptor potential vanilloid 4 (TRPV4), a ciliary mechanosensory channel, suggests that OCRL may act through regulation of this channel. A novel disease-causing OCRL allele prevents TRPV4-mediated calcium signaling. In addition, TRPV4 agonist GSK 1016790A treatment reduced intraocular pressure in mice; TRPV4 knockout animals exhibited elevated intraocular pressure and shortened cilia. Thus, mechanotransduction by primary cilia in TM cells is implicated in how the eye senses pressure changes and highlights OCRL and TRPV4 as attractive therapeutic targets for the treatment of glaucoma. Implications of OCRL and TRPV4 in primary cilia function may also shed light on mechanosensation in other organ systems

Autopercepción de las habilidades para la cesación del tabaquismo entre los residentes de cardiología en Argentina

Objective. To evaluate the self-perception of cardiology residents in Argentina regarding their abilities to help their patients stop smoking, as well as their opinions about their knowledge and skills in this area. Materials and methods. A cross-sectional study was carried out using secondary data from a study carried out in five Latin American countries and Spain, focusing on the information provided by cardiology residents in Argentina. Discrete variables were expressed as median and interquartile range, and categorical variables were expressed as percentages, and were analyzed using the chi-square test or Fisher’s exact test, depending on the relative frequency of the expected values. Results. 447 residents participated; 87.5% routinely provided brief advice to quit smoking, and 11.6% used validated questionnaires to assess the degree of addiction. Furthermore, 32.1% stated that they prescribed pharmacological treatment, but 53.1% were only familiar with a single drug. When asked about their self-perception of getting their patients to stop smoking, the median response was 5 (scale from 1 to 10); only 13.7% responded with a score of 8 or more. Conclusions. The present study suggests that cardiology residents in Argentina recognize the importance of carrying out smoking cessation interventions, but a high proportion of them do not feel qualified to do so.Objetivo. Evaluar la autopercepción de los residentes de cardiología en Argentina con respecto a sus habilidades para que sus pacientes dejen de fumar, así como sus opiniones sobre sus conocimientos y habilidades en esta área. Materiales y métodos. Estudio transversal a partir de los datos secundarios de un estudio realizado en cinco países de Latinoamérica y España, centrándose en la información proporcionada por los residentes de cardiología en Argentina. Las variables discretas se expresaron como mediana y rango intercuartílico, las variables categóricas se expresaron como porcentajes, y se analizaron mediante la prueba de chi cuadrado o la prueba exacta de Fisher, según la frecuencia relativa de los valores esperados. Resultados. Participaron 447 residentes; el 87,5% proporcionaba rutinariamente consejos breves para dejar de fumar y el 11,6% utilizaba cuestionarios validados para evaluar el grado de adicción. Además, el 32,1% manifestó que prescribía tratamiento farmacológico, pero el 53,1% solo estaba familiarizado con un solo fármaco. Cuando se les preguntó sobre su autopercepción para lograr que sus pacientes dejen de fumar, la mediana de respuesta fue 5 (escala del 1 al 10); solo el 13,7% respondió con un puntaje de 8 o más. Conclusiones. El presente estudio sugiere que los residentes de cardiología en Argentina reconocen la importancia de realizar intervenciones para dejar de fumar, pero una alta proporción de ellos no se sienten capacitados para hacerlo

CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

Entry and exit of proteins into flagella is gauged by CEP290 in the transition zone

The ubiquitin conjugation system is involved in the disassembly of cilia and flagella

The disassembly of cilia and flagella is linked to the cell cycle and environmental cues. We have found that ubiquitination of flagellar proteins is an integral part of flagellar disassembly. Free ubiquitin and the ubiquitin-conjugating enzyme CrUbc13 are detected in flagella, and several proteins are ubiquitinated in isolated flagella when exogenous ubiquitin and adenosine triphosphatase are added, suggesting that the ubiquitin conjugation system operates in flagella. Levels of ubiquitinated flagellar proteins increase during flagellar resorption, especially in intraflagellar transport (IFT) mutants, suggesting that disassembly products are labeled with ubiquitin and transported to the cell body by IFT. Substrates of the ubiquitin conjugation system include α-tubulin (but not β-tubulin), a dynein subunit (IC2), two signaling proteins involved in the mating process, cyclic guanosine monophosphate–dependent kinase, and the cation channel polycystic kidney disease 2. Ubiquitination of flagellar proteins is enhanced early in mating, suggesting that ubiquitination also plays an active role in regulating signaling pathways in flagella

Primary cilia sensitize endothelial cells to BMP and prevent excessive vascular regression

Blood flow shapes vascular networks by orchestrating endothelial cell behavior and function. How endothelial cells read and interpret flow-derived signals is poorly understood. Here, we show that endothelial cells in the developing mouse retina form and use luminal primary cilia to stabilize vessel connections selectively in parts of the remodeling vascular plexus experiencing low and intermediate shear stress. Inducible genetic deletion of the essential cilia component intraflagellar transport protein 88 (IFT88) in endothelial cells caused premature and random vessel regression without affecting proliferation, cell cycle progression, or apoptosis. IFT88 mutant cells lacking primary cilia displayed reduced polarization against blood flow, selectively at low and intermediate flow levels, and have a stronger migratory behavior. Molecularly, we identify that primary cilia endow endothelial cells with strongly enhanced sensitivity to bone morphogenic protein 9 (BMP9), selectively under low flow. We propose that BMP9 signaling cooperates with the primary cilia at low flow to keep immature vessels open before high shear stress-mediated remodeling