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This article considers the relationship between the decriminalization of abortion and women’s access to abortion services. It focuses on the four Australian jurisdictions which are, with Canada, the only jurisdictions in the world where abortion has been removed from the criminal law. This paper draws on documentary evidence and an oral history project to give a “before and after” account of each jurisdiction. The paper assumes that the meaning and impact of decriminalization must be assessed in each local context. Understanding the conditions that shape access must incorporate analysis of the broader social, political and economic environment as well as the law. The article finds that decriminalization does not necessarily deliver any improvement in women’s access to abortion, at least in the short term. Further, it is not inconsistent with the neoliberal policy environment that characterizes the provision of abortion care in Australia, where most abortions are provided through the private sector at financial cost to women. If all women are to enjoy their human rights to full reproductive health care, the public health system must take responsibility for the adequate provision of abortion services; ongoing and vigilant activism is central if this is to be achieved.

In their letter, Bussy and Kostarelos reproduced aspects of our recent in vitro study of graphene oxide (GO)-induced mammalian cell plasma membrane (PM) ruffling and shedding and performed additional tests on the effects of different culture media.1 GO-induced acute responses of NIH/3T3 fibroblast cells were less pronounced in their selected media than in two buffered salt solutions (balanced salt solution [BSS] and phosphate-buffered saline [PBS]) commonly used in immunofluorescence and other cell studies in vitro.

Recent feminist scholarship about abortion in Australia has focused on the politics of abortion as it is played out in the law, the media, public discourse and political debate. This article seeks to offer new angles on the politics of abortion by focusing on the provision of abortion services and in particular the position of doctors in relation to abortion in Australia since the early 1990s. It crafts a theoretical framework to think about the place of doctors, and their patients, in abortion provision and sketches a history of doctors over the last two decades. It is based in a small interview project conducted by the author in 2013 and uses interviews to supplement documentary evidence with ‘inside information’ about local histories and the micro-politics of decision-making in order to build an account of the multifaceted position of doctors in relation to abortion. It also uses the interviews to build a picture of the subjectivity of doctors who play a crucial part in the provision of abortio...

The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes. The fabrication and implementation of this technique are demonstrated for both model membranes and live cells. Membrane-bound proteins were observed to cluster upon the addition of a multivalent cross-linker: On supported lipid bilayers, clusters of cholera toxin subunit B were formed upon cross-linking by an antibody specific for this protein; on living cells, immunoglobulin E bound to its receptor (FcεRI) on the plasma membranes of RBL mast cells was observed to form clusters upon exposure to a trivalent antigen. The formation of membrane clusters was quantified via fluorescence intensity vs time and changes in the temporal auto- and cross-correlations above a single nanoscale aperture. The illumination profile from a single aperture is analyzed experimentally and computationally with a rim-dominated illumination profile, yielding no change in the autocorrelation dwell time with changes in aperture diameter from 60 to 250 nm. This near-field fluorescence cross-correlation methodology provides access to nanoscale details of dynamic membrane interactions and motivates further development of near-field optical methods.

Infectious diseases, such as influenza, present a prominent global problem including the constant threat of pandemics that initiate in avian or other species and then pass to humans. We report a new sensor that can be specifically functionalized to detect antibodies associated with a wide range of infectious diseases in multiple species. This biosensor is based on electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies: the antibody catalyzed water oxidation pathway (ACWOP). Our platform includes a polymer brush-modified surface where specific antibodies bind to conjugated haptens with high affinity and specificity. Hydrogen peroxide provides an electrochemical signal that is mediated by Resorufin/Amplex Red. We characterize the biosensor platform, using model anti-DNP antibodies, with the ultimate goal of designing a versatile device that is inexpensive, portable, reliable, and fast. We demonstrate detection of antibodies at concentrations that fall well within clinically relevant levels.

The cell surface receptor for epidermal growth factor (EGFR), a receptor tyrosine kinase, is a key player in normal cell growth and proliferation. Mutations in this receptor often lead to oncological transformation and other pathologies. Because of its representation of the receptor tyrosine kinase family and its important role in health and disease, a broad range of studies have been carried out in many laboratories to investigate the structural basis for transmembrane receptor activation and the resulting assembly of cytosolic signaling components. This review highlights two approaches our laboratory has taken to gain more detailed information about both aspects: Surface patterned ligands to examine recruitment of the signaling machinery, and mutational analysis to examine the regulatory role of EGFR's juxtamembrane segment. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Graphene oxide (GO) has attracted intense interest for use in living systems and environmental applications. GO's compatibility with mammalian cells is sometimes inferred from its low cytotoxicity, but such conclusions ignore non-lethal effects that will influence GO's utility. Here we demonstrate, with rat basophilic leukemia (RBL) cells, profound plasma membrane (PM) ruffling and shedding induced by GO using confocal and live cell fluorescence microscopy, as well as scanning electron microscopy. These membrane structures contain immunoglobulin E receptors, are resistant to detergents, and lack detectable fluorescence labeling of F-actin and fibronectin. The formation of these membrane structures correlates with a loss of contact inhibition between RBL cells. We observe similar cellular responses towards GO for NIH-3T3 fibroblast cells and MDA-MB-231 human breast cancer cells. These findings reveal a previously unreported cellular response towards foreign nanomaterials. Membrane ruffling and shedding raise fundamental questions about how GO interacts with the PM, as well as its potential to modulate cellular mechanosensing for tissue engineering, stem cell differentiation, and other biomedical applications.

Most studies of the media sexualisation of children debate focus on its significance in terms of discourses of sexuality and gender. This article makes observations about public meetings called to express concern about media sexualisation held in several Australian capital cities 2009–2010 to argue that media sexualisation is also meaningful in relation to discourses of nation. Drawing on the claim made by Valerie Walkerdine that concern with the eroticisation of girls is a defence against acknowledgement of difficult sexual desires and Gayle Rubin’s observation that concerns with children’s sexuality often carry other political agendas, this article argues that the concern with media sexualisation of children in the Australian context carries meaning about national identity and national virtue. It places the emergence of the discourse in the recent history of revelations about the systemic abuse of children by the state and the Christian churches throughout the 20th century and into this century, and argues that the media sexualisation discourse constitutes subjects who, via their investment in innocence, are exonerated from any culpability in this compromising national history.

Electroactive nanofibers decorated with α,ω-bi-(dinitrophenylcaproic acid), abbreviated as DNP, have been prepared by electrospinning a solution of α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)], polystyrene and single-walled carbon nanotubes (SWCNTs). The α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethyleneoxide)] polymers were synthesized by living anionic polymerization and subsequent esterification to obtain the DNP-functionalized polymers. The nanofibers (300 nm) were electrospun onto a silicon wafer substrate at a voltage of 10 kV using dimethylformamide and chlorobenzene as solvents. The nanofibers were characterized by Raman spectroscopy, fluorescence microscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM) and optical microscopy. The DNP groups, therein, were tethered to the nanofibers via oligo(oxyethylene) spacers. The DNP groups decorating the nanofibers were capable of specifically binding with anti-DNP immunoglobulin E (IgE). The binding specificity of nanofibers containing 1% SWCNTs with anti-DNP IgE was studied via fluorescently labeled (Fluorescein IsoThioCyanate) FITC-IgE. Electronic activity of the nanofibers was studied by I–V plots from Kelvin sensing. The I–V plots before sensitizing with IgE differed from the I–V plots after binding with IgE. The results of the studies suggest the possibility of developing functional nanofibers as the active component in biosensors. Copyright © 2013 John Wiley & Sons, Ltd.

This article describes the politics of abortion during the Howard years (1996–2007) as a time of significant turbulence. It considers four separate significant developments in abortion discourse in the actions of federal and state governments and parliaments and in the publication of an influential anti-abortion text. It argues that while support for women's choice as a framework for understanding abortion prevailed during the Howard years, the definition of what an abortion is changed significantly towards an experience that is contrary to, rather than consistent with, women's health and well-being. This shift is attributed to the Howard government, the most pro-actively anti-abortion federal government since the early 1970s, and broader white patriarchal discourses of gender that elevated maternity as a moral imperative for women and constructed abortion as a harm.

The ability to spatially deposit multiple biomolecules onto a single surface with high-resolution while retaining biomolecule stability and integrity is critical to the development of micro- and nanoscale bio-devices. While conventional lithographic patterning methods are attractive for this application, they typically require the use of UV exposure and/or harsh solvents and imaging materials, which may be damaging to fragile biomolecules. Here, we report the development of a new patterning process based on a fluorinated patterning material that is soluble in hydrofluoroether solvents, which we show to be benign to biomolecules, including proteins and DNA. We demonstrate the implementation of these materials into an orthogonal processing system for patterning multi-biomolecule arrays by imprint lithography at room temperature. We further showcase this method’s capacity for fabricating patterns of receptor-specific ligands for fundamental cell studies.

Store operated Ca2+ entry through the channel protein Orai1 contributes to mast cell spontaneous motility and antigen-mediated chemotaxis. Migration is a fundamental function of immune cells, and a role for Ca2+ in immune cell migration has been an interest of scientific investigations for many decades. Mast cells are the major effector cells in IgE-mediated immune responses, and cross-linking of IgE-FcɛRI complexes at the mast cell surface by antigen activates a signaling cascade that causes mast cell activation, resulting in Ca2+ mobilization and granule exocytosis. These cells are known to accumulate at sites of inflammation in response to parasite and bacterial infections. Using real-time imaging, we monitored chemotactic migration of RBL and rat BMMCs in response to a gradient of soluble multivalent antigen. Here, we show that Ca2+ influx via Orai1 plays an important role in regulating spontaneous motility and directional migration of mast cells toward antigen via IgER complexes. Inhibition of Ca2+ influx or knockdown of the Ca2+ entry channel protein Orai1 by shRNA causes inhibition of both of these processes. In addition, a mutant Syk− shows impaired spontaneous motility and chemotaxis toward antigen that is rescued by expression of Syk. Our findings identify a novel Ca2+ influx-mediated, Orai1-dependent mechanism for mast cell migration.

This article compares two key moments in the Australian emergence of the political identity of ‘lesbian mothers’. It documents the first national lesbian mothers conference in 1984 and then the eruption of lesbian mothers onto the front pages of major Australian newspapers in 2000. From its founding in a lesbian-feminist politics of ambivalence about motherhood, the identity became increasingly shaped by a postmodern discourse of self-invention, an embrace of motherhood and a liberal politics of equal rights. The article argues that contemporary lesbian mothers could usefully recover ambivalence about motherhood as a key strategic response to the challenges of neo-liberalism.

Stimulated exocytic events provide a means for physiological communication and are a hallmark of the mast cell-mediated allergic response. In mast cells these processes are triggered by antigen crosslinking of IgE bound to its high-affinity receptor, FcϵRI, on the cell surface. Here we use the endosomal v-SNARE VAMP8, and the lysosomal hydrolase β-hexosaminidase (β-Hex), each C-terminally fused to super-ecliptic pHluorin, to monitor stimulated exocytosis. Using these pHluorin-tagged constructs, we monitor stimulated exocytosis by fluorimetry and visualize individual exocytic events with total internal reflection (TIRF) microscopy. Similar to constitutive recycling endosome (RE) trafficking, we find that stimulated RE exocytosis, monitored by VAMP8, is attenuated by expression of dominant negative (S25N) Rab11. Stimulated β-Hex exocytosis is also reduced in the presence of S25N Rab11, suggesting that expression of this mutant broadly impacts exocytosis. Interestingly, pretreatment with inhibitors of actin polymerization, cytochalasin D or latrunculin A, substantially restores both RE and lysosome exocytosis in cells expressing S25N Rab11. Conversely, stabilizing F-actin with jasplakinolide inhibits antigen-stimulated exocytosis but is not additive with S25N Rab11-mediated inhibition, suggesting that these reagents inhibit related processes. Together, our results suggest that Rab11 participates in the regulation necessary for depolymerization of the actin cytoskeleton during stimulated exocytosis in mast cells.

Cells respond to their physical environment and to chemical stimuli in terms of collective molecular interactions that are regulated in time and space. Small molecules may engage specific receptors to initiate a transmembrane signal, and the system amplifies this nanoscale interaction to microscale assemblies within the cell and often to longer length scales involving surrounding tissue and ultimately the whole organism. A striking example of signal integration over multiple length scales is the allergic immune response. IgE receptors (FceRI) on mast cells are the gatekeepers of this response, and this system has proven to be a valuable model for investigating receptor-mediated cellular activation. Spanning the range of cellular responses we use super resolution fluorescence localization microscopy to investigate the earliest signaling events and ligands patterned in micron size features together with confocal microscopy to investigate early and later events. We are characterizing distinctive regulatory roles played by the actin cytoskeleton at different stages. We are further investigating spatial redistributions of the cellular integrins that relate to the enhancement of signaling observed for cells spreading on surfaces.

A series of 2,4-dinitrophenyl (DNP) functionalized polypyrrole terpolymers, capable of specific binding to IgE antibodies (proteins), have been synthesized using oxidizing initiator, ammonium persulfate and were characterized by 1H-NMR, IR, DSC, Light Scattering etc. The terpolymers were composed of monomers and macromonomers: monomer (pyrrole), macromonomer A (pyrrole with pendant ethylene glycol) and macromonomer B (pyrrole with pendant DNP) with specific functionality of conductance, processiblity and binding, respectively. The terpolymers are found to be semiconductive, (5 × 10−6 S cm−1) by itself without the addition of doping agents. Molecular dynamics simulation of terpolymer shows that the DNP-(2,4-dinitrophenyl) functional group extends out from the polymer backbone and thus, is available for binding. The DNP functional groups on the terpolymer achieve steady state binding with anti-DNP IgE proteins at nanomolar concentrations in solution. The terpolymer was blended with sulfonated polystyrene an...

We use patterned poly(acrylic acid) (PAA) polymer brushes to explore the effects of surface chemistry and topography on cell-surface interactions. Most past studies of surface topography effects on cell adhesion have focused on patterned feature sizes that are larger than the dimensions of a cell, and PAA brushes have been characterized as cell repellent. Here we report cell adhesion studies for RBL mast cells incubated on PAA brush surfaces patterned with a variety of different feature sizes. We find that when patterned at sub-cellular dimensions on silicon surfaces, PAA brushes that are 30 or 15 nm thick facilitate cell adhesion. This appears to be mediated by fibronectin, which is secreted by the cells, adsorbing to the brushes and then engaging cell surface integrins. The result is detectable accumulation of plasma membrane within the brushes, and this involves cytoskeletal remodeling at the cell-surface interface. By decreasing brush thickness, we find that PAA can be ‘tuned’ to promote cell adhesion with down-modulated membrane accumulation. We exemplify the utility of patterned PAA brush arrays for spatially controlling the activation of cells by modifying brushes with ligands that specifically engage IgE bound to high affinity receptors on mast cells.

Mesoporous silica with cubic symmetry has attracted interest from researchers for some time. Here, we present the room temperature synthesis of mesoporous silica nanoparticles possessing cubic Pm3n symmetry with very high molar ratios (>50%) of 3-aminopropyl triethoxysilane. The synthesis is robust allowing, for example, co-condensation of organic dyes without loss of structure. By means of pore expander molecules, the pore size can be enlarged from 2.7 to 5 nm, while particle size decreases. Adding pore expander and co-condensing fluorescent dyes in the same synthesis reduces average particle size further down to 100 nm. After PEGylation, such fluorescent aminated mesoporous silica nanoparticles are spontaneously taken up by cells as demonstrated by fluorescence microscopy.

This response to Hester Eisenstein's article focuses on the question of religion. In the section of her article under the heading ‘Islamophobia and the Global War on Terror’ Eisenstein joins other ...

Delivered at the ‘Manifesting Literary Feminisms’ conference in Melbourne, December 2008. My qualification to speak, and now write, about Sue and her contribution to feminist scholarship is multi-s...

Mast cell stimulation via IgE receptors causes activation of multiple processes, including Ca(2+) mobilization, granule exocytosis, and outward trafficking of recycling endosomes to the plasma membrane. We used fluorescein-conjugated cholera toxin B (FITC-CTxB) to label GM(1) in recycling endsomes and to monitor antigen-stimulated trafficking to the plasma membrane in both fluorimeter and imaging-based assays. We find that the sphingosine derivatives D-sphingosine and N,N'-dimethylsphingosine effectively inhibit this outward trafficking response, whereas a quarternary ammonium derivative, N,N',N″-trimethylsphingosine, does not inhibit. This pattern of inhibition is also found for Ca(2+) mobilization and secretory lysosomal exocytosis, indicating a general effect on Ca(2+)-dependent signaling processes. This inhibition correlates with the capacity of sphingosine derivatives to flip to the inner leaflet of the plasma membrane that is manifested as changes in plasma membrane-associated FITC-CTxB fluorescence and cytoplasmic pH. Using a fluorescently labeled MARCKS effector domain to monitor plasma membrane-associated polyphosphoinositides, we find that these sphingosine derivatives displace the electrostatic binding of this MARCKS effector domain to the plasma membrane in parallel with their capacity to inhibit Ca(2+)-dependent signaling. Our results support roles for plasma membrane polyphosphoinositides in Ca(2+) signaling and stimulated exocytosis, and they illuminate a mechanism by which D-sphingosine regulates signaling responses in mammalian cells.

Ca2+ mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca2+ sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca2+ waves that originate most frequently from the tips of extended cell protrusions, as well as Ca2+ oscillations throughout the cell that usually follow the initiating Ca2+ wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca2+ puffs at the region of cell contact. Initiating Ca2+ waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca2+ release from endoplasmic reticulum stores and to extracellular Ca2+, but they do not depend on store-operated Ca2+ entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca2+ signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca2+ response.

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

Signaling in mast cells and basophils is mediated through IgE and its high affinity cell surface receptor, FcϵRI. Crosslinking of the receptors by a cognate multivalent antigen leads to degranulation and release of mediators of the allergic immune response. Using multicolor fluorescence confocal microscopy, we probed the spatio-temporal dynamics of early events in the IgE receptor signal cascade. We monitored the recruitment of GFP-/CFP-labeled signaling proteins by acquiring sequential images with time resolution of 3s during stimulation of RBL-2H3 mast cells with multivalent antigen. A fluorescent tag on the antigen allowed us to visualize the plasma membrane localization of crosslinked receptors, and fluorescent cholera toxin B served as a plasma membrane marker. We developed an automated image analysis scheme to quantify the recruitment of fluorescent intracellular proteins to the plasma membrane and to assess the time-dependent colocalization of these and other membrane-associated proteins with crosslinked receptors as measured by cross-correlation between the plasma membrane distributions of the two fluorophores. This automated method permits analysis of thousands of individual images from multiple experiments for each cross-correlation pair. We systematically applied this analysis to characterize stimulated interactions of IgE receptors with several signaling proteins, including the tyrosine kinases Lyn and Syk, and the adaptor protein LAT. Notably, for Syk-CFP we observed a rapid stimulated translocation to the plasma membrane but very little colocalization with aggregated receptors. Our results demonstrate the utility of this simple, automated method to monitor protein interactions quantitatively during cell signaling.

Advances in microfabrication and nanofabrication are opening new opportunities to investigate complicated questions of cell biology in ways not before possible. In particular, the spatial regulation of cellular processes can be examined by engineering the chemical and physical environment to which the cell responds. Lithographic methods and selective chemical modification schemes can provide biocompatible surfaces that control cellular interactions on the micron and submicron scales on which cells are organized. Combined with fluorescence microscopy and other approaches of cell biology, a widely expanded toolbox is becoming available. This review illustrates the potential of these integrated engineering tools, with an emphasis on patterned surfaces, for investigating fundamental mechanisms of receptor-mediated signaling in cells. We highlight progress made with immune cells and in particular with the IgE receptor system, which has been valuable for developing technology to gain new information about spatial regulation in signaling events.

Two series of functional polymers, α,ω‐bi[2,4‐dinitrophenyl][poly(ethylene oxide)‐b‐poly(2‐methoxystyrene)‐b‐poly(ethylene oxide)] (DNP‐PEO‐P2MS‐PEO‐DNP) and α,ω‐bi[2,4‐dinitrophenyl caproic][poly(ethylene oxide)‐b‐poly(2‐methoxystyrene)‐b‐poly(ethylene oxide)] (CDNP‐PEO‐P2MS‐PEO‐CDNP), were synthesized by anionic living polymerization. The polymers were characterized by FT‐IR, 1H‐NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions for the lower molecular weight functional polymers were slightly broad (1.3–1.5). However, the molecular weight distributions for higher molecular weight polymers were narrower (1.1–1.2). Differential scanning calorimetry (DSC) studies showed thermal transitions indicative of the presence of microphases in the polymer solid state. The polymers were white powders and soluble in tetrahydrofuran. The binding affinity of DNP‐PEO‐P2MS‐PEO‐DNP ligands towards anti DNP IgE was determined by titrations with fluorescently labeled FITC‐IgE. A water soluble CDN...

Antigen-mediated cross-linking of IgE bound to its receptor, FcRI, stimulates degranulation, phospholipid metabolism, and cytokine production in mast cells and basophils to initiate inflammatory and allergic responses. Previous studies suggested that spatial organization of the clustered receptors affects the assembly of the transmembrane signaling complexes. To investigate systematically the structural constraints in signal initiation, we utilized rigid double-stranded DNA scaffolds to synthesize ligands with tunable lengths. We characterized a series of symmetric trivalent DNA ligands with rigid spacing between 2,4-dinitrophenyl (DNP) haptenic groups in the range of 5-15 nm. These ligands all bind to anti-DNP IgE on RBL mast cells with similar avidity, and they all cross-link IgE-FcRI complexes effectively. We observe length-dependent stimulation of tyrosine phosphorylation of FcRI beta and gamma subunits and the adaptor protein LAT: the shortest ligand is approximately 5-10-fold more potent than the longest. Stimulated Ca2+ mobilization and degranulation also exhibits kinetics and magnitudes that differ as a function of ligand length. In contrast, tyrosine phosphorylation of phospholipase Cgamma1 and consequent Ca2+ release from intracellular stores do not show this dependence on ligand length. Our results with these rigid, DNA-based ligands provide direct support for receptor transphosphorylation as a key step in amplified signaling leading to degranulation, and they further reveal branching of pathways in signaling events.

Novel biophysical approaches combined with modeling and new biochemical data have helped to recharge the lipid raft field and have contributed to the generation of a refined model of plasma membrane organization. In this review, we summarize new information in the context of previous literature to provide new insights into the spatial organization and dynamics of lipids and proteins in the plasma membrane of live cells. Recent findings of large-scale separation of liquid-ordered and liquid-disordered phases in plasma membrane vesicles demonstrate this capacity within the complex milieu of plasma membrane proteins and lipids. Roles for membrane heterogeneity and reorganization in immune cell activation are discussed in light of this new information.

Fluorescence resonance energy transfer (FRET) between matched carbocyanine lipid analogs in the plasma membrane outer leaflet of RBL mast cells was used to investigate lateral distributions of lipids and to develop a general method for quantitative measurements of lipid heterogeneity in live cell membranes. FRET measured as fluorescence quenching of long-chain donor probes such as DiO-C18 is greater with long-chain, saturated acceptor probes such as DiI-C16 than with unsaturated or shorter-chain acceptors with the same chromophoric headgroup compared at identical concentrations. FRET measurements between these lipid probes in model membranes support the conclusion that differential donor quenching is not caused by nonideal mixing or spectroscopic differences. Sucrose gradient analysis of plasma membrane-labeled, Triton X-100-lysed cells shows that proximity measured by FRET correlates with the extent of lipid probe partitioning into detergent-resistant membranes. FRET between DiO-C16 and DiI-C16 is sensitive to cholesterol depletion and disruption of liquid order (Lo) by short-chain ceramides, and it is enhanced by cross linking of Lo-associated proteins. Consistent results are obtained when homo-FRET is measured by decreased fluorescence anisotropy of DiI-C16. These results support the existence of nanometer-scale Lo/liquid disorder heterogeneity of lipids in the outer leaflet of the plasma membrane in live cells.

Immune receptors that specifically recognize foreign antigens to activate leukocytes in adaptive immune responses belong to a family of multichain cell surface proteins. All of these contain immunoreceptor tyrosine-based activation motifs in one or more subunits that initiate signaling cascades following stimulated tyrosine phosphorylation by Src-family kinases. As highlighted in this review, lipids participate in this initial activation step, as well as in more downstream signaling steps. We summarize evidence for cholesterol-dependent ordered lipids serving to regulate the store-operated Ca(2+) channel, Orai1, and we describe the sensitivity of Orai1 coupling to the ER Ca(2+) sensor, STIM1, to inhibition by polyunsaturated fatty acids. Phosphoinositides play key roles in regulating STIM1-Orai1 coupling, as well as in the stimulated Ca(2+) oscillations that are a consequence of IgE receptor signaling in mast cells. They also participate in the coupling between the plasma membrane and the actin cytoskeleton, which regulates immune receptor responses in T cells, B cells, and mast cells, both positively and negatively, depending on the cellular context. Recent studies show that other phospholipids with mostly saturated acylation also participate in coupling between receptors and the actin cytoskeleton. Lipid heterogeneity is a central feature of the intimate relationship between the plasma membrane and the actin cytoskeleton. The detailed nature of these interactions and how they are dynamically regulated to initiate and propagate receptor-mediated cell signaling are challenging questions for further investigation. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Surgical abortion has been provided liberally in Australia since the early 1970s, mainly in privately owned specialist clinics. The introduction of medical abortion, however, was deliberately obstructed and consequently significantly delayed when compared to similar countries. Mifepristone was approved for commercial import only in 2012 and listed as a government subsidised medicine in 2013. Despite optimism from those who seek to improve women's access to abortion, the increased availability of medical abortion has not yet addressed the disadvantage experienced by poor and non-metropolitan women. After telling the story of medical abortion in Australia, this paper considers the context through which it has become available since 2013. It argues that the integration of medical abortion into primary health care, which would locate abortion provision in new settings and expand women's access, has been constrained by the stigma attached to abortion, overly cautious institutionalised frameworks, and the lack of public health responsibility for abortion services. The paper draws on documentary sources and oral history interviews conducted in 2013 and 2015.

Synaptic transmission and cell to cell communication in the human body are frequently characterized by the release of charged transmitters and other chemical mediators from secretory vesicles or granules which then impinge on specific receptor molecules expressed on target cells1,2,3. Depending on the excitable nature, the initiating cells respond to chemical inputs by releasing vesicular granules containing specific compounds or by inducing an electrical wave such as an action potential (AP). The process of vesicle fusion with the cell plasma membrane upon stimulation and subsequent release of the granular contents (i.e. in the form of quanta) into the extracellular environment is termed exocytosis4. When measured electrochemically such release events reveal a distinctive temporal response5,6. Exocytosis recordings are also often employed to characterize the mechanism of drug action on cells. For example, amperometric recordings have shown that the Parkinson’s drug L-Dopa increases the quantal size7, i.e. the total released charge increases, a consequence of increase vesicle size. There is thus a need to develop high throughput, scalable and multi-functional electronic instrumentation in order to characterize the action of various pharmacological inhibitors, toxins and stimulants on vesicle release. Transmitter and granular release can be specifically stimulated or inhibited depending on the cell type under study. In neurons, electrical excitations in the form of action potentials (AP) propagate along the axon and stimulate neurotransmitter release in the region between the axon terminus of the pre-synaptic neuron and the dendritic spine of the post-synaptic neuron [Fig. 1(a)] called the synapse. The released transmitters impinge on specific receptors on the post-synaptic neuron exciting or inhibiting action potential generation. In immune cells such as mast cells on the contrary, exocytosis can be induced through a receptor effector function where a specific antigen-receptor interaction causes a signal cascade within the cell, culminating in the release of chemical mediators which causes an allergic response. The released compounds from mast cells impinge on cells expressing specific receptors (such as the histamine receptor on smooth muscle cells) [Fig. 1(c)] and elicit a downstream response. In this study we seek to create a CMOS bio-sensor capable of detecting granule release from mast cells as a function of transmitter-receptor induced signaling. We then extend the approach to measuring depolarization induced activity from chromaffin cells where it can function as an electronic post-synaptic sensor [Fig. 1(d)]. Such a system not only provides a test bench for fundamental exocytotic analysis by monitoring release from vesicles and action potential’s with high temporal resolution, which is paramount in understanding cellular kinetics and establishing rapid screening procedures but also sets a promising route towards future artificial synapse systems and ionic-electronic interfacing circuitry. Figure 1 The cell-transistor synapse. The rat basophilic leukemia cell (RBL-2H3) is a tumor cell line used frequently as an experimental model for mucosal mast cells8. The release of inflammatory mediators from mast cells is the primary event in an allergic response9. These cells serve as a robust model for understanding the underlying biophysical and biochemical mechanism which couples signals originating at the membrane receptor with a biological effector function. Immunoglobulins of the IgE class serve as antigenic receptors which are anchored to cells via the membrane protein complex FceRI. Upon stimulation with multivalent antigen, the receptors crosslink causing a signal cascade within the cell, which eventually culminates in the secretion of preformed mediators stored in the cellular granules. Mast cells form a specialized niche of the immune system, because the triggered cellular activity is immediate. Depending on the particular type of mast cells or basophil’s, secretion occurs within seconds to minutes following the IgE cross linking step. Mast cells also provide a meaningful model for cell activation by an immunological stimulus, i.e., by an antibody-antigen reaction. We further demonstrate the device detection capability using chromaffin cells of the bovine adrenal medulla to detect neurotransmitter release and related electrical activity. The chromaffin cell is an excitable cell and allows us to study stimulus secretion coupling as mediated by both calcium entry and voltage gated channels, i.e., exocytosis induced by depolarization. Current methods of monitoring exocytosis include fluorescent techniques1,8,10,11, carbon-fiber amperometry12 and membrane capacitance measurements13. Fluorescence techniques often require labels and sophisticated optics, which increases the complexity of the experiment. On the other hand, amperometry may be prone to noise due to low current levels involved, relies on faradaic chemistry for detection, and is challenging to miniaturize in terms of pixel density, although recent efforts have resulted in significant improvements14,15,16. Non-faradaic transistor-based measurements on the contrary extend the detection capability to electrochemically inactive molecules, are extremely sensitive to surface adsorption, record cellular signals with a high degree of temporal sensitivity, present a naturally occurring high impedance node due to the gate oxide and can render sub-cellular spatial resolution17,18,19,20 with very low input referred noise characteristics21. Previous work on transistor-based cellular sensing has primarily focused on recording electrical activity from excitable cells such cardiac myocytes22,23 and nerve cells21,24. Recently Stern and co-workers25 extended transistor based sensing to detect antigen-stimulated T-cell activation detection by CMOS-compatible semiconducting nanowire sensors, however the exact signal generating mechanism was not investigated. In another report, transistor recording of vesicle release from chromaffin cells26 was demonstrated using open-gate ISFET’s. The recorded signal was attributed solely to the change in the local pH across the double-layer interface which leads to protonation of the surface and hence a change in surface potential. In this work, we extend this understanding and demonstrate the use of CMOS compatible floating gate transistors as cell based biosensors. Specifically we demonstrate that transistors can detect degranulation from electrically non-excitable cells such as the RBL-2H3 cell line and exocytosis from chromaffin cells. In addition to extracellular pH variation, we provide preliminary evidence suggesting direct molecular binding to the sensor surface as a signal generating mechanism. Since the nature of the FET interface allows one to record both electrochemical and ionic activities simultaneously we demonstrate the detection of AP’s and vesicle release simultaneously. Furthermore, we demonstrate simultaneous quasi-static and impedimetric based signal detection suggesting that FET’s could be used to readout passive membrane properties in addition to secreted charge.

Our long-term efforts to elucidate receptor-mediated signalling in immune cells, particularly transmembrane signalling initiated by FcɛRI, the receptor for IgE in mast cells, led us unavoidably to contemplate the role of the heterogeneous plasma membrane. Our early investigations with fluorescence microscopy revealed co-redistribution of certain lipids and signalling components with antigen-cross-linked IgE–FcɛRI and pointed to participation of ordered membrane domains in the signalling process. With a focus on this function, we have worked along with others to develop diverse and increasingly sophisticated tools to analyse the complexity of membrane structure that facilitates regulation and targeting of signalling events. The present chapter describes how initial membrane interactions of clustered IgE–FcɛRI lead to downstream cellular responses and how biochemical information integrated with nanoscale resolution spectroscopy and imaging is providing mechanistic insights at the level of molecular complexes.

The importance of membrane-based compartmentalization in eukaryotic cell function has become broadly appreciated, and a number of studies indicate that these eukaryotic cell membranes contain coexisting liquid-ordered (L(o)) and liquid-disordered (L(d)) lipid domains. However, the current evidence for such phase separation is indirect, and so far there has been no direct demonstration of differences in the ordering and dynamics for the lipids in these two types of regions or their relative amounts in the plasma membranes of live cells. In this study, we provide direct evidence for the presence of two different types of lipid populations in the plasma membranes of live cells from four different cell lines by electron spin resonance. Analysis of the electron spin resonance spectra recorded over a range of temperatures, from 5 to 37 degrees C, shows that the spin-labeled phospholipids incorporated experience two types of environments, L(o) and L(d), with distinct order parameters and rotational diffusion coefficients but with some differences among the four cell lines. These results suggest that coexistence of lipid domains that differ significantly in their dynamic order in the plasma membrane is a general phenomenon. The L(o) region is found to be a major component in contrast to a model in which small liquid-ordered lipid rafts exist in a 'sea' of disordered lipids. The results on ordering and dynamics for the live cells are also compared with those from model membranes exhibiting coexisting L(o) and L(d) phases.

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcepsilonRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.

Antigen-decorated shell cross-linked knedel-like nanoparticles (SCKs) were synthesized and studied as multivalent nanoscale surfaces from which antibody-binding units were presented in a manner that was designed to approach virus particle surfaces. The SCK nanostructures were fabricated with control over the number of antigenic groups, from mixed micellization of amphiphilic diblock copolymer building blocks that contained either an antigen (2,4-dinitrophenyl) or an ethylpropionate group at the hydrophilic alpha-chain terminus. Amphiphilic diblock copolymers were synthesized by atom transfer radical polymerization of tert-butyl acrylate and methyl acrylate sequentially from either a 2,4-dinitrophenyl-functionalized initiator or ethyl 2-bromopropionate, followed by selective removal of the tert-butyl groups to afford 2,4-dinitrophenyl-poly(acrylic acid)60-b-poly(methyl acrylate)60 (DNP-PAA(60)-b-PMA60) and poly(acrylic acid)70-b-poly(methyl acrylate) (PAA70-b-PMA70). Micelles were assembled via addition of water to THF solutions of the polymers in 0:1, 1:1, and 1:0 molar ratios of DNP-PAA60-b-PMA60 to PAA70-b-PMA70, followed by dialysis against water. The acrylic acid groups of the micelle coronas were partially cross-linked (nominally 50%) with 2,2'-(ethylenedioxy)bis(ethylamine), in the presence of 1-(3'-dimethylaminopropyl)-3-ethylcarbodiimide methiodide. Following extensive dialysis against water, the 0%, 50%, and 100% dinitrophenylated shell cross-linked nanoparticles (DNP-SCKs) were characterized with dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), infrared and UV-vis spectroscopies, and analytical ultracentrifugation (AU). The surface accessibility and bioavailability of the DNP units upon the DNP-SCKs were investigated by performing quenching titrations of fluorescein-labeled IgE antibody in solution and degranulation of IgE sensitized RBL-2H3 cells. The DNP antigens proved to be surface-available and able to form multivalent bonds with IgE antibodies, causing degranulation.

The mechanisms by which a cell uses and adapts its functional membrane organization are poorly understood and are the subject of ongoing investigation and discussion. Here, we study one proposed mechanism: the crosslinking of membrane components. In immune cell signaling (and other membrane-associated processes), a small change in the clustering of specific membrane proteins can lead to large-scale reorganizations that involve numerous other membrane components. We have investigated the large-scale physical effect of crosslinking a minor membrane component, the ganglioside GM 1 , in simple lipid models of the plasma membrane containing sphingomyelin, cholesterol, and phosphatidylcholine. We observe that crosslinking GM 1 can cause uniform membranes to phase-separate into large, coexistent liquid ordered and liquid disordered membrane domains. We also find that this lipid separation causes a dramatic redistribution of a transmembrane peptide, consistent with a raft model of membrane organization. These experiments demonstrate a mechanism that could contribute to the effects of crosslinking observed in cellular processes: Domains induced by clustering a small number of proteins or lipids might rapidly reorganize many other membrane proteins.

Protein patterning has become an important topic as advances are made in biologically integrated devices and protein chip technology. Versatile and effective patterning requires substrates that can be quantified, with active presentation of proteins and control over protein density and orientation. Herein we describe a model system and the use of low-energy electron beam lithography to pattern molecular templates for immobilization of antibodies through ligand recognition. The templates were patterned over a background of poly(ethylene glycol) (PEG) modified silicon oxide (SiO x ). These substrates were exposed to a low-voltage (2 keV) electron beam to remove PEG selectively from exposed regions. These regions were then functionalized with a dinitrophenyl (DNP) ligand and tested for specific binding of fluorescently labeled anti-DNP antibodies. The PEG modified regions in conjunction with ligand-presenting regions in the patterned arrays substantially reduces non-specific adsorption of proteins, yielding a specific/nonspecific ratio of approx 10. The surface coverage of the biologically active DNP groups on SiO x and the amount of immobilized antibody on DNP were measured with a fluorescence-based, enzyme-linked immunosorbent assay. The specificity of the interaction between DNP ligand and fluorescently labeled anti-DNP antibodies was evaluated with fluorescence microscopy. This approach to patterning of molecular templates and assays for quantification are generally applicable to immobilization of any ligand-receptor pair on a wide range of substrates.

Cross-linking of the high-affinity IgE receptor (FceRI) by multivalent antigen on mast cells initiates transmembrane signaling by coupling of FceRI with the cytoplasmic tyrosine kinase Lyn in coalesced protein−lipid rafts. As part of our ongoing investigation of these membrane-based interactions, we employed fluorescence correlation spectroscopy and fluorescence photobleaching recovery to compare lateral mobilities of representative components: Cy3−IgE−FceRI, a transmembrane protein; DiI−C16, an outer leaflet lipid probe with two saturated acyl chains; PM−EGFP, a Lyn analogue anchored to the inner leaflet via the saturated acyl chains palmitate and myristate; and EGFP−GG, a control protein anchored to the inner leaflet via an unsaturated geranylgeranyl chain and an adjacent polybasic amino acid sequence. Interpreting the data with both free diffusion and anomalous subdiffusion models, we find that EGFP−GG diffuses faster than PM−EGFP on average. Both inner leaflet probes are sensitive to day-to-day varia...

Four chain spin labels and a spin-labeled cholestane were used to study the dynamic structure of plasma membrane vesicles (PMV) prepared from RBL-2H3 mast cells at temperatures ranging from 22 degrees C to 45 degrees C. Analysis shows that the spectra from most labels consist of two components. The abundant spectral components exhibit substantial ordering that is intermediate between that of a liquid-ordered (Lo) phase, and that of a liquid-crystalline (Lc) phase as represented by model membranes. Also, rotational diffusion rates of the spin labels are comparable to those in the Lo phase. In contrast, the ordering for the less abundant components is much lower. These results indicate that a Lo-like region or phase (the abundant component) and an Lc-like region or phase (the less abundant component) coexist in the PMV. In contrast, membranes reconstituted from extracted lipids exhibit the more ordered phase only. This suggests that membrane-associated proteins are important for the coexistence of Lo-like and Lc-like regions in the plasma membrane. In addition, binding of the myristoylated protein, ARF6 to PMV, leads to a new spectral component for a headgroup lipid spin label that indicates the formation of plasma membrane defects by this low molecular weight GTPase.

The plasma membrane contains ordered lipid domains, commonly called lipid rafts, enriched in cholesterol, sphingolipids, and certain signaling proteins. Lipid rafts play a structural role in signal initiation by the high affinity receptor for IgE. Cross-linking of IgE-receptor complexes by antigen causes their coalescence with lipid rafts, where they are phosphorylated by the Src family tyrosine kinase, Lyn. To understand how lipid rafts participate in functional coupling between Lyn and FcepsilonRI, we investigated whether the lipid raft environment influences the specific activity of Lyn. We used differential detergent solubility and sucrose gradient fractionation to isolate Lyn from raft and nonraft regions of the plasma membrane in the presence or absence of tyrosine phosphatase inhibitors. We show that Lyn recovered from lipid rafts has a substantially higher specific activity than Lyn from nonraft environments. Furthermore, this higher specific activity correlates with increased tyrosine phosphorylation at the active site loop of the kinase domain. Based on these results, we propose that lipid rafts exclude a phosphatase that negatively regulates Lyn kinase activity by constitutive dephosphorylation of the kinase domain tyrosine residue of Lyn. In this model, cross-linking of FcepsilonRI promotes its proximity to active Lyn in a lipid raft environment.

Binding of fluorescein isothiocyanate (FITC)-conjugated cholera toxin B subunit to ganglioside GM1 on RBL-2H3 cells at 37 °C results in labeling of the plasma membrane as well as a pool of perinuclear intracellular membranes identified as the endosomal recycling compartment. Antigen-mediated activation of IgE receptor signaling causes rapid, sustained outward trafficking of these labeled endosomes, that is monitored as an increase in FITC fluorescence due to relief of quenching in the acidic endosomes upon delivery to the plasma membrane. Stimulation of this process depends on the integrity of cholesterol-dependent lipid rafts and occurs in response to Ca2+-mobilizing thapsigargin as well as antigen. Inhibitors of some early signaling enzymes stimulated by FceRI, including Syk tyrosine kinase and phosphoinositide 3-kinase, have little or no effect on this trafficking response. Other signaling pathways, including activation of phospholipase C and Ca2+ influx, do not appear to be necessary for the initiation of the outward trafficking response, but they contribute to maintaining the sustained phase of this process. Consistent with this, antigen-stimulated ruffles are labeled with FITC-cholera toxin B in a Ca2+-dependent manner. Thus, this trafficking response provides a mechanism by which an internal membrane pool can contribute to plasma membrane remodeling during stimulated membrane ruffling, cell motility, and phagocytosis.