Autophagy is a quality-control mechanism that helps to maintain cellular homeostasis by removing damaged proteins and organelles through lysosomal degradation. During autophagy, signaling events lead to the formation of a cup-shaped structure called the phagophore that matures into the autophagosome. Recruitment of the autophagy-associated Atg12-5-16L1 complex to Wipi2-positive phagophores is crucial for producing microtubule-associated protein 1 light chain 3-II (LC3-II), which is required for autophagosome formation. Here, we explored the role of the autophagy receptor optineurin (Optn) in autophagosome formation. Fibroblasts from Optn knock-out mouse showed reduced LC3-II formation and a lower number of autophagosomes and autolysosomes during both basal and starvation-induced autophagy. However, the number of Wipi2-positive phagophores was not decreased in Optn-deficient cells. We also found that the number of Atg12/16L1-positive puncta and recruitment of the Atg12-5-16L1 complex to Wipi2-positive puncta are reduced in Optn-deficient cells. Of note, Optn was recruited to Atg12-5-16L1-positive puncta, and interacted with Atg5 and also with Atg12-5 conjugate. A disease-associated Optn mutant, E478G, defective in ubiquitin binding, was also defective in autophagosome formation and recruitment to the Atg12-5-16L1-positive puncta. Moreover, we noted that Optn phosphorylation at Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores.

WD-repeat proteins are very diverse, yet these are structurally related proteins that participate in a wide range of cellular functions. WDR13, a member of this family, is conserved from fishes to humans and localizes into the nucleus. To understand the in vivo function(s) of Wdr13 gene, we have created and characterized a mutant mouse strain lacking this gene. The mutant mice had higher serum insulin levels and increased pancreatic islet mass as a result of enhanced beta cell proliferation. While a known cell cycle inhibitor, p21, was downregulated in the mutant islets, over expression of WDR13 in the pancreatic beta cell line (MIN6) resulted in upregulation of p21, accompanied by retardation of cell proliferation. We suggest that WDR13 is a novel negative regulator of the pancreatic beta cell proliferation. Given the higher insulin levels and better glucose clearance in Wdr13 gene deficient mice, we propose that this protein may be a potential candidate drug target for ameliorating impaired glucose metabolism in diabetes.

Acquisition of milk production capabilities by an ancestor of mammals is at the root of mammalian evolution. Milk casein micelles are a primary source of amino acids and calcium phosphate to neonates. To understand the role of -casein in lactation, we have created and characterized a null mouse strain (Csnk ؊/؊ ) lacking this gene. The mutant -casein allele did not affect the expression of other milk proteins in Csnk ؊/؊ females. However, these females did not suckle their pups and failed to lactate because of destabilization of the micelles in the lumina of the mammary gland. Thus, -casein is essential for lactation and, consequently, for the successful completion of the process of reproduction in mammals. In view of the extreme structural conservation of the casein locus, as well as the phenotype of Csnk ؊/؊ females, we propose that the organization of a functional -casein gene would have been one of the critical events in the evolution of mammals. Further, -casein variants are known to affect the industrial properties of milk in dairy animals. Given the expenses and the time scale of such experiments in livestock species, it is desirable to model the intended genetic modifications in mice first. The mouse strain that we have created would be a useful model to study the effect of -casein variants on the properties of milk and͞or milk products.casein locus ͉ evolution ͉ lactation ͉ milk ͉ micelle M ilk production is an essential component of the reproductive strategy of mammals. The development of a zygote into an organism is accompanied by the preparation of milk synthesis potential by the mammary gland. Perinatal survival in mammals is solely dependent on the availability of milk from the mother. Caseins account for Ͼ80% of the total milk proteins (1). These proteins exist as micelles, consisting of three to four phosphoproteins (i.e., the calcium-sensitive caseins: ␣-, ␤-, ␥-, and ␦-casein in mice) and a calcium-insensitive phosphoglycoprotein, -casein (2). In addition to functioning as the primary source of amino acids, one of the key functions of micelles is to sequester large amounts of calcium phosphate from the maternal diet or bodily stores and make it readily available to the newborn. Calcium-sensitive casein genes seem to have evolved from a primitive casein gene through gene duplication and exon shuffling (3); however, it appears that -casein is not related structurally and evolutionarily to these genes. Instead, it appears to be related to ␥-fibrinogen (4, 5). Recently, it has also been speculated that both the calcium-sensitive and the calciuminsensitive casein gene belong to one gene family (6). The null mutation of the ␤-casein gene in mice (7) and that of ␣ S1 -casein in goats (8) do not show any significant effect on milk micelle formation and lactation. The lack of adverse effects on micelle formation in these mutants suggests functional redundancy amongst calcium-sensitive caseins. However, the absence of ␣ S1 -casein in goats does affect the rate of transport of caseins. The experimental ev...

RNA interference (RNAi) pathways regulate self-renewal and differentiation of embryonic stem (ES) cells. Argonaute 2 (Ago2) is a vital component of RNA-induced silencing complex (RISC) and the only Ago protein with slicer activity. We generated Ago2-deficient ES cells by conditional gene targeting. Ago2-deficient ES cells are defective in the small-RNA-mediated gene silencing and are significantly compromised in biogenesis of mature microRNA. The self-renewal rate of Ago2-deficient ES cells is affected due to failure of silencing of Cdkn1a by EScell- specific microRNAs (miRNA) in the absence of Ago2. Interestingly, unlike Dicer- and Dgcr8-deficient ES cells, they differentiate to all three germ layers both in vivo and in vitro. However, early differentiation of Ago2-deficient ES cells is delayed by 2-4 days as indicated by persistence of higher levels of self-renewal/ pluripotency markers during differentiation. Further, appearance of morphological and differentiation markers is also delayed during the differentiation. In this study we show that Ago2 is essential for normal self-renewal and differentiation. Also, our data suggest that self-renewal and differentiation of ES cells are regulated by both siRNA and miRNA pathways.