Notes and tips for improving quality of lipid-protein overlay assays

To reduce costs of lipid-binding assays, allow for multiple lipids to be screened for protein binding simultaneously, and to make lipid binding more user friendly, lipids have been dotted onto membranes to investigate lipid-protein interactions. These assays are similar to a western blot where the membrane is blocked, incubated with a protein of interest and detected using antibodies. Although the assay is inexpensive and straightforward, problems with promiscuous or poor binding, as well as insufficient blocking occur frequently. In this technical note, we share several specific improvements to ensure lipid-protein overlay assays are of high quality and contain proper controls

The Mechanism of Membrane Targeting of Human Sphingosine Kinase 1

Sphingosine 1-phosphate is a bioactive sphingolipid that regu- lates cell growth and suppresses programmed cell death. The bio- synthesis of sphingosine 1-phosphate is catalyzed by sphingosine kinase (SK) but the mechanism by which the subcellular localization and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural determi- nant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK1 (hSK1) and selected mutants. In vitro measurements showed that hSK1 selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr54 and Asn89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr54 and Asn89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK1 is mediated largely by specific lipid-protein interactions

Cysteine mutations in the ebolavirus matrix protein VP40 promote phosphatidylserine binding by increasing the flexibility of a lipid-binding loop

Ebolavirus (EBOV) is a negative-sense RNA virus that causes severe hemorrhagic fever in humans. The matrix protein VP40 facilitates viral budding by binding to lipids in the host cell plasma membrane and driving the formation of filamentous, pleomorphic virus particles. The C-terminal domain of VP40 contains two highly-conserved cysteine residues at positions 311 and 314, but their role in the viral life cycle is unknown. We therefore investigated the properties of VP40 mutants in which the conserved cysteine residues were replaced with alanine. The C311A mutation significantly increased the affinity of VP40 for membranes containing phosphatidylserine (PS), resulting in the assembly of longer virus-like particles (VLPs) compared to wild-type VP40. The C314A mutation also increased the affinity of VP40 for membranes containing PS, albeit to a lesser degree than C311A. The double mutant behaved in a similar manner to the individual mutants. Computer modeling revealed that both cysteine residues restrain a loop segment containing lysine residues that interact with the plasma membrane, but Cys311 has the dominant role. Accordingly, the C311A mutation increases the flexibility of this membrane-binding loop, changes the profile of hydrogen bonding within VP40 and therefore binds to PS with greater affinity. This is the first evidence that mutations in VP40 can increase its affinity for biological membranes and modify the length of Ebola VLPs. The Cys311 and Cys314 residues therefore play an important role in dynamic interactions at the plasma membrane by modulating the ability of VP40 to bind PS

A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria

Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination

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

Determination of EGFR Endocytosis Kinetic by Auto-Regulatory Association of PLD1 with mu 2

Background: Upon ligand binding, cell surface signaling receptors are internalized through a process tightly regulated by endocytic proteins and adaptor protein 2 (AP2) to orchestrate them. Although the molecular identities and roles of endocytic proteins are becoming clearer, it is still unclear what determines the receptor endocytosis kinetics which is mainly regulated by the accumulation of endocytic apparatus to the activated receptors. Methodology/Principal Findings: Here we employed the kinetic analysis of endocytosis and adaptor recruitment to show that ??2, a subunit of AP2 interacts directly with phospholipase D (PLD)1, a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). We also demonstrate that the PLD1-??2 interaction requires the binding of PLD1 with phosphatidic acid, its own product. Conclusions/Significance: These results suggest that the temporal regulation of EGFR endocytosis is achieved by auto-regulatory PLD1 which senses the receptor activation and triggers the translocation of AP2 near to the activated receptor.open3

A Common Ca2+-Driven Interdomain Module Governs Eukaryotic NCX Regulation

Na+/Ca2+ exchanger (NCX) proteins mediate Ca2+-fluxes across the cell membrane to maintain Ca2+ homeostasis in many cell types. Eukaryotic NCX contains Ca2+-binding regulatory domains, CBD1 and CBD2. Ca2+ binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca2+. To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca2+ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca2+-driven interdomain switch controls slow dissociation of “occluded” Ca2+ from the primary sensor and thus dictates Ca2+ sensing dynamics. In the Ca2+-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca2+-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants

A comparison of alternative assays to measure DNA damage in stallion spermatozoa: TUNEL test versus ‘Nicoletti assay’

The aberrations of sperm DNA may cause various problems and have negative consequences on fertility. These influence embryonic development or might lead to early embryo loss. Sperm Chromatin Structure Assay (SCSA) is the flow cytometric method most often used for the detection of DNA lesions; however, some studies using that method reached confusing conclusions. The aim of this pilot study was to adjust and compare two alternative tests, namely the TUNEL test and the Nicoletti assay. The above-mentioned two flow cytometric methods capable of detecting the fragmented DNA of sperm were tested on 12 frozen-thawed stallion semen samples. The TUNEL test demonstrated much higher DNA fragmentation ratio than the Nicoletti assay (mean ± SD: 30.77 ± 13.03% vs. 1.93 ± 0.89%, respectively). A fluorescent microscopic check of the samples showed that TUNEL labelled the plasma membrane and the mitochondria in a nonspecific way, rather than detecting only the fragmented DNA, thus eventually resulting in a false positive sign. The Nicoletti assay is simpler, quicker and does not detect nonspecific binding; however, further analyses are required to determine its diagnostic value

Soft tissue damage after minimally invasive THA: A comparison of 5 approaches

Methods 5 surgeons each performed a total hip arthroplasty on 5 fresh frozen cadaver hips, using either a MIS anterior, MIS anterolateral, MIS 2-incision, MIS posterior, or lateral transgluteal approach. Postoperatively, the hips were dissected and muscle damage color-stained. We measured proportional muscle damage relative to the midsubstance cross-sectional surface area (MCSA) using computerized color detection. The integrity of external rotator muscles, nerves, and ligaments was assessed by direct observation. Results None of the other MIS approaches resulted in less gluteus medius muscle damage than the lateral transgluteal approach. However, the MIS anterior approach completely preserved the gluteus medius muscle in 4 cases while partial damage occurred in 1 case. Furthermore, the superior gluteal nerve was transected in 4 cases after a MIS anterolateral approach and in 1 after the lateral transgluteal approach. The lateral femoral cutaneous nerve was transected once after both the MIS anterior approach and the MIS 2-incision approach. Interpretation The MIS anterior approach may preserve the gluteus medius muscle during total hip arthroplasty, but with a risk of damaging the lateral femoral cutaneous nerv

Differential Virulence Gene Expression of Group A Streptococcus Serotype M3 in Response to Co-Culture with Moraxella catarrhalis

Streptococcus pyogenes (group A Streptococcus, GAS) and Moraxella catarrhalis are important colonizers and (opportunistic) pathogens of the human respiratory tract. However, current knowledge regarding colonization and pathogenic potential of these two pathogens is based on work involving single bacterial species, even though the interplay between respiratory bacterial species is increasingly important in niche occupation and the development of disease. Therefore, to further define and understand polymicrobial species interactions, we investigated whether gene expression (and hence virulence potential) of GAS would be affected upon co-culture with M. catarrhalis. For co-culture experiments, GAS and M. catarrhalis were cultured in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) at 37°C with 5% CO2aeration. Each strain was grown in triplicate so that triplicate experiments could be performed. Bacterial RNA was isolated, cDNA synthesized, and microarray transcriptome expression analysis performed. We observed significantly increased (≥4-fold) expression for genes playing a role in GAS virulence such as hyaluronan synthase (hasA), streptococcal mitogenic exotoxin Z (smeZ) and IgG endopeptidase (ideS). In contrast, significantly decreased (≥4-fold) expression was observed in genes involved in energy metabolism and in 12 conserved GAS two-component regulatory systems. This study provides the first evidence that M. catarrhalis increases GAS virulence gene expression during co-culture, and again shows the importance of polymicrobial infections in directing bacterial virulence