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Refereed/Peer-reviewed Extracellular vesicles (EVs) are important players in cell to cell communication in reproductive systems. Notably, EVs have been found and characterized in the male reproductive tract, however, direct functional evidence for their importance in mediating sperm function is lacking. We have previously demonstrated that Arrdc4, a member of the α-arrestin protein family, is involved in extracellular vesicle biogenesis and release. Here we show that Arrdc4-mediated extracellular vesicle biogenesis is required for proper sperm function. Sperm from Arrdc4–/– mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as observed by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and two-cell embryo production. We found a significant reduction in extracellular vesicle production by Arrdc4–/- epididymal epithelial cells, and further, supplementation of Arrdc4–/– sperm with additional vesicles dampened the acrosome reaction defect and restored zona pellucida binding. These results indicate that Arrdc4 is important for proper sperm maturation through the control of extracellular vesicle biogenesis. © 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.

Within the reproductive tract, distinct cell types must have precisely controlled communication for complex processes such as gamete production, fertilisation and implantation. Intercellular communication in many physiological processes involves extracellular vesicles (EVs). In reproductive systems, EVs have been implicated in many aspects, from gamete maturation to embryo development. Sperm develop within the testis and then exit into the epididymis in an immature form, lacking motility and fertilising capabilities. Due to their small size, compact nature of the nucleus and the lack of specific organelles, sperm are unable to perform de novo protein synthesis, and thus rely on extrinsic signals delivered from the external milieu to gain full function. Mounting evidence points to EVs as being a major provider of these signals, not just within the male reproductive tract but also within the female as the sperm make their way through a seemingly hostile environment to the oocyte. In this chapter, we review the current knowledge on EVs as mediators of sperm maturation and function and highlight their potential roles in male fertility.

Extracellular vesicles (EVs) are lipid-bilayered vesicles that are released by multiple cell types and contain nucleic acids and proteins. Very little is known about how the cargo is packaged into EVs. Ubiquitination of proteins is a key posttranslational modification that regulates protein stability and trafficking to subcellular compartments including EVs. Recently, arrestin-domain containing protein 1 (Arrdc1), an adaptor for the Nedd4 family of ubiquitin ligases, has been implicated in the release of ectosomes, a subtype of EV that buds from the plasma membrane. However, it is currently unknown whether Arrdc1 can regulate the release of exosomes, a class of EVs that are derived endocytically. Furthermore, it is unclear whether Arrdc1 can regulate the sorting of protein cargo into the EVs. Exosomes and ectosomes are isolated from mouse embryonic fibroblasts isolated from wild type and Arrdc1-deficient (Arrdc1-/- ) mice. Nanoparticle tracking analysis-based EV quantitation shows that Arrdc1 regulates the release of both exosomes and ectosomes. Proteomic analysis highlights the change in protein cargo in EVs upon deletion of Arrdc1. Functional enrichment analysis reveals the enrichment of mitochondrial proteins in ectosomes, while proteins implicated in apoptotic cleavage of cell adhesion proteins and formation of cornified envelope are significantly depleted in exosomes upon knockout of Arrdc1.

Newly synthesized transmembrane proteins undergo a series of steps to ensure that only the required amount of correctly folded protein is localized to the membrane. The regulation of protein quality and its abundance at the membrane are often controlled by ubiquitination, a multistep enzymatic process that results in the attachment of ubiquitin, or chains of ubiquitin to the target protein. Protein ubiquitination acts as a signal for sorting, trafficking, and the removal of membrane proteins via endocytosis, a process through which multiple ubiquitin ligases are known to specifically regulate the functions of a number of ion channels, transporters, and signaling receptors. Endocytic removal of these proteins through ubiquitin-dependent endocytosis provides a way to rapidly downregulate the physiological outcomes, and defects in such controls are directly linked to human pathologies. Recent evidence suggests that ubiquitination is also involved in the shedding of membranes and associated proteins as extracellular vesicles, thereby not only controlling the cell surface levels of some membrane proteins, but also their potential transport to neighboring cells. In this review, we summarize the mechanisms and functions of ubiquitination of membrane proteins and provide specific examples of ubiquitin-dependent regulation of membrane proteins. Refereed/Peer-reviewed

The regulation of divalent metal ion transporter DMT1, the primary non-heme iron importer in mammals, is critical for maintaining iron homeostasis. Previously we identified ubiquitin-dependent regulation of DMT1 involving the Nedd4 family of ubiquitin ligases and the Ndfip1 and Ndfip2 adaptors. We also established the in vivo function of Ndfip1 in the regulation of DMT1 in the duodenum of mice. Here we have studied the function of Ndfip2 using Ndfip2-deficient mice. The DMT1 protein levels in the duodenum were comparable in wild type and Ndfip2−/− mice, as was the transport activity of isolated enterocytes. A complete blood examination showed no significant differences between wild type and Ndfip2−/− mice in any of the hematological parameters measured. However, when fed a low iron diet, female Ndfip2−/− mice showed a decrease in liver iron content, although they maintained normal serum iron levels and transferrin saturation, compared to wild type female mice that showed a reduction in serum iron and transferrin saturation. Ndfip2−/− female mice also showed an increase in DMT1 expression in the liver, with no change in male mice. We suggest that Ndfip2 controls DMT1 in the liver with female mice showing a greater response to altered dietary iron than the male mice.

The release of extracellular vesicles (EVs) is important for both normal physiology and disease. However, a basic understanding of the targeting of EV cargoes, composition and mechanism of release is lacking. Here we present evidence that the divalent metal ion transporter (DMT1) is unexpectedly regulated through release in EVs. This process involves the Nedd4-2 ubiquitin ligase, and the adaptor proteins Arrdc1 and Arrdc4 via different budding mechanisms. We show that mouse gut explants release endogenous DMT1 in EVs. Although we observed no change in the relative amount of DMT1 released in EVs from gut explants in Arrdc1 or Arrdc4 deficient mice, the extent of EVs released was significantly reduced indicating an adaptor role in biogenesis. Furthermore, using Arrdc1 or Arrdc4 knockout mouse embryonic fibroblasts, we show that both Arrdc1 and Arrdc4 are non-redundant positive regulators of EV release. Our results suggest that DMT1 release from the plasma membrane into EVs may represent a novel mechanism for the maintenance of iron homeostasis, which may also be important for the regulation of other membrane proteins.

Ubiquitination is essential in mediating diverse cellular functions including protein degradation and trafficking. Ubiquitin-protein (E3) ligases determine the substrate specificity of the ubiquitination process. The Nedd4 family of E3 ligases is an evolutionarily conserved family of proteins required for the ubiquitination of a large number of cellular targets. As a result, this family regulates a wide variety of cellular processes including transcription, stability and trafficking of plasma membrane proteins, and the degradation of misfolded proteins. The modular architecture of the proteins, comprising a C2 domain, multiple WW domains and a catalytic domain, enables diverse intermolecular interactions and recruitment to various subcellular locations. The WW domains commonly mediate interaction with substrate proteins; however, an increasing number of Nedd4 targets do not contain obvious WW domain-interaction motifs suggesting the involvement of accessory proteins. This review discusses recent insights into how accessory and adaptor proteins modulate the activities of Nedd4 family members, including recruitment of novel substrates, alteration of subcellular localisation and effects on ubiquitination.

In the Drosophila wing, the Nedd4 ubiquitin ligases (E3s), dNedd4 and Su(dx), are important negative regulators of Notch signaling; they ubiquitinate Notch, promoting its endocytosis and turnover. Here, we show that Drosophila Nedd4 family interacting protein (dNdfip) interacts with the Drosophila Nedd4-like E3s. dNdfip expression dramatically enhances dNedd4 and Su(dx)-mediated wing phenotypes and further disrupts Notch signaling. dNdfip colocalizes with Notch in wing imaginal discs and with the late endosomal marker Rab7 in cultured cells. In addition, dNdfip expression in the wing leads to ectopic Notch signaling. Supporting this, expression of dNdfip suppressed Notchþ/ wing phenotype and knockdown of dNdfip enhanced the Notchþ/ wing phenotype. The increase in Notch activity by dNdfip is ligand independent as dNdfip expression also suppressed deltex RNAi and Serrateþ/ wing phenotypes. The opposing effects of dNdfip expression on Notch signaling and its late endosomal localization support a model whereby dNdfip promotes localization of Notch to the limiting membrane of late endosomes allowing for activation, similar to the model previously shown with ectopic Deltex expression. When dNedd4 or Su(dx) are also present, dNdfip promotes their activity in Notch ubiquitination and internalization to the lysosomal lumen for degradation. Refereed/Peer-reviewed

The divalent metal ion transporter DMT1 is critical for nonheme iron import. We have previously shown that DMT1 is regulated in vitro by ubiquitination that is facilitated by the adaptor proteins Ndfip1 and Ndfip2. Here we report that in Ndfip1−/− mice fed a low- iron diet, DMT1 expression and activity in duodenal enterocytes are significant higher than in the wild-type animals. This correlates with an increase in serum iron levels and transferrin saturation. Liver and spleen iron stores were also increased in Ndfip1−/− mice fed a normal diet. Counterintuitive to the increase in iron uptake, Ndfip1−/− mice fed a low iron diet develop severe microcytic, hypochromic anemia. We demonstrate that this is due to a combination of iron deficiency and inflammatory disease in Ndfip1−/− mice, because Ndfip1−/−/Rag1−/− immunodeficient mice fed a low iron diet did not develop anemia and showed an iron overload phenotype. These data demonstrate that Ndfip1 is a critical mediator of DMT1 regulation in vivo, particularly under iron restricted conditions.

Recent findings are reported about certain aspects of the structure and function of the mammalian and avian lungs that include(a) the architecture of the air capillaries (ACs) and the blood capillaries (BCs); (b) the pulmonary blood capillary circulatory dynamics; (c) the adaptive molecular, cellular, biochemical,compositional, and developmental characteristics of the surfactant system; (d) the mechanisms of the translocation of fine and ultra fine particles across the airway epithelial barrier; and (e) the particle-cell interactions in the pulmonary airways. In the lung of the Muscovy duck Cairina moschata, at least, the ACs are rotund structures that are interconnected by narrow cylindrical sections, while the BCs comprise segments that are almost as long as they are wide. In contrast to the mammalian pulmonary BCs, which are highly compliant, those of birds practically behave like rigid tubes. Diving pressure has been a very powerful directional selection force that has influenced phenotypic changes in surfactant composition and function in lungs of marine mammals. After nanosized particulates are deposited on the respiratory tract of healthy human subjects, some reach organs such as the brain with potentially serious health implications. Finally, in the mammalian lung, dendritic cells of the pulmonary airways are powerful agents in engulfing deposited particles, and in birds, macrophages and erythrocytes are ardent phagocytizing cellular agents. The morphology of the lung that allows it to perform different functions - including gas exchange, ventilation of the lung by being compliant, defense, and secretion of important pharmacological factors - is reflected in its "compromise design." Refereed/Peer-reviewed

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50-90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress.

Many ion channels and transporters are regulated by ubiquitination mediated by the Nedd4 family of HECT-type ubiquitin ligases (E3s). These E3s commonly interact with substrates via their WW domains that bind to specific motifs in target proteins. However, not all potential targets of these E3s contain WW-binding motifs. Therefore, accessory proteins may mediate the interaction between Nedd4 family members and their targets. Here we report that the divalent metal ion transporter DMT1, the primary nonheme iron transporter in mammals, is regulated by ubiquitination mediated by the Nedd4 family member WWP2. DMT1 interacts with 2 WW domain-interacting proteins, Ndfip1 and Ndfip2, previously proposed to have roles in protein trafficking. This promotes DMT1 ubiquitination and degradation by WWP2. Consistent with these observations, Ndfip1−/− mice show increased DMT1 activity and a concomitant increase in hepatic iron deposition, indicating an essential function of Ndfip1 in iron homeostasis. This novel mechanism of regulating iron homeostasis suggests that Ndfips and WWP2 may contribute to diseases involving aberrant iron transport.