PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

A Scheme for Solving the Plane–Plane Challenge in Force Measurements at the Nanoscale

Non-contact interaction between two parallel flat surfaces is a central paradigm in sciences. This situation is the starting point for a wealth of different models: the capacitor description in electrostatics, hydrodynamic flow, thermal exchange, the Casimir force, direct contact study, third body confinement such as liquids or films of soft condensed matter. The control of parallelism is so demanding that no versatile single force machine in this geometry has been proposed so far. Using a combination of nanopositioning based on inertial motors, of microcrystal shaping with a focused-ion beam (FIB) and of accurate in situ and real-time control of surface parallelism with X-ray diffraction, we propose here a “gedanken” surface-force machine that should enable one to measure interactions between movable surfaces separated by gaps in the micrometer and nanometer ranges

Peer influence in network markets: a theoretical and empirical analysis

Network externalities spur the growth of networks and the adoption of network goods in two ways. First, they make it more attractive to join a network the larger its installed base. Second, they create incentives for network members to actively recruit new members. Despite indications that the latter "peer effect" can be more important for network growth than the installed-base effect, it has so far been largely ignored in the literature. We address this gap using game-theoretical models. When all early adopters can band together to exert peer influence-an assumption that fits, e.g., the case of firms supporting a technical standard-we find that the peer effect induces additional growth of the network by a factor. When, in contrast, individuals exert peer influence in small groups of size n, the increase in network size is by an additive constant-which, for small networks, can amount to a large relative increase. The difference between small, local, personal networks and large, global, anonymous networks arises endogenously from our analysis. Fundamentally, the first type of networks is "tie-reinforcing," the other, "tie-creating". We use survey data from users of the Internet services, Skype and eBay, to illustrate the main logic of our theoretical results. As predicted by the model, we find that the peer effect matters strongly for the network of Skype users-which effectively consists of numerous small sub-networks-but not for that of eBay users. Since many network goods give rise to small, local networks

Temperature Tolerance and Stress Proteins as Mechanisms of Invasive Species Success

Invasive species are predicted to be more successful than natives as temperatures increase with climate change. However, few studies have examined the physiological mechanisms that theoretically underlie this differential success. Because correlative evidence suggests that invasiveness is related to the width of a species' latitudinal range, it has been assumed – but largely untested – that range width predicts breadth of habitat temperatures and physiological thermotolerances. In this study, we use empirical data from a marine community as a case study to address the hypotheses that (1) geographic temperature range attributes are related to temperature tolerance, leading to greater eurythermality in invasive species, and (2) stress protein expression is a subcellular mechanism that could contribute to differences in thermotolerance. We examined three native and six invasive species common in the subtidal epibenthic communities of California, USA. We assessed thermotolerance by exposing individuals to temperatures between 14°C and 31°C and determining the temperature lethal to 50% of individuals (LT50) after a 24 hour exposure. We found a strong positive relationship between the LT50 and both maximum habitat temperatures and the breadth of temperatures experience across the species' ranges. In addition, of the species in our study, invasives tended to inhabit broader habitat temperature ranges and higher maximum temperatures. Stress protein expression may contribute to these differences: the more thermotolerant, invasive species Diplosoma listerianum expressed higher levels of a 70-kDa heat-shock protein than the less thermotolerant, native Distaplia occidentalis for which levels declined sharply above the LT50. Our data highlight differences between native and invasive species with respect to organismal and cellular temperature tolerances. Future studies should address, across a broader phylogenetic and ecosystem scope, whether this physiological mechanism has facilitated the current success of invasive species and could lead to greater success of invasives than native species as global warming continues

VAMP7 modulates ciliary biogenesis in kidney cells

Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis. © 2014 Szalinski et al

MicroRNA-sequence profiling reveals novel osmoregulatory microRNA expression patterns in catadromous eel anguilla marmorata

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate gene expression by post-transcriptional repression of mRNAs. Recently, several miRNAs have been confirmed to execute directly or indirectly osmoregulatory functions in fish via translational control. In order to clarify whether miRNAs play relevant roles in the osmoregulation of Anguilla marmorata, three sRNA libraries of A. marmorata during adjusting to three various salinities were sequenced by Illumina sRNA deep sequencing methods. Totally 11,339,168, 11,958,406 and 12,568,964 clear reads were obtained from 3 different libraries, respectively. Meanwhile, 34 conserved miRNAs and 613 novel miRNAs were identified using the sequence data. MiR-10b-5p, miR-181a, miR-26a-5p, miR-30d and miR-99a-5p were dominantly expressed in eels at three salinities. Totally 29 mature miRNAs were significantly up-regulated, while 72 mature miRNAs were significantly down-regulated in brackish water (10‰ salinity) compared with fresh water (0‰ salinity); 24 mature miRNAs were significantly up-regulated, while 54 mature miRNAs were significantly down-regulated in sea water (25‰ salinity) compared with fresh water. Similarly, 24 mature miRNAs were significantly up-regulated, while 45 mature miRNAs were significantly down-regulated in sea water compared with brackish water. The expression patterns of 12 dominantly expressed miRNAs were analyzed at different time points when the eels transferred from fresh water to brackish water or to sea water. These miRNAs showed differential expression patterns in eels at distinct salinities. Interestingly, miR-122, miR-140-3p and miR-10b-5p demonstrated osmoregulatory effects in certain salinities. In addition, the identification and characterization of differentially expressed miRNAs at different salinities can clarify the osmoregulatory roles of miRNAs, which will shed lights for future studies on osmoregulation in fish

Abnormal Changes in NKT Cells, the IGF-1 Axis, and Liver Pathology in an Animal Model of ALS

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing fatal neurodegenerative disorder characterized by the selective death of motor neurons (MN) in the spinal cord, and is associated with local neuroinflammation. Circulating CD4+ T cells are required for controlling the local detrimental inflammation in neurodegenerative diseases, and for supporting neuronal survival, including that of MN. T-cell deficiency increases neuronal loss, while boosting T cell levels reduces it. Here, we show that in the mutant superoxide dismutase 1 G93A (mSOD1) mouse model of ALS, the levels of natural killer T (NKT) cells increased dramatically, and T-cell distribution was altered both in lymphoid organs and in the spinal cord relative to wild-type mice. The most significant elevation of NKT cells was observed in the liver, concomitant with organ atrophy. Hepatic expression levels of insulin-like growth factor (IGF)-1 decreased, while the expression of IGF binding protein (IGFBP)-1 was augmented by more than 20-fold in mSOD1 mice relative to wild-type animals. Moreover, hepatic lymphocytes of pre-symptomatic mSOD1 mice were found to secrete significantly higher levels of cytokines when stimulated with an NKT ligand, ex-vivo. Immunomodulation of NKT cells using an analogue of α-galactosyl ceramide (α-GalCer), in a specific regimen, diminished the number of these cells in the periphery, and induced recruitment of T cells into the affected spinal cord, leading to a modest but significant prolongation of life span of mSOD1 mice. These results identify NKT cells as potential players in ALS, and the liver as an additional site of major pathology in this disease, thereby emphasizing that ALS is not only a non-cell autonomous, but a non-tissue autonomous disease, as well. Moreover, the results suggest potential new therapeutic targets such as the liver for immunomodulatory intervention for modifying the disease, in addition to MN-based neuroprotection and systemic treatments aimed at reducing oxidative stress

Male oxidative stress infertility (MOSI): proposed terminology and clinical practice guidelines for management of idiopathic male infertility

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm's potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause

Male Oxidative Stress Infertility (MOSI):proposed terminology and clinical practice guidelines for management of idiopathic male infertility

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm's potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause