Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy.

Autophagosome formation is stimulated by VPS34-dependent PI(3)P formation and by alternative VPS34-independent pathways. We recently described that PI(5)P regulates autophagosome biogenesis and rescues autophagy in VPS34-inactivated cells, suggesting that PI(5)P contributes to canonical autophagy. Our analysis revealed a hitherto unknown functional interplay between PIKfyve and PIPK type II in controlling PI(5)P levels in the context of autophagy. Among phosphoinositides, visualization of PI(5)P in intact cells has remained difficult. While PI(5)P has been implicated in signaling pathways, chromatin organization, bacterial invasion, and cytoskeletal remodeling, our study is the first report showing PI(5)P localization on autophagosomes and early autophagosomal structures when autophagy is induced by nutrient deprivation (amino acids or glucose starvation). We provided a detailed analysis of PI(5)P distribution by the use of super-resolution structured illuminated microscopy. Here, we present a set of tools for detection of PI(5)P during autophagy by confocal microscopy, live-cell imaging, and super-resolution microscopy.

Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator.

Mismanaged protein trafficking by the proteostasis network contributes to several conformational diseases, including cystic fibrosis, the most frequent lethal inherited disease in Caucasians. Proteostasis regulators, as cystamine, enable the beneficial action of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators in ΔF508-CFTR airways beyond drug washout. Here we tested the hypothesis that functional CFTR protein can sustain its own plasma membrane (PM) stability. Depletion or inhibition of wild-type CFTR present in bronchial epithelial cells reduced the availability of the small GTPase Rab5 by causing Rab5 sequestration within the detergent-insoluble protein fraction together with its accumulation in aggresomes. CFTR depletion decreased the recruitment of the Rab5 effector early endosome antigen 1 to endosomes, thus reducing the local generation of phosphatidylinositol-3-phosphate. This diverts recycling of surface proteins, including transferrin receptor and CFTR itself. Inhibiting CFTR function also resulted in its ubiquitination and interaction with SQSTM1/p62 at the PM, favoring its disposal. Addition of cystamine prevented the recycling defect of CFTR by enhancing BECN1 expression and reducing SQSTM1 accumulation. Our results unravel an unexpected link between CFTR protein and function, the latter regulating the levels of CFTR surface expression in a positive feed-forward loop, and highlight CFTR as a pivot of proteostasis in bronchial epithelial cells.

Phosphoinositides as regulators of membrane trafficking in health and disease.

Membrane trafficking is crucial in the homeostasis of the highly compartmentalized eukaryotic cells. This compartmentalization occurs both at the organelle level, with distinct organelles maintaining their identities while also intensely interchanging components, and at a sub-organelle level, with adjacent subdomains of the same organelle containing different sets of lipids and proteins. A central question in the field is thus how this compartmentalization is established and maintained despite the intense exchange of components and even physical continuities within the same organelle. The phosphorylated derivatives of phosphatidylinositol, known as the phosphoinositides, have emerged as key components in this context, both as regulators of membrane trafficking and as finely tuned spatial and temporal landmarks for organelle and sub-organelle domains. The central role of the phosphoinositides in cell homeostasis is highlighted by the severe consequences of the derangement of their metabolism caused by genetic deficiencies of the enzymes involved, and from the systematic hijacking of phosphoinositide metabolism that pathogens operate to promote their entry and/or survival in host cells.

Delta-CHr improves the identification of anemic syndromes and the evaluation of hemoglobin synthesis.

Reticulocyte hemoglobin content (CHr) is considered an index of iron status, helpful in the differential diagnosis of microcytoses. Its potential can be enhanced by comparing CHr dynamic reference values (CHr-e: expected CHr), which are proportional to the MCVr variations occurring in micro- or macrocytosis, with measured CHr values. We demonstrate that the difference between measured CHr and CHr-e (DeltaCHr) is helpful to differentiate the anemic syndromes and, in particular, beta-thalassemia vs. presumable sideropenia. DeltaCHr can also indicate when to interrupt iron supplementation. DeltaCHr allows an insight into the erythropoiesis of thalassemic and sideropenic subjects, pointing out the reduced hemoglobin production and ineffective erythroid activity in these conditions.