FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4246529395> ?p ?o ?g. }

• W4246529395 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique ‘cationic grip’ configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. https://doi.org/10.7554/eLife.01808.001 eLife digest It is often said that attack is the best form of defense; and the immune systems of plants and animals will often target the cell membranes of microbes and other pathogens in order to defend themselves. Disrupting the cell membrane causes essential contents to leak from the cell, and eventually, the cell will burst and die. Most plants and animals produce small proteins called defensins that kill microbes by attacking their cell membranes. These defensins are thought to either destabilize the cell membrane by coating its outer surface or to insert themselves into the membrane to form open pores that allow vital biomolecules to leak out of the cell. However, the exact mechanism by which defensins attack microbial membranes is not understood. In this study, Poon, Baxter, Lay et al. show that a defensin called NaD1—which was isolated from the ornamental tobacco Nicotiana alata—binds to a molecule from the cell membrane called phosphatidylinositol 4,5-bisphosphate, or PIP2 for short. By working out the three-dimensional structure of this complex, Poon, Baxter, Lay et al. show that it contains 14 PIP2 molecules and 14 NaD1 molecules in an arch-shaped structure and suggest that sequestering large numbers of PIP2 molecules in this way destabilizes the cell membrane of the microbe. These findings raise a number of questions: are there other small proteins that can destabilize cell membranes in a similar manner to defensins? Do the immune systems of other organisms also recognize molecules from microbial cell membranes to trigger this kind of counterattack? Furthermore, since defensins can also kill tumor cells, a better understanding of how they work might also lead to new treatments for cancer and other diseases in humans. https://doi.org/10.7554/eLife.01808.002 Introduction Host defense peptides, which include cationic antimicrobial peptides (CAPs), are a group of innate immune molecules produced by essentially all plant and animal species that act as a first line of defense against microbial invasion. In common with most innate immunity peptides, they are relatively small (typically <100 amino acid residues), are predominantly cationic, and typically harbor a substantial number of hydrophobic amino acids (Hancock and Lehrer, 1998; Brogden, 2005; Lai and Gallo, 2009). Although originally identified due to their potent activity against microbial pathogens, several CAPs also exhibit cytolytic activity against a range of mammalian tumor cells (Lichtenstein et al., 1986; Cruciani et al., 1991; Hancock and Sahl, 2006; Schweizer, 2009). The defensins are a family of CAPs that are ubiquitously expressed in plants, animals, insects, and fungi that play an important role in innate immune defense against microbial threats (Brogden, 2005; Lay and Anderson, 2005; Hancock and Sahl, 2006; Lai and Gallo, 2009). The plant defensins belong to a large family of molecules that are highly variable in sequence but have a conserved structure. The sequence variability leads to several biological functions including antimicrobial activity, regulation of plant development, and pollen tube guidance (Carvalho and Gomes, 2009; De Coninck et al., 2013). Even those plant defensins that have been ascribed antifungal activity have large differences in sequence and are likely to act by different mechanisms (van der Weerden and Anderson, 2013). The plant defensins are small (∼5 kDa, 45–54 amino acids), basic, cysteine-rich proteins that display a family-defining disulfide bond array (in a CI–CVIII, CII–CV, CIII–CVI, and CIV–CVII configuration) known as the cysteine-stabilized αβ (CSαβ) motif. This motif consists of a triple-stranded antiparallel β-sheet, which is cross-braced via three disulfide bonds at the core of the molecule to an α-helix (in a βαββ arrangement). The fourth conserved disulfide bond further rigidifies the protein by linking together the N- and C-terminal regions of the molecule, effectively generating a highly stable pseudocyclic molecule (Janssen et al., 2003; Lay et al., 2003b, 2012; Lay and Anderson, 2005). This CSαβ fold is also conserved in defensins found in other organisms, including insects and fungi (Lay and Anderson, 2005). NaD1, a plant defensin isolated from the flowers of the ornamental tobacco (Nicotiana alata), exhibits potent antifungal activity against pathogenic fungi, including Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cryptococcus species, as well as the yeasts Saccharomyces cerevisiae and Candida albicans (Lay et al., 2003a, 2003b, 2012; van der Weerden et al., 2008, 2010; Hayes et al., 2013). NaD1 inhibits fungal growth in a three-stage process that involves specific interaction with the cell wall and entry into the cytoplasm before cell death (van der Weerden et al., 2008, 2010). Interaction with NaD1 also leads to hyper-production of reactive oxygen species, inducing oxidative damage that contributes to its fungicidal activity on Candida albicans (Hayes et al., 2013). Many CAPs have been postulated to act at the level of the plasma membrane of target cells. Suggested mechanisms of action for membrane permeabilization are based on the (1) carpet, (2) barrel-stave, and (3) toroidal-pore models (reviewed in Brogden, 2005). In the carpet model, the CAPs act like classic detergents, accumulating and forming a carpet layer on the membrane outer surface, leading to local disintegration (including membrane micellization or fragmentation) upon reaching a critical concentration. Other CAPs are suggested to aggregate on the membrane surface before inserting into the bilayer forming a ‘barrel-stave’ pore where the hydrophobic peptide regions align with the lipid core and the hydrophilic peptide regions form the interior of the pore. Alternatively, in the toroidal pore model, the CAPs induce the lipid monolayers to bend continuously through the pore, with the polar peptide faces associating with the polar lipid head groups (Brogden, 2005). Although these models have been useful for describing potential mechanisms underlying the antimicrobial activity of various CAPs, it is not clear how well they represent the actual configuration of CAPs at the membrane. Furthermore, the oligomeric state of CAPs required for their activity based on the postulated models remains unknown. Indeed, it has long been hypothesized that the molecules could form proteinaceous pores and function through insertion into membranes (Brogden, 2005). However, to date, the structural basis of CAP activity at the target membrane has not been defined. In addition to the uncertainty about the configuration of CAPs at the membrane, the role of ligands in modulating the recognition of target surfaces by CAPs remains unclear. One class of ligands that has been linked to plant defensin antifungal activity are sphingolipids (Wilmes et al., 2011), a key component of fungal cell walls and membranes. Plant defensins that bind sphingolipids include RsAFP2 from radish (binds glucosylceramide, GlcCer) (Thomma et al., 2003; Thevissen et al., 2004), DmAMP1 from dahlia (binds mannose-(inositol-phosphate)2-ceramide, M(IP)2C) (Thevissen et al., 2000, 2003), as well as the pea defensin Psd1 (Goncalves et al., 2012) and sugarcane defensin Sd5 (de Paula et al., 2011) that both bind membranes enriched for specific glycosphingolipids. MsDef1, a defensin from Medicago sativa, has also been implicated in binding sphingolipids, as a mutant of the fungus Fusarium graminearum that is depleted in glucosylceramide, is highly resistant to MsDef1 (Ramamoorthy et al., 2007). In this report, we have identified the cellular phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) as a key ligand that is recognized during membrane permeabilization of fungal and mammalian plasma membranes. Using X-ray crystallography, we have defined the molecular interaction of NaD1 with PIP2 and demonstrate that NaD1 forms oligomeric complexes with PIP2. Structure-guided mutagenesis revealed a critical arginine residue (R40) that is pivotal for NaD1:PIP2 oligomer formation and that oligomerization is required for plasma membrane permeabilization. Engagement of PIP2 is mediated by NaD1 dimers that form a distinctive PIP2-binding ‘cationic grip’ that interacts with the head groups of two PIP2 molecules. Functional assays using NaD1 mutants reveal that the mechanism of membrane permeabilization by NaD1 is likely to be conserved between fungal and mammalian tumor cells. Together, these data lead to a new perspective on the role of ligand binding and oligomer formation of defensins during membrane permeabilization. Results NaD1 binds phospholipids including phosphatidylinositol 4,5-bisphosphate (PIP2) To define the molecular basis of NaD1 target cell membrane permeabilization activity, we set out to identify potential ligands for NaD1. Membrane lipids represent an attractive target for NaD1; therefore, we investigated whether NaD1 interacts with cellular lipids using protein–lipid overlay assays based on lipid strips immobilized with 100 pmoles of various biologically active lipids (Poon et al., 2010; Patel et al., 2013). NaD1 specifically bound to certain phospholipids, including several phosphatidylinositol mono-/bis-/tri-phosphates, phosphatidylserine, phosphatidic acid, cardiolipin, and sulfatide (Figure 1A). Interestingly, NaD1 bound the functionally important plasma membrane phospholipid PIP2 (Figure 1A) but did not bind to a panel of other membrane lipids or sphingolipids. To confirm that the ability of NaD1 to engage PIP2 was not a result of immobilization on the lipid strip, we confirmed that NaD1 also bound PIP2 in the context of a membrane bilayer using a liposome pull-down assay (Figure 1B). Figure 1 with 1 supplement see all Download asset Open asset Interaction of NaD1 with lipids. (A) Detection of NaD1 binding to cellular lipids by protein-lipid overlay assay. Blots are representative of at least two independent experiments for each strip. (B) Binding of NaD1 to PIP2-containing liposomes. NaD1 in A and B was detected using a rabbit anti-NaD1 antibody. https://doi.org/10.7554/eLife.01808.003 PIP2 binding to NaD1 leads to the formation of an arch-shaped oligomer To gain insight into the NaD1:PIP2 interaction at the atomic level, we determined the crystal structure of NaD1 in complex with PIP2. The structure of monomeric NaD1 (Lay et al., 2012) was used to solve the structure of a NaD1:PIP2 complex by molecular replacement and refined to a resolution of 1.6 Å with values of Rwork/Rfree of 0.155/0.184 (Table 1). Upon PIP2 binding, NaD1 forms an arch composed of 14 NaD1 molecules (Figure 2A), with a final arch diameter of 90 Å and a width of 35 Å. The asymmetric unit contains all 14 NaD1 molecules that form the final arch, with the symmetry of the arch being entirely non-crystallographic. Fourteen PIP2 molecules are bound in an extended binding groove (Figure 2B) on the inside of the arch (Figure 2A). The entire oligomeric complex is held together by a complex network of interactions, which include numerous NaD1:NaD1 (Figure 3A,B) and NaD1:PIP2 interactions (Figure 3C,D). Notably, the arch-shaped oligomer displays a small degree of pitch, which although noticeable is not sufficient to allow the formation of an extended coil in the crystal (Figure 2B). Table 1 Data collection and refinement statistics https://doi.org/10.7554/eLife.01808.005 NaD1:PIP2 nativeData collection Space groupC2221 Cell dimensions a, b, c (Å)79.64, 132.04, 153.01 α, β, γ (°)90.00, 90.00, 90.00 Wavelength (Å)0.9537 Resolution (Å)*40.84–1.6 (1.69–1.60) Rsym or Rmerge*0.092 (0.617) I/σI*11.6 (2.2) Completeness (%)*99.7 (94.7) Redundancy*6.7 (5.4)Refinement Resolution (Å)40.37–1.6 No. reflections105745 Rwork/Rfree0.155/0.184 No. atoms Protein10326 Ligand/ion845 Water816 B-factors Protein21.5 Ligand/ion28.9 Water31.2 R.m.s. deviations Bond lengths (Å)0.010 Bond angles (°)1.651 * Values in parentheses are for highest resolution shell. Figure 2 Download asset Open asset Crystal structure of the NaD1:PIP2 complex. (A) Two orthogonal views of a cartoon representation of the NaD1:PIP2 oligomer comprising 14 NaD1 monomers (shown as ribbons) and 14 PIP2 molecules (shown as green sticks). The surface of the NaD1 oligomer is shown in gray. (B) Surface representation of the NaD1 14-mer, displaying the extended binding groove on the inside of the arch. Coloring is by atom type (N in blue, O in red, S in yellow, and C in gray). For clarity the 14 bound PIP2 molecules were omitted. https://doi.org/10.7554/eLife.01808.006 Figure 3 with 2 supplements see all Download asset Open asset Detailed view of the crystal structure of the NaD1:PIP2 complex. In all panels, hydrogen bonds and salt bridges are shown as black dotted lines. (A) View of the interface of two NaD1 monomers revealing the hydrogen bonding pattern, with monomer I shown in cyan and monomer II in magenta. Secondary structure elements are labeled in black. For clarity bound PIP2 molecules are omitted. (B) Cartoon diagram of four molecules of NaD1 forming a dimer of dimers. (C) PIP2 binding site on monomer I. Cartoon diagram of the PIP2 binding site in monomer I on dimeric NaD1. (D) PIP2 binding site on monomer II. Cartoon diagram of the PIP2 binding site on monomer II on dimeric NaD1. https://doi.org/10.7554/eLife.01808.007 The NaD1:PIP2 oligomer contains two distinct NaD1:NaD1 interfaces The observed NaD1:PIP2 oligomer can be described as an assembly of seven NaD1 dimers, which comprise two distinct NaD1:NaD1 interfaces. The first interface is formed by an antiparallel alignment of the β1-strand from each of two NaD1 molecules (monomers I and II) and exhibits two-fold symmetry between the associated monomers (Figure 3A). It comprises an average buried surface area of 430 Å2 and is formed by a network of six hydrogen bonds involving R1, K4, E6, E27, K45, and C47. This dimeric arrangement leads to the formation of a ‘cationic grip’ (Figure 4A,B), which is able to accommodate two PIP2 head groups simultaneously (Figure 3—figure supplement 1). A second interface is formed by the dimeric NaD1 (comprising monomers I and II) and adjacent NaD1 monomers III and IV (Figure 3B). This interface is formed by hydrogen bonds involving N8 of monomer I, R1, E2, K17; D31 of monomer II, R1, K17; D31 of monomer III; and N8 of monomer IV, effectively forming a dimer of dimers (Figure 3—figure supplement 1). The interactions between two dimers are repeated seven times to allow formation of the observed 14-mer. The full 14-mer is thus constructed using two different interfaces. Figure 4 Download asset Open asset The dimeric NaD1 ‘cationic grip‘ with two bound PIP2 molecules. (A) Surface view in two orientations of a NaD1 dimer (monomer I in cyan and monomer II in magenta) with two bound PIP2 molecules (yellow and green). (B) The same as in A except that the surface shows a qualitative electrostatic representation (blue is positive, red in negative, and white is uncharged or hydrophobic). Figure generated using Pymol. https://doi.org/10.7554/eLife.01808.010 PIP2 is bound in an extended binding groove In addition to NaD1:NaD1 interactions, oligomer formation requires the presence of PIP2. NaD1 binds PIP2 primarily via a ‘cationic grip’ that is created by a NaD1 dimer, which results in the formation of a distinct binding site (Figure 3—figure supplement 2) formed by K4 together with residues 33–40, which comprise a characteristic ‘KILRR’ motif (Figure 3C,D). PIP2 forms a dense network of hydrogen bonds involving K4, H33, K36, I37, L38, and R40 of a single NaD1 monomer. In oligomeric NaD1:PIP2, a single PIP2 binding site also contains interactions with neighboring NaD1 monomers (Figure 3C,D; Figure 3—figure supplement 1). Bound PIP2 forms additional hydrogen bonds with R40 from monomer II and K36 from monomer IV′, with the full PIP2 binding site in the oligomer comprising contributions from three different NaD1 molecules (Figure 3C,D). Consequently, oligomer formation appears to be highly cooperative, with multiple interactions between adjacent NaD1 and PIP2 molecules required to form the observed 14-mer (Figure 3). NaD1:PIP2 oligomers form readily in solution To confirm that oligomer formation is not a crystallization artifact, we treated mixtures of NaD1 and PIP2 in aqueous solution with the crosslinker BS3, which resulted in covalent cross-linking of multiple NaD1 molecules that occurred only in the presence of PIP2 (Figure 5A), whereas NaD1 on its own only formed a dimer as reported previously (Lay et al., 2012). Figure 5 Download asset Open asset NaD1 forms oligomers with PIP2. (A) Ability of NaD1 to form multimers in the presence of PIP2 as determined by protein–protein cross-linking with BS3 followed by SDS-PAGE and Coomassie Brilliant Blue staining. (B) TEM of NaD1:PIP2 complexes. TEM micrographs of NaD1 alone, PIP2 alone, or NaD1 in complex with PIP2. Data in A and B are representative of at least two independent experiments. https://doi.org/10.7554/eLife.01808.011 NaD1:PIP2 forms fibrils We next imaged NaD1:PIP2 oligomers using transmission electron microscopy (TEM). Complexes of NaD1:PIP2 (1:1.2 molar ratio) were applied to a carbon-coated copper grid and imaged. Strikingly, long string-like fibrillar structures were observed when both NaD1 and PIP2 were present, whereas they were absent on grids bearing either NaD1 or PIP2 in isolation (Figure 5B). Although the NaD1:PIP2 oligomer we observed by crystallography displays a subtle pitch, it is not sufficient to allow continuous addition or concatenation of 14-mers to form the fibrils observed by TEM, with the ends of two 14-mers running into each other. However, given that the crystal structure of the oligomer reveal an outer diameter of 90 Å, with a corresponding diameter of the fibrils under TEM of 10 nM, additional twisting of the 14-mer could allow for the formation of continuous coils with a diameter to match the fibrils observed under TEM. PIP2 binding and oligomerization of NaD1 are critical for fungal cell killing Based on our oligomeric NaD1:PIP2 structure, we performed site-directed mutagenesis on NaD1 to confirm the role of proposed key amino acid residues in PIP2 binding, oligomerization, and fungal cell killing. Examination of the PIP2 binding pockets in the NaD1:PIP2 oligomer suggests that R40, which contacts two adjacent PIP2 molecules simultaneously and interacts with the phosphate moiety at position 4, is critical for cooperative binding of PIP2 and therefore formation of the NaD1:PIP2 oligomer (Figure 6A). Mutation of R40 should not lead to loss of PIP2 binding, since PIP2 would still form five hydrogen bonds and ionic interactions with NaD1 and should only impact oligomerization. In contrast, I37 contributes to PIP2 binding but not oligomerization. We generated recombinant proteins of NaD1 (rNaD1) and NaD1 mutants (rNaD1(R40E) and rNaD1(I37F)) and confirmed their correct folding by CD spectroscopy (data not shown) and evaluated the ability of the mutant NaD1 to bind phospholipids, undergo PIP2-induced oligomerization, and kill the filamentous fungus F. oxysporum f. sp. vasinfectum. Figure 6 with 1 supplement see all Download asset Open asset Multimerization of the NaD1:PIP2 complex. (A) Schematic representation of residues from neighboring NaD1 monomers involved in binding two PIP2 molecules. Ability of rNaD1, rNaD1(R40E), and rNaD1(I37F) to (B) bind cellular lipids by protein-lipid overlay assay, (C) form multimers in the presence of PIP2 as determined by protein–protein cross-linking with BS3 followed by SDS-PAGE and Coomassie Brilliant Blue staining, and (D) to inhibit fungal cell growth. Error bars in D indicate SEM (n = 3). Data in B–D are representative of at least two independent experiments. https://doi.org/10.7554/eLife.01808.012 As predicted, mutation of R40 to glutamic acid led to a largely unchanged binding to PIP2, with the remaining five hydrogen bonds and ionic interactions formed between PIP2 and rNaD1(R40E) compensating for the loss of two ionic interactions as well as the charge repulsion. However, it did result in reduced binding to PI(4)P (Figure 6B) and oligomerization (Figure 6C) that correlated with substantially reduced fungal cell killing (Figure 6D). It is important to note that although PIP2 binding was maintained, the loss of interaction with the 4-phosphate moiety of PIP2 results in loss of cooperative binding and therefore ablation of oligomerization, which is critically dependent on R40 forming ‘bridging’ interactions between two neighboring PIP2 molecules. In contrast, mutation of I37 to phenylalanine had little effect on PIP2 binding specificity, oligomerization, and fungal cell killing (Figure 6B–D). These data support our defined NaD1-PIP2 structure and demonstrate that the coordinated oligomerization of NaD1 by interaction with PIP2 is an important event during fungal cell killing. NaD1 permeabilizes the plasma membrane of mammalian tumor cells Since PIP2 is a critical component of mammalian plasma membranes, we investigated whether NaD1 also harbored permeabilization activity against mammalian cells. To this end, we performed a flow cytometry-based cell permeabilization assay on U937 monocytic lymphoma cells to measure uptake of the membrane-impermeable nucleic acid dye propidium iodide (PI). NaD1 permeabilized the plasma membrane of the U937 cells and induced a change in cell morphology in a concentration-dependent manner (Figure 7A). Furthermore, rapid leakage of intracellular ATP from U937 cells was observed within the first 200 s following exposure to NaD1 (Figure 7—figure supplement 1). Figure 7 with 3 supplements see all Download asset Open asset NaD1 kills mammalian tumor cells by membrane permeabilization. (A) Forward scatter, side scatter, and PI uptake analysis of U937 cells treated with NaD1. (B) Binding of FITC-dextran and (C) LDH release by NaD1-treated U937 cells. Error bars in C indicate SEM (n = 3). Data in A–C are representative of at least two independent experiments. https://doi.org/10.7554/eLife.01808.014 NaD1-mediated lysis of U937 cells was confirmed by the uptake of FITC-dextran (up to 40 kDa) and the release of lactate dehydrogenase (140 kDa) into the supernatant after NaD1 treatment (Figure 7B,C). In contrast, reduced and alkylated NaD1 (NaD1R&A) showed no cytotoxic activities against U937 cells (Figure 7—figure supplement 2), confirming the importance of the NaD1 tertiary structure for the ability to induce membrane permeabilization. It should be noted that NaD1 also permeabilized a diverse range of normal primary human cells and tumor cell lines (Figure 7—figure supplement 3), with the highest levels of activity exhibited against tumor cell lines. Collectively, these data suggest that, in addition to antifungal activity, NaD1 also exhibits antiproliferative properties against mammalian cells. We then sought to examine changes in cell morphology upon NaD1 treatment. Live confocal laser scanning microscopy (CLSM) revealed rapid changes on the cell surface of NaD1-permeabilized tumor cells and showed the formation of large plasma membrane blebs, with adherent cells (HeLa and PC3) forming multiple blebs of different sizes (Video 1) and non-adherent cells (U937) forming typically one to two large blebs (Video 2; Figure 8A). Moreover, bleb size was frequently larger than the actual cell (diameter >20 μm) and did not retract over a period of 20 min (Figure 8—figure supplement 1). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg NaD1 rapidly induces membrane blebbing and permeabilization of HeLa cells. Live CLSM of PKH67-stained HeLa cells in the presence of PI. Cells were imaged over a period of 10 min (5 s/frame), with NaD1 (10 μM) being added to cells at 1 min. https://doi.org/10.7554/eLife.01808.018 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Formation of a single large membrane bleb on a U937 cell following NaD1 treatment. Three-dimensional reconstruction of CLSM images of a NaD1-treated (10 μM) PKH67-/PI-stained U937 cell. https://doi.org/10.7554/eLife.01808.019 Figure 8 with 2 supplements see all Download asset Open asset NaD1 induces membrane blebbing of tumor cells. (A) CLSM of PKH67-stained NaD1 (10 μM) permeabilized HeLa and U937 cells. (B) CLSM of U937 cells treated with NaD1 (20 μM) in the presence of PI and 4 kDa FITC-dextran. Arrows indicate entry of PI. It should be noted that in this experiment the detector gain on the helium–neon laser (red channel) was increased compared to that used in A to enable visualization of the cell lysis events. Scale bars represent 10 μm. Data in A and B are representative of at least two independent experiments. https://doi.org/10.7554/eLife.01808.020 In our CLSM studies, we noticed that NaD1-induced membrane blebbing typically coincided with PI uptake (Video 1). To determine whether membrane blebbing occurs prior to, during, or following membrane permeabilization, we treated U937 cells with NaD1 in the presence of PI and 4 kDa FITC-dextran to monitor the entry of these molecules into NaD1-sensitive cells (Figure 8B; Video 3). FITC-dextran and NaD1 were added at 00:35 min, with FITC-dextran being excluded from cells with an intact membrane. Bleb formation was first observed for the cell located at the center of the panel at 03:25 min, with PI staining appearing at a specific point at the edge of the bleb. From 03:25 to 04:15 min, PI staining was observed in the bleb and the cytoplasm with FITC-dextran also entering the cell from the bleb site. At 04:20 min, PI-stained molecules were ‘expelled’ out of the cell, possibly at the same region that PI first entered the bleb. Similar results were also observed for PC3 cells (Figure 8—figure supplement 2). These data suggest that (i) small molecules such as PI can enter the cell initially at a ‘weakened’ point at the membrane bleb, (ii) the bleb continues to enlarge while PI and 4 kDa FITC-dextran enters, and (iii) intracellular contents are released at the bleb site, representing cytolysis. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg NaD1-mediated membrane permeabilization occurs at the blebs of U937 cells. Live CLSM of U937 cells treated with NaD1 in the presence of PI and 4 kDa FITC-dextran. Cells were imaged over a period of 10 min (5 s/frame), with NaD1 (20 μM) and 4 kDa FITC-dextran (100 μg/ml) being added to cells at 30 s. https://doi.org/10.7554/eLife.01808.023 NaD1 interacts with phosphoinositides in cellular membranes of tumor cells We next determined the specific mechanism by which NaD1 permeabilizes mammalian cells. Firstly, we tested the binding of BODIPY-labeled NaD1 to U937 cells. BODIPY-NaD1 permeabilized U937 cells at a level comparable to unlabeled NaD1 and bound to both viable (7AAD-negative) and permeabilized (7AAD-positive) cells, with more BODIPY-NaD1 bound to membrane-damaged cells (Figure 9A). These data suggest that NaD1 can interact with U937 cells prior to membrane permeabilization and accumulates on/within NaD1-sensitive cells. Figure 9 Download asset Open asset Subcellular localisation of BODIPY-NaD1 in tumor cells. (A) Detection of BODIPY-NaD1 binding to viable and permeabilized U937" @default.
• W4246529395 created "2022-05-12" @default.
• W4246529395 date "2013-12-20" @default.
• W4246529395 modified "2023-09-26" @default.
• W4246529395 title "Decision letter: Phosphoinositide-mediated oligomerization of a defensin induces cell lysis" @default.
• W4246529395 doi "https://doi.org/10.7554/elife.01808.033" @default.
• W4246529395 hasPublicationYear "2013" @default.
• W4246529395 type Work @default.
• W4246529395 citedByCount "0" @default.
• W4246529395 crossrefType "peer-review" @default.
• W4246529395 hasBestOaLocation W42465293951 @default.
• W4246529395 hasConcept C1491633281 @default.
• W4246529395 hasConcept C185592680 @default.
• W4246529395 hasConcept C2776498113 @default.
• W4246529395 hasConcept C2779281246 @default.
• W4246529395 hasConcept C2781322055 @default.
• W4246529395 hasConcept C55493867 @default.
• W4246529395 hasConcept C57409179 @default.
• W4246529395 hasConcept C62478195 @default.
• W4246529395 hasConcept C70721500 @default.
• W4246529395 hasConcept C86554907 @default.
• W4246529395 hasConcept C86803240 @default.
• W4246529395 hasConcept C95444343 @default.
• W4246529395 hasConceptScore W4246529395C1491633281 @default.
• W4246529395 hasConceptScore W4246529395C185592680 @default.
• W4246529395 hasConceptScore W4246529395C2776498113 @default.
• W4246529395 hasConceptScore W4246529395C2779281246 @default.
• W4246529395 hasConceptScore W4246529395C2781322055 @default.
• W4246529395 hasConceptScore W4246529395C55493867 @default.
• W4246529395 hasConceptScore W4246529395C57409179 @default.
• W4246529395 hasConceptScore W4246529395C62478195 @default.
• W4246529395 hasConceptScore W4246529395C70721500 @default.
• W4246529395 hasConceptScore W4246529395C86554907 @default.
• W4246529395 hasConceptScore W4246529395C86803240 @default.
• W4246529395 hasConceptScore W4246529395C95444343 @default.
• W4246529395 hasLocation W42465293951 @default.
• W4246529395 hasOpenAccess W4246529395 @default.
• W4246529395 hasPrimaryLocation W42465293951 @default.
• W4246529395 hasRelatedWork W112407786 @default.
• W4246529395 hasRelatedWork W116785052 @default.
• W4246529395 hasRelatedWork W1551485561 @default.
• W4246529395 hasRelatedWork W1966120122 @default.
• W4246529395 hasRelatedWork W2043827740 @default.
• W4246529395 hasRelatedWork W2067571365 @default.
• W4246529395 hasRelatedWork W2335830501 @default.
• W4246529395 hasRelatedWork W2606158061 @default.
• W4246529395 hasRelatedWork W2921628574 @default.
• W4246529395 hasRelatedWork W4246529395 @default.
• W4246529395 isParatext "false" @default.
• W4246529395 isRetracted "false" @default.
• W4246529395 workType "peer-review" @default.