PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

Adducins Regulate Remodeling of Apical Junctions in Human Epithelial Cells

This article identifies membrane skeleton proteins, adducins, as important regulators of epithelial cell–cell adhesions that promote assembly and antagonize stimulus-induced disassembly of adherens and tight junctions

Association of the PHACTR1/EDN1 genetic locus with spontaneous coronary artery dissection

Background: Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of acute coronary syndromes (ACS) afflicting predominantly younger to middle-aged women. Observational studies have reported a high prevalence of extracoronary vascular anomalies, especially fibromuscular dysplasia (FMD) and a low prevalence of coincidental cases of atherosclerosis. PHACTR1/EDN1 is a genetic risk locus for several vascular diseases, including FMD and coronary artery disease, with the putative causal noncoding variant at the rs9349379 locus acting as a potential enhancer for the endothelin-1 (EDN1) gene. Objectives: This study sought to test the association between the rs9349379 genotype and SCAD. Methods: Results from case control studies from France, United Kingdom, United States, and Australia were analyzed to test the association with SCAD risk, including age at first event, pregnancy-associated SCAD (P-SCAD), and recurrent SCAD. Results: The previously reported risk allele for FMD (rs9349379-A) was associated with a higher risk of SCAD in all studies. In a meta-analysis of 1,055 SCAD patients and 7,190 controls, the odds ratio (OR) was 1.67 (95% confidence interval [CI]: 1.50 to 1.86) per copy of rs9349379-A. In a subset of 491 SCAD patients, the OR estimate was found to be higher for the association with SCAD in patients without FMD (OR: 1.89; 95% CI: 1.53 to 2.33) than in SCAD cases with FMD (OR: 1.60; 95% CI: 1.28 to 1.99). There was no effect of genotype on age at first event, P-SCAD, or recurrence. Conclusions: The first genetic risk factor for SCAD was identified in the largest study conducted to date for this condition. This genetic link may contribute to the clinical overlap between SCAD and FMD

Doppler sonography and liver focal tumors

But : évaluer l'intérêt de l'écho-Doppler dans l'étude des tumeurs du foie chez l'adulte. L'écho-Doppler a été volontiers sous-estimée. ces dernières années, au profit d'autres techniques d'imagerie. Matériel et méthodes : les techniques ultrasonores en couleur font appel au Doppler fréquentiel, au Doppler énergie, voire à l'utilisation des produits de contraste spécifiques. L'opérateur a recherché un flux circulant en contraste spontané afin de mesurer des vitesses ou des index. Nous avons étudié de manière prospective 72 tumeurs focales et avons confronté nos résultats à ceux de la littérature. Résultats et discussion : ils sont exprimés sans chereber à mesurer sensibilité ou spécificité. On identifie un certain nombre de signes évocateurs de telle ou telle autre affection, en utilisant des faisceaux d'arguments, car aucun des signes pris isolément ne semble pathognomoniq ue. Conclusion : il existe des signes évocateurs de pathologies diverses telles le carcinome hépato-cellulaire, la métastase, l'angiome, l'hyerplasie nodulaire focale ou l'adénome. Ce n'est que lorsque l'examen n'est pas contributif qu'il convient de faire appel aux autres techniques d'examen. De même lorsque le traitement en découle, on ne peut se passer de la vérification histologique. A l'heure où un effort en économie de santé est demandé à tous, le corps médical devrait s'interroger sur la nécessité de faire plus appel à l'échographie, dans de telles affections

The scaffolding domain of caveolin 2 is responsible for its Golgi localization in Caco-2 cells

In this work, we showed that in Caco-2 cells, a polarized cell line derived from human colon cancer that does not express caveolin 1 (Cav-1), there was no detectable expression of caveolin 2 (Cav-2). When Cav-2 was reintroduced in these cells, it accumulated in the Golgi complex. A chimera, in which the scaffolding domain of Cav-1 was replaced by the one from Cav-2, induced a prominent Golgi staining of Cav-1, strongly indicating that this domain was responsible for the accumulation of Cav-2 in the Golgi complex. Cav-2 was able to interact with Cav-1 in the Golgi complex but this interaction was not sufficient to export it from this compartment. Several chimeras between Cav-1 and 2 were used to show that surface expression of caveolin was necessary but not sufficient to promote caveolae formation. Interestingly, levels of incorporation of the chimeras into Triton insoluble rafts correlated with their ability to trigger caveolae formation raising the possibility that a critical concentration of caveolins to discrete domains of the plasma membrane might be necessary for caveolae formation

Role of aPKC isoforms and their binding partners Par3 and Par6 in epidermal barrier formation

The skin water barrier, essential for terrestrial life, is formed by a multilayered stratifying epithelium, which shows a polarized distribution of both differentiation and intercellular junction markers. Recently, several reports showed the crucial importance of tight junctions for the in vivo water barrier function of the skin. In simple epithelial cells, intercellular junction formation is closely coupled to the establishment of polarity. However, if and how polarity proteins contribute to epidermal differentiation and junction formation is not yet known. Here, we have characterized the localization and isoform expression of the polarity protein atypical PKC (aPKC) and its binding partners Par3 and Par6 in epidermis and primary keratinocytes of mice. Their distribution is only partially overlapping in the granular layer, the site of functional tight junctions, suggesting that next to a common Par3/Par6/aPKC function they also may have functions independent of each other. Both aPKCζ and aPKCι/λ, are expressed in the epidermis but only aPKCι/λ showed a strong enrichment in the junctions, suggesting that this aPKC isoform is important for epidermal tight junction function. Indeed, inhibition of aPKC function showed that endogenous aPKC is crucial for in vitro barrier function and this required the presence of both the Par3 and Par6 binding sites