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Funder: Maratona da Saúde, Funder: Royal Society Wolfson, Funder: Wellcome; Id: http://dx.doi.org/10.13039/100010269, Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.

96 p.-20 fig.-7 tab., Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion., This research was funded by the European Union's Horizon 2020 (EU) Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No.721906-TRACT (TK, SM, MG, NMcC, GO’S, RT, VK, SG, DZ, GC, SB); Tuscany strategic project POR-FSE 2014-2020, ‘Medicina di Precisione e Malattie Rare’(MePreMaRe), (ACE-ESCC) (NR, GC, SB), and by “Epigenetic Hallmarks of Multiple Sclerosis” (acronym Epi-MS) (id:415, Merit Ranking Area ERC LS) in VALERE 2019 Program; V:ALERE 2020 – Progetto competitivo “CIRCE” in risposta al bando D.R. n. 138 del February 17, 2020 Program; Blueprint 282510; EPICHEMBIO CM1406; AIRC-17217; VALERE: Vanvitelli per la Ricerca; Campania Regional Government Technology Platform Lotta alle Patologie Oncologiche: iCURE; Campania Regional Government FASE2: IDEAL. MIUR, Proof of Concept POC01_00043. POR Campania FSE 2014-2020 ASSE III.

The development of specific and individualized training programs is a possible way to improve athletic performance and minimize injuries in professional athletes. The information regarding the sport's physical demands and the athletes' physical profile have been, so far, considered as exhaustive for the design of effective training programs. However, it is currently emerging that the genetic profile has to be also taken into consideration. By merging medical and genetic data, it is thus possible to identify the athlete's specific attitude to respond to training, diet, and physical stress. In this context, we performed a study in which 30 professional soccer players, subjected to standard sport medical evaluation and practices, were also screened for genetic polymorphism in five key genes (ACTN3, COL5A1, MCT1, VEGF, and HFE). This genetic analysis represents the central point of a multidisciplinary method that can be adopted by elite soccer teams to obtain an improvement in athletic performance and a concomitant reduction of injuries by tailoring training and nutritional programs. The genetic fingerprinting of single athletes led to the identification of two performance-enhancing polymorphisms (ACTN3 18705C>T, VEGF-634C>G) significantly enriched. Moreover, we derived a genetic model based on the gene set analyzed, which was tentatively used to reduce athletes' predisposition to injuries, by dictating a personalized nutrition and training program. The potential usefulness of this approach is concordant with data showing that this team has been classified as the healthiest and least injured team in Europe while covering the highest distance/match with the highest number of high-intensity actions/match.

Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.

Autophagy is widely considered as a housekeeping mechanism that enables cells to survive stress conditions and, in particular, nutrient deprivation. Autophagy begins with the formation of the phagophore that expands and closes around cytosolic material and damaged organelles destined for degradation. The execution of this complex machinery is guaranteed by the coordinated action of more than 40 ATG (autophagy-related) proteins that control the entire process at different stages from the biogenesis of the autophagosome to cargo sequestration and fusion with lysosomes. Autophagosome biogenesis occurs at multiple intracellular sites, such as the endoplasmic reticulum (ER) and the plasma membrane. Soon after the formation of the phagophore, the nascent autophagosome progressively grows in size and ultimately closes by recruiting intracellular membranes. In this review, we focus on the contribution of three membrane sources - the ER, the ER-Golgi intermediate compartment, and the Golgi complex - to autophagosome biogenesis and expansion. We also highlight the interplay between the secretory pathway and autophagy in cells when nutrients are scarce.

Work by R. E. in the laboratory of H. Y. was supported by a European Molecular Biology Organisation (EMBO) short term fellowship (ASTF 534–2014). Work by P. L. and E. F. was supported by CNRS and the Agence Nationale de la Recherche (ANR-13-BSV2–0011-01,TED; ANR-08-BLAN-0067, Chor-Evo-Net). Work by R. E., A. P. and L. S. in the laboratory of A. S. was supported by SZN PhD fellowships. The group of D. E. K. F. is supported by the Leverhulme Trust (RPG-2016-351). Isolation of the Gsx containing cosmid was conducted in the laboratory of Peter Holland and supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (no. G09218). The ascidian neural plate consists of a defined number of identifiable cells organized in a grid of rows and columns, representing a useful model to investigate the molecular mechanisms controlling neural patterning in chordates. Distinct anterior brain lineages are specified via unique combinatorial inputs of signalling pathways with Nodal and Delta-Notch signals patterning along the medial-lateral axis and FGF/MEK/ERK signals patterning along the anterior-posterior axis of the neural plate. The Ciona Gsx gene is specifically expressed in the a9.33 cells in the row III/column 2 position of anterior brain lineages, characterised by a combinatorial input of Nodal-OFF, Notch-ON and FGF-ON. Here, we identify the minimal cis-regulatory element (CRE) of 376 bp, which can recapitulate the early activation of Gsx. We show that this minimal CRE responds in the same way as the endogenous Gsx gene to manipulation of FGF- and Notch-signalling pathways and to overexpression of Snail, a mediator of Nodal signals, and Six3/6, which is required to demarcate the anterior boundary of Gsx expression at the late neurula stage. We reveal that sequences proximal to the transcription start site include a temporal regulatory element required for the precise transcriptional onset of gene expression. We conclude that sufficient spatial and temporal information for Gsx expression is integrated in 376 bp of non-coding cis-regulatory sequences. Postprint

Mutations in OCRL encoding the inositol polyphosphate 5-phosphatase OCRL (Lowe oculocerebrorenal syndrome protein) disrupt phosphoinositide homeostasis along the endolysosomal pathway causing dysfunction of the cells lining the kidney proximal tubule (PT). The dysfunction can be isolated (Dent disease 2) or associated with congenital cataracts, central hypotonia and intellectual disability (Lowe syndrome). The mechanistic understanding of Dent disease 2/Lowe syndrome remains scarce due to limitations of animal models of OCRL deficiency. Here, we investigate the role of OCRL in Dent disease 2/Lowe syndrome by using OcrlY/− mice, where the lethal deletion of the paralogue Inpp5b was rescued by human INPP5B insertion, and primary culture of proximal tubule cells (mPTCs) derived from OcrlY/− kidneys. The OcrlY/− mice show muscular defects with dysfunctional locomotricity and present massive urinary losses of low-molecular-weight proteins and albumin, caused by selective impairment of receptor-mediated endocytosis in PT cells. The latter was due to accumulation of phosphatidylinositol 4,5–bisphosphate PI(4,5)P2 in endolysosomes, driving local hyper-polymerization of F-actin and impairing trafficking of the endocytic LRP2 receptor, as evidenced in OcrlY/− mPTCs. The OCRL deficiency was also associated with a disruption of the lysosomal dynamic and proteolytic activity. Partial convergence of disease-pathways and renal phenotypes observed in OcrlY/− and Clcn5Y/− mice suggest shared mechanisms in Dent diseases 1 and 2. These studies substantiate the first mouse model of Lowe syndrome and give insights into the role of OCRL in cellular trafficking of multiligand receptors. These insights open new avenues for therapeutic interventions in Lowe syndrome and Dent disease., Human Molecular Genetics, 28 (12), ISSN:0964-6906, ISSN:1460-2083

Phosphoinositides (PIs) play pivotal roles in the regulation of many biological processes. The quality and quantity of PIs is regulated in time and space by the activity of PI-kinases and PI-phosphatases. The number of PI metabolizing enzymes exceeds the number of PIs with, in many cases, more than one enzyme controlling the same biochemical step. This would suggest that the PI system has an intrinsic ability to buffer and compensate for the absence of a specific enzymatic activity. However, there are several examples of severe inherited human diseases caused by mutations in one of the PI-enzymes, although other enzymes with the same activity are fully functional. The kidney depends strictly on PIs for physiological processes such as cell polarization, filtration, solute reabsorption and extracellular signal transduction. Indeed, alteration of the PI system in the kidney very often results in pathological conditions, both inherited and acquired. Most of the knowledge of the roles that PIs play in the kidney comes from the study of knock-out animal models for genes encoding PI-enzymes and from the study of human genetic diseases such as Lowe syndrome/Dent disease 2 and Joubert syndrome caused by mutations in the genes encoding the PI-phosphatases OCRL and INPP5E, respectively.

Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER.

Lowe syndrome is an X-linked disease that is characterized by congenital cataracts, central hypotonia, intellectual disability and renal Fanconi syndrome. The disease is caused by mutations in OCRL, which encodes an inositol polyphosphate 5-phosphatase (OCRL) that acts on phosphoinositides - quantitatively minor constituents of cell membranes that are nonetheless pivotal regulators of intracellular trafficking. In this Review we summarize the considerable progress made over the past decade in understanding the cellular roles of OCRL in regulating phosphoinositide balance along the endolysosomal pathway, a fundamental system for the reabsorption of proteins and solutes by proximal tubular cells. We discuss how studies of OCRL have led to important discoveries about the basic mechanisms of membrane trafficking and describe the key features and limitations of the currently available animal models of Lowe syndrome. Mutations in OCRL can also give rise to a milder pathology, Dent disease 2, which is characterized by renal Fanconi syndrome in the absence of extrarenal pathologies. Understanding how mutations in OCRL give rise to two clinical entities with differing extrarenal manifestations represents an opportunity to identify molecular pathways that could be targeted to develop treatments for these conditions.

More than twenty different genetic diseases have been described that are caused by mutations in phosphoinositide metabolizing enzymes, mostly in phosphoinositide phosphatases. Although generally ubiquitously expressed, mutations in these enzymes, which are mainly loss-of-function, result in tissue-restricted clinical manifestations through mechanisms that are not completely understood. Here we analyze selected disorders of phosphoinositide metabolism grouped according to the principle tissue affected: the nervous system, muscle, kidney, the osteoskeletal system, the eye, and the immune system. We will highlight what has been learnt so far from the study of these disorders about not only the cellular and molecular pathways that are involved or are governed by phosphoinositides, but also the many gaps that remain to be filled to gain a full understanding of the pathophysiological mechanisms underlying the clinical manifestations of this steadily growing class of diseases, most of which still remain orphan in terms of treatment. This article is part of a Special Issue entitled Phosphoinositides.

Historically, mutations have had a significant impact on the study of developmental processes and phenotypic evolution. Lesions in DNA are created by artificial methods or detected by natural genetic variation. Random mutations are then ascribed to genetic change by direct sequencing or positional cloning. Tunicate species of the ascidian genus Ciona represent nearly fully realized model systems in which gene function can be investigated in depth. Additionally, tunicates are valuable organisms for the study of naturally occurring mutations due to the capability to exploit genetic variation down to the molecular level. Here, we summarize the available information about how mutations are studied in ascidians with examples of insights that have resulted from these applications. We also describe notions and methodologies that might be useful for the implementation of easy and tight procedures for mutations studies in Ciona.

Sea lampreys (Petromyzon marinus) are members of the ancient class Agnatha and undergo a metamorphosis that transforms blind, sedentary, filter-feeding larvae into free-swimming, parasitic juveniles. Thyroid hormones (THs) appear to be important for lamprey metamorphosis, however, serum TH concentrations are elevated in the larval phase, decline rapidly during early metamorphosis and remain low until metamorphosis is complete; these TH fluctuations are contrary to those of other metamorphosing vertebrates. Moreover, thyroid hormone synthesis inhibitors (goitrogens) induce precocious metamorphosis and exogenous TH treatments disrupt natural metamorphosis in P. marinus. Given that THs exert their effects by binding to TH nuclear receptors (TRs) that often act as heterodimers with retinoid X receptors (RXRs), we cloned and characterized these receptors from P. marinus and examined their expression during metamorphosis. Two TRs (PmTR1 and PmTR2) and three RXRs (PmRXRs) were isolated from P. marinus cDNA. Phylogenetic analyses group the PmTRs together on a branch prior to the gnathostome TRα/β split. The three RXRs also group together, but our data indicated that these transcripts are most likely either allelic variants of the same gene locus, or the products of a lamprey-specific duplication event. Importantly, these P. marinus receptors more closely resemble vertebrate as opposed to invertebrate chordate receptors. Functional analysis revealed that PmTR1 and PmTR2 can activate transcription of TH-responsive genes when treated with nanomolar concentrations of TH and they have distinct pharmacological profiles reminiscent of vertebrate TRβ and TRα, respectively. Also similar to other metamorphosing vertebrates, expression patterns of the PmTRs during lamprey metamorphosis suggest that PmTR1 has a dynamic, tissue-specific expression pattern that correlates with tissue morphogenesis and biochemical changes and PmTR2 has a more uniform expression pattern. This TR expression data suggests that THs, either directly or via a metabolite, may function to positively modulate changes at the tissue or organ levels during lamprey metamorphosis. Collectively the results presented herein support the hypothesis that THs have a dual functional role in the lamprey life cycle whereby high levels promote larval feeding, growth and lipogenesis and low levels promote metamorphosis.