Ankyrin repeat and zinc-finger domain-containing 1 mutations are associated with infantile-onset inflammatory bowel disease.

Infantile-onset inflammatory bowel disease (IO IBD) is an invalidating illness with an onset before 2 years of age and has a complex pathophysiology in which genetic factors are important. Homozygosity mapping and whole-exome sequencing in an IO IBD patient and subsequent sequencing of the candidate gene in 12 additional IO IBD patients revealed two patients with two mutated ankyrin repeat and zinc-finger domain-containing 1 (ANKZF1) alleles (homozygous ANKZF1 R585Q mutation and compound heterozygous ANKZF1 E152K and V32_Q87del mutations, respectively) and two patients with one mutated ANKZF1 allele. Although the function of ANKZF1 in mammals had not been previously evaluated, we show that ANKZF1 has an indispensable role in the mitochondrial response to cellular stress. ANKZF1 is located diffusely in the cytoplasm and translocates to the mitochondria upon cellular stress. ANKZF1 depletion reduces mitochondrial integrity and mitochondrial respiration under conditions of cellular stress. The ANKZF1 mutations identified in IO IBD patients with two mutated ANKZF1 alleles result in dysfunctional ANKZF1, as shown by an increased level of apoptosis in patients' lymphocytes, a decrease in mitochondrial respiration in patient fibroblasts with a homozygous ANKZF1 R585Q mutation, and an inability of ANKZF1 R585Q and E152K to rescue the phenotype of yeast deficient in Vms1, the yeast homologue of ANKZF1. These data indicate that loss-of-function mutations in ANKZF1 result in deregulation of mitochondrial integrity, and this may play a pathogenic role in the development of IO IBD.

Understanding chronic neutropenia: life is short.

The pathophysiological mechanisms underlying chronic neutropenia are extensive, varying from haematopoietic stem cell disorders resulting in defective neutrophil production, to accelerated apoptosis of neutrophil progenitors or circulating mature neutrophils. While the knowledge concerning genetic defects associated with congenital neutropenia or bone marrow failure is increasing rapidly, the functional role and consequences of these genetic alterations is often not well understood. In addition, there is a large group of diseases, including primary immunodeficiencies and metabolic diseases, in which chronic neutropenia is one of the symptoms, while there is no clear bone marrow pathology or haematopoietic stem cell dysfunction. Altogether, these disease entities illustrate the complexity of normal neutrophil development, the functional role of the (bone marrow) microenvironment and the increased propensity to undergo apoptosis, which is typical for neutrophils. The large variety of disorders associated with chronic neutropenia makes classification almost impossible and possibly not desirable, based on the clinical phenotypes. However, a better understanding of the regulation of normal myeloid differentiation and neutrophil development is of great importance in the diagnostic evaluation of unexplained chronic neutropenia. In this review we propose insights in the pathophysiology of chronic neutropenia in the context of the functional role of key players during normal neutrophil development, neutrophil release and neutrophil survival.

Atg18 function in autophagy is regulated by specific sites within its ß-propeller.

Autophagy is a conserved degradative transport pathway. It is characterized by the formation of double-membrane autophagosomes at the phagophore assembly site (PAS). Atg18 is essential for autophagy but also for vacuole homeostasis and probably endosomal functions. This protein is basically a ß-propeller, formed by seven WD40 repeats, that contains a conserved FRRG motif that binds to phosphoinositides and promotes Atg18 recruitment to the PAS, endosomes and vacuoles. However, it is unknown how Atg18 association with these organelles is regulated, as the phosphoinositides bound by this protein are present on the surface of all of them. We have investigated Atg18 recruitment to the PAS and found that Atg18 binds to Atg2 through a specific stretch of amino acids in the ß-propeller on the opposite surface to the FRRG motif. As in the absence of the FRRG sequence, the inability of Atg18 to interact with Atg2 impairs its association with the PAS, causing an autophagy block. Our data provide a model whereby the Atg18 ß-propeller provides organelle specificity by binding to two determinants on the target membrane.

Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores.

The autophagy-related (Atg) proteins play a key role in the formation of autophagosomes, the hallmark of autophagy. The function of the cluster composed by Atg2, Atg18, and transmembrane Atg9 is completely unknown despite their importance in autophagy. In this study, we provide insights into the molecular role of these proteins by identifying and characterizing Atg2 point mutants impaired in Atg9 binding. We show that Atg2 associates to autophagosomal membranes through lipid binding and independently from Atg9. Its interaction with Atg9, however, is key for Atg2 confinement to the growing phagophore extremities and subsequent association of Atg18. Assembly of the Atg9-Atg2-Atg18 complex is important to establish phagophore-endoplasmic reticulum (ER) contact sites. In turn, disruption of the Atg2-Atg9 interaction leads to an aberrant topological distribution of both Atg2 and ER contact sites on forming phagophores, which severely impairs autophagy. Altogether, our data shed light in the interrelationship between Atg9, Atg2, and Atg18 and highlight the possible functional relevance of the phagophore-ER contact sites in phagophore expansion.

Phosphatidylinositol-3-phosphate clearance plays a key role in autophagosome completion.

BACKGROUND: The biogenesis of autophagosomes, the hallmark of autophagy, depends on the function of the autophagy-related (Atg) proteins and the generation of phosphatidylinositol-3-phosphate (PtdIns3P) at the phagophore assembly site (PAS), the location where autophagosomes arise. The current model is that PtdIns3P is involved primarily in the recruitment of Atg proteins to the PAS and that once an autophagosome is complete, the Atg machinery is released from its surface back into the cytoplasm and reused for the formation of new vesicles. RESULTS: We have identified a PtdIns3P phosphatase, Ymr1, that is essential for the normal progression of both bulk and selective types of autophagy. This protein is recruited to the PAS at an early stage of formation of this structure through a process that requires both its GRAM domain and its catalytic activity. In the absence of Ymr1, Atg proteins fail to dissociate from the limiting membrane of autophagosomes, and these vesicles accumulate in the cytoplasm. CONCLUSIONS: Our data thus reveal a key role for PtdIns3P turnover in the regulation of the late steps of autophagosome biogenesis and indicate that the disassembly of the Atg machinery from the surface of autophagosomes is a requisite for their fusion with the vacuole.

An ENU-mutagenesis screen in the mouse: identification of novel developmental gene functions.

BACKGROUND: Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development. METHODOLOGY/PRINCIPAL FINDINGS: Mice were mutagenized with N-ethyl-N-nitrosourea (ENU) and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila]) and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib. CONCLUSIONS/SIGNIFICANCE: Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.

An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.

Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.

Multiple roles of the cytoskeleton in autophagy.

Autophagy is involved in a wide range of physiological processes including cellular remodeling during development, immuno-protection against heterologous invaders and elimination of aberrant or obsolete cellular structures. This conserved degradation pathway also plays a key role in maintaining intracellular nutritional homeostasis and during starvation, for example, it is involved in the recycling of unnecessary cellular components to compensate for the limitation of nutrients. Autophagy is characterized by specific membrane rearrangements that culminate with the formation of large cytosolic double-membrane vesicles called autophagosomes. Autophagosomes sequester cytoplasmic material that is destined for degradation. Once completed, these vesicles dock and fuse with endosomes and/or lysosomes to deliver their contents into the hydrolytically active lumen of the latter organelle where, together with their cargoes, they are broken down into their basic components. Specific structures destined for degradation via autophagy are in many cases selectively targeted and sequestered into autophagosomes. A number of factors required for autophagy have been identified, but numerous questions about the molecular mechanism of this pathway remain unanswered. For instance, it is unclear how membranes are recruited and assembled into autophagosomes. In addition, once completed, these vesicles are transported to cellular locations where endosomes and lysosomes are concentrated. The mechanism employed for this directed movement is not well understood. The cellular cytoskeleton is a large, highly dynamic cellular scaffold that has a crucial role in multiple processes, several of which involve membrane rearrangements and vesicle-mediated events. Relatively little is known about the roles of the cytoskeleton network in autophagy. Nevertheless, some recent studies have revealed the importance of cytoskeletal elements such as actin microfilaments and microtubules in specific aspects of autophagy. In this review, we will highlight the results of this work and discuss their implications, providing possible working models. In particular, we will first describe the findings obtained with the yeast Saccharomyces cerevisiae, for long the leading organism for the study of autophagy, and, successively, those attained in mammalian cells, to emphasize possible differences between eukaryotic organisms.