A palmitoylation code controls PI4KIIIα complex formation and PI(4,5)P2 homeostasis at the plasma membrane.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the major enzyme responsible for generating phosphatidylinositol (4)-phosphate [PI(4)P] at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-cysteine motif in EFR3B (Cys5, Cys7 and Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B 'lipoforms' affects both interactions between EFR3B and TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] resynthesis following phospholipase C signaling, and EFR3B partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code involved in controlling protein-protein and protein-lipid interactions that affect a plasma membrane-resident lipid biosynthetic pathway.

Lipid-based and protein-based interactions synergize transmembrane signaling stimulated by antigen clustering of IgE receptors.

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-FcεRI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered FcεRI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and FcεRI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of FcεRI that it initially phosphorylates for amplified activity. Net phosphorylation above a nonfunctional threshold is achieved in the stimulated state but not in the resting state, and current evidence supports the hypothesis that this relies on Ag crosslinking to disrupt a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to suprathreshold phosphorylation of Ag-crosslinked IgE-FcεRI in live rat basophilic leukemia mast cells. Our high-precision diffusion measurements by imaging fluorescence correlation spectroscopy on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains in the membrane inner leaflet and concomitant sharpening of segregation from liquid disordered (Ld)-like regions. With other structural variants, we determine that lipid-based interactions are essential for access by Lyn, leading to phosphorylation of and protein-based binding to clustered FcεRI. By contrast, TM tyrosine phosphatase, PTPα, is excluded from these regions due to its Ld-preference and steric exclusion of TM segments. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells.

Lipid-driven interleaflet coupling of plasma membrane order regulates FcεRI signaling in mast cells.

Interleaflet coupling-the influence of one leaflet on the properties of the opposing leaflet-is a fundamental plasma membrane organizational principle. This coupling is proposed to participate in maintaining steady-state biophysical properties of the plasma membrane, which in turn regulates some transmembrane signaling processes. A prominent example is antigen (Ag) stimulation of signaling by clustering transmembrane receptors for immunoglobulin E (IgE), FcεRI. This transmembrane signaling depends on the stabilization of ordered regions in the inner leaflet for sorting of intracellular signaling components. The resting inner leaflet has a lipid composition that is generally less ordered than the outer leaflet and that does not spontaneously phase separate in model membranes. We propose that interleaflet coupling can mediate ordering and disordering of the inner leaflet, which is poised in resting cells to reorganize upon stimulation. To test this in live cells, we first established a straightforward approach to evaluate induced changes in membrane order by measuring inner leaflet diffusion of lipid probes by imaging fluorescence correlation spectroscopy, by imaging fluorescence correlation spectroscopy (ImFCS), before and after methyl-α-cyclodexrin (mαCD)-catalyzed exchange of outer leaflet lipids (LEX) with exogenous order- or disorder-promoting phospholipids. We examined the functional impact of LEX by monitoring two Ag-stimulated responses: recruitment of cytoplasmic Syk kinase to the inner leaflet and exocytosis of secretory granules (degranulation). Based on the ImFCS data in resting cells, we observed global increase or decrease of inner leaflet order when outer leaflet is exchanged with order- or disorder-promoting lipids, respectively. We find that the degree of both stimulated Syk recruitment and degranulation correlates positively with LEX-mediated changes of inner leaflet order in resting cells. Overall, our results show that resting-state lipid ordering of the outer leaflet influences the ordering of the inner leaflet, likely via interleaflet coupling. This imposed lipid reorganization modulates transmembrane signaling stimulated by Ag clustering of IgE-FcεRI.

My path in the company of chemistry.

Experiencing the honor of this international recognition in chemistry, I wonder how this came to be. I reflect on my imperfect but rewarding path to where I am now, and on those who have helped me along the way.

Going Viral: Researching Safely on Social Media.

Safety issues for researchers conducting and disseminating research on social media have been inadequately addressed in institutional policies and practice globally, despite posing significant challenges to research staff and student well-being. In the context of the COVID-19 pandemic and given the myriad of advantages that web-based platforms offer researchers over traditional recruitment, data collection, and research dissemination methods, developing a comprehensive understanding of and guidance on the safe and effective conduct of research in web-based spaces has never been more pertinent. In this paper, we share our experience of using social media to recruit participants for a study on abortion stigma in Australia, which brought into focus the personal, professional, and institutional risks associated with conducting web-based research that goes viral. The lead researcher (KV), a postgraduate student, experienced a barrage of harassment on and beyond social media. The supportive yet uncoordinated institutional response highlighted gaps in practice, guidance, and policy relating to social media research ethics, researcher safety and well-being, planning for and managing web-based and offline risk, and coordinated organizational responses to adverse events. We call for and provide suggestions to inform the development of training, guidelines, and policies that address practical and ethical aspects of using social media for research, mental and physical health and safety risks and management, and the development of coordinated and evidence-based institutional- and individual-level responses to cyberbullying and harassment. Furthermore, we argue the case for the urgent development of this comprehensive guidance around researcher safety on the web, which would help to ensure that universities have the capacity to maximize the potential of social media for research while better supporting the well-being of their staff and students.

Abortion at the edges: Politics, practices, performances.

This article provides a brief overview of the state of discourse, politics and provision of abortion in the Anglophone West, including developments in the wake of the COVID-19 pandemic. It then surveys three promising directions for feminist abortion scholarship. The first is work inspired by the Reproductive Justice Movement, that points to the intersectional axes of inequality that shape abortion discourse and position us in relation to reproductive choice and access issues. The second is work that examines the particularity of the constitution of the aborting body, reflecting the particularity of the pregnant body. This is a specific body, with a specific history; abortion discourse draws from and makes a significant contribution to the meaning and lived experience of this body. The third area of scholarship we highlight is that which seeks to amplify the meaning of abortion as a social good. Much abortion scholarship is attuned to a critique of negative aspects of abortion-from its representation in popular culture to restrictive law and access issues. This is critical work but/and the performative nature of abortion scholarship, like all discourse, means that it can amplify the association of negativity with abortion. The article concludes by introducing the articles contained in the special section of Women's Studies International Forum, 'Abortion at the edges: Politics, practices, performances'.

Molecular anatomy of the early events in STIM1 activation - oligomerization or conformational change?

Decreased luminal endoplasmic reticulum (ER) Ca2+ concentration triggers oligomerization and clustering of the ER Ca2+ sensor STIM1 to promote its association with plasma membrane Orai1 Ca2+ channels leading to increased Ca2+ influx. A key step in STIM1 activation is the release of its SOAR domain from an intramolecular clamp formed with the STIM1 first coiled-coil (CC1) region. Using a truncated STIM1(1-343) molecule that captures or releases the isolated SOAR domain depending on luminal ER Ca2+ concentrations, we analyzed the early molecular events that control the intramolecular clamp formed between the CC1 and SOAR domains. We found that STIM1 forms constitutive dimers, and its CC1 domain can bind the SOAR domain of another STIM1 molecule in trans. Artificial oligomerization failed to liberate the SOAR domain or activate STIM1 unless the luminal Ca2+-sensing domains were removed. We propose that the release of SOAR from its CC1 interaction is controlled by changes in the orientation of the two CC1 domains in STIM1 dimers. Ca2+ unbinding in the STIM1 luminal domains initiates the conformational change allowing SOAR domain liberation and clustering, leading to Orai1 channel activation.

Rab11 Regulates the Mast Cell Exocytic Response.

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 ß-hexosaminidase (ß-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 ß-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.

Roles for lipid heterogeneity in immunoreceptor signaling.

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.

Mechanisms of epidermal growth factor receptor signaling as characterized by patterned ligand activation and mutational analysis.

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.

Roles for Ca2+ mobilization and its regulation in mast cell functions: recent progress.

Ca(2+)mobilization in response to cross-linking of IgE bound to its high affinity receptor, FcεRI, on mast cells is central to immune allergic responses. Stimulated tyrosine phosphorylation caused by this cross-linking activates store-operated Ca(2+)entry that results in sustained Ca(2+)oscillations dependent on Rho family GTPases and phosphoinositide synthesis. Coupling of the endoplasmic reticulum (ER) Ca(2+)sensor, stromal interaction molecule 1 (STIM1), to the Ca(2+)-selective channel, Orai1, is regulated by these elements and depends on membrane organization, both at the plasma membrane and at the ER. Mitochondria also contribute to the regulation of Ca(2+)mobilization, and we describe recent evidence that the ER membrane protein vesicle-associated membrane protein-associated protein (VAP) plays a significant role in the coupling between ER and mitochondria in this process. In addition to granule exocytosis, Ca(2+)mobilization in these cells also contributes to stimulated outward trafficking of recycling endosomes and to antigen-stimulated chemotaxis, and it is pathologically regulated by protozoan parasitic invasion.

Polyunsaturated fatty acids inhibit stimulated coupling between the ER Ca(2+) sensor STIM1 and the Ca(2+) channel protein Orai1 in a process that correlates with inhibition of stimulated STIM1 oligomerization.

Polyunsaturated fatty acids (PUFAs) have been found to be effective inhibitors of cell signaling in numerous contexts, and we find that acute addition of micromolar PUFAs such as linoleic acid effectively inhibit of Ca(2+) responses in mast cells stimulated by antigen-mediated crosslinking of FcεRI or by the SERCA pump inhibitor, thapsigargin. In contrast, the saturated fatty acid, stearic acid, with the same carbon chain length as linoleic acid does not inhibit these responses. Consistent with this inhibition of store-operated Ca(2+) entry (SOCE), linoleic acid inhibits antigen-stimulated granule exocytosis to a similar extent. Using the fluorescently labeled plasma membrane Ca(2+) channel protein, AcGFP-Orai1, together with the labeled ER Ca(2+) sensor protein, STIM1-mRFP, we monitor stimulated coupling of these proteins that is essential for SOCE with a novel spectrofluorimetric resonance energy transfer method. We find effective inhibition of this stimulated coupling by linoleic acid that accounts for the inhibition of SOCE. Moreover, we find that linoleic acid induces some STIM1-STIM1 association, while inhibiting stimulated STIM1 oligomerization that precedes STIM1-Orai1 coupling. We hypothesize that linoleic acid and related PUFAs inhibit STIM1-Orai1 coupling by a mechanism that involves perturbation of ER membrane structure, possibly by disrupting electrostatic interactions important in STIM1 oligomerization. Thisarticle is part of a Special Issue entitled Tools to study lipid functions.

A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase IIIα is important for protein trafficking from the endoplasmic reticulum to the plasma membrane.

BACKGROUND: Biosynthetic trafficking of receptors and other membrane-associated proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) underlies the capacity of these proteins to participate in crucial cellular roles. Phosphoinositides have been shown to mediate distinct biological functions in cells, and phosphatidylinositol 4-phosphate (PI4P), in particular, has emerged as a key regulator of biosynthetic trafficking. RESULTS: To investigate the source of PI4P that orchestrates trafficking events, we developed a novel flow cytometry based method to monitor biosynthetic trafficking of transiently transfected proteins. We demonstrated that our method can be used to assess the trafficking of both type-1 transmembrane and GPI-linked proteins, and that it can accurately monitor the pharmacological disruption of biosynthetic trafficking with brefeldin A, a well-documented inhibitor of early biosynthetic trafficking. Furthermore, utilizing our newly developed method, we applied pharmacological inhibition of different isoforms of PI 4-kinase to reveal a role for a distinct pool of PI4P, synthesized by PI4KIIIα, in ER-to-PM trafficking. CONCLUSIONS: Taken together, these findings provide evidence that a specific pool of PI4P plays a role in biosynthetic trafficking of two different classes of proteins from the ER to the Golgi complex. Furthermore, our simple, flow cytometry-based biosynthetic trafficking assay can be widely applied to the study of multiple classes of proteins and varied pharmacological and genetic perturbations.

Medical abortion in Australia: a short history.

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.

Spatially defined EGF receptor activation reveals an F-actin-dependent phospho-Erk signaling complex.

We investigated the association of signaling proteins with epidermal growth factor (EGF) receptors (EGFR) using biotinylated EGF bound to streptavidin that is covalently coupled in an ordered array of micron-sized features on silicon surfaces. Using NIH-3T3 cells stably expressing EGFR, we observe concentration of fluorescently labeled receptors and stimulated tyrosine phosphorylation that are spatially confined to the regions of immobilized EGF and quantified by cross-correlation analysis. We observe recruitment of phosphorylated paxillin to activated EGFR at these patterned features, as well as ß1-containing integrins that preferentially localize to more peripheral EGF features, as quantified by radial fluorescence analysis. In addition, we detect recruitment of EGFP-Ras, MEK, and phosphorylated Erk to patterned EGF in a process that depends on F-actin and phosphoinositides. These studies reveal and quantify the coformation of multiprotein EGFR signaling complexes at the plasma membrane in response to micropatterned growth factors.

Mutations in the polybasic juxtamembrane sequence of both plasma membrane- and endoplasmic reticulum-localized epidermal growth factor receptors confer ligand-independent cell transformation.

Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling.

Generalized platform for antibody detection using the antibody catalyzed water oxidation pathway.

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.

Spatiotemporal resolution of mast cell granule exocytosis reveals correlation with Ca2+ wave initiation.

Mast cell activation initiated by antigen-mediated crosslinking of IgE receptors results in stimulated exocytosis of secretory lysosomes in the process known as degranulation. Much has been learned about the molecular mechanisms important for this process, including the crucial role of Ca(2+) mobilization, but spatio-temporal relationships between stimulated Ca(2+) mobilization and granule exocytosis are incompletely understood. Here we use a novel imaging-based method that uses fluorescein isothiocyanate (FITC)-dextran as a reporter for granule exocytosis in RBL mast cells and takes advantage of the pH sensitivity of FITC. We demonstrate the selectivity of FITC-dextran, accumulated by fluid-phase uptake, as a marker for secretory lysosomes, and we characterize its capacity to delineate different exocytotic events, including full fusion, kiss-and-run transient fusion and compound exocytosis. Using this method, we find strong dependence of degranulation kinetics on the duration of cell to substrate attachment. We combine imaging of degranulation and Ca(2+) dynamics to demonstrate a spatial relationship between the sites of Ca(2+) wave initiation in extended cell protrusions and exocytosis under conditions of limited antigen stimulation. In addition, we find that the spatially proximal Ca(2+) signaling and secretory events correlate with participation of TRPC1 channels in Ca(2+) mobilization.

Overcoming Barriers Associated with Oral Delivery of Differently Sized Fluorescent Core-Shell Silica Nanoparticles.

Oral delivery, while a highly desirable form of nanoparticle-drug administration, is limited by challenges associated with overcoming several biological barriers. Here, the authors study how fluorescent and poly(ethylene glycol)-coated (PEGylated) core-shell silica nanoparticles sized 5 to 50 nm interact with major barriers including intestinal mucus, intestinal epithelium, and stomach acid. From imaging fluorescence correlation spectroscopy studies using quasi-total internal reflection fluorescence microscopy, diffusion of nanoparticles through highly scattering mucus is progressively hindered above a critical hydrodynamic size around 20 nm. By studying Caco-2 cell monolayers mimicking the intestinal epithelia, it is observed that ultrasmall nanoparticles below 10 nm diameter (Cornell prime dots, [C' dots]) show permeabilities correlated with high absorption in humans from primarily enhanced passive passage through tight junctions. Particles above 20 nm diameter exclusively show active transport through cells. After establishing C' dot stability in artificial gastric juice, in vivo oral gavage experiments in mice demonstrate successful passage through the body followed by renal clearance without protein corona formation. Results suggest C' dots as viable candidates for oral administration to patients with a proven pathway towards clinical translation and may generate renewed interest in examining silica as a food additive and its effects on nutrition and health.