Membrane Apposition Research Articles

The Immune Synapse: Past, Present, and Future.

Immunological synapses are specialized cell-cell junctions characterized by (1) close apposition of the immune cell membrane with the membrane of another cell driven by adaptive or innate immune recognition, (2) adhesion, (3) stability, and (4) directed secretion. This phenomenon was first recognized in the 1970s and the early 1980s through electron microscopy of ex vivo functioning immune cells. Progressive advances in fluorescence microscopy and molecular immunology in the past 20 years have led to rapid progress on understanding the modes of cell-cell interaction and underlying molecular events. This volume contains a diverse range of protocols that can be applied to the study of the immunological synapses and related immune cell junctions both in vitro and in vivo; and in disease settings in animal models and humans. We have also included chapters on critical molecular tools such as protein expression and mRNA electroporation that underpin or expand imaging approaches, although they are not specific to the study of immune synapses. We hope that these chapters will be of use to people entering the field as well as seasoned practitioners looking to expand their repertoire of methods.

Signaling hubs at ER/mitochondrial membrane associations

Signaling between organelles has profound implications for our understanding of organelle structural organization and regulatory processes. Close regional apposition of endoplasmic reticulum (ER) and mitochondrial membranes has been known for 50 years, but only in the past 20 years have scientists begun to unravel the nature and purpose of these quasi-synaptic contact points. At these sites of association, membranes have been shown to be of unique molecular composition, defining raft domains that are densely populated with membrane proteins, sphingolipids, and cholesterol. These associations are now referred to as mitochondrial associated membranes (MAMs). MAM domains mediate a complex array of cellular processes including; exchange of macromolecules, Ca2+ transfer, physical tethering, regulation of mitochondrial division, and signaling pathways that control autophagy and apoptosis. Dysfunction of MAM function is known to have profound cellular influence, and including the activation of several neurodegenerative disorders.

Abstract 13129: Acute Inhibition of Sodium Channel Beta Subunit (β1) -mediated Adhesion is Highly Proarrhythmic

Introduction: The sodium channel auxiliary subunit and cell adhesion molecule β1 (SCN1b) is enriched at cardiomyocyte intercalated disks (ID) along with the pore-forming subunit Na V 1.5. We recently demonstrated that the perinexus, an Na V 1.5-rich ID nanodomain located near connexin43 (Cx43) gap junctions (GJ), is important for cell-to-cell electrical coupling. Disrupting close membrane apposition within the perinexus was highly proarrhythmic. Hypothesis: β1-mediated adhesion is key to close membrane apposition within the perinexus; therefore, inhibiting β1-mediated adhesion may disrupt perinexal structure, impair conduction and precipitate arrhythmias. Methods and Results: Na V 1.5 and β1 localization within ID nanodomains was quantitatively assessed from super-resolution STochastic Optical Reconstruction Microscopy (STORM) images of en face IDs from adult guinea pig ventricles. Na V 1.5 and β1 were located 63±15 and 75±25 nm from Cx43 GJ; 31% of both Na V 1.5 and β1 localized to GJ perinexi. β1-mediated adhesion was acutely inhibited by βadp1, a novel peptide mimetic of β1’s putative extracellular adhesion domain. βadp1 decreased cellular adhesion as assessed by Electric Cell-Substrate Impedance Spectroscopy (ECIS) in β1-overexpressing 1610 cells in a dose-dependent manner but not in native 1610 cells. Perinexal intermembrane spacing as measured by Transmission Electron Microscopy (TEM) increased 3-fold from 17±1 nm in vehicle controls (0.25% DMSO) to 49±5 nm (p<0.05) in 100 μM βadp1-treated hearts; however, intermembrane spacing at other ID locations was unaltered (22±1 vs. 24±1 nm). Optical mapping revealed preferential slowing of transverse conduction (8.0±0.7 vs. 16.5±1.2 cm/s, p<0.05) and increased anisotropy (3.9±0.2 vs. 2.6±0.2, p<0.05) in βadp1-treated hearts relative to vehicle controls. Further, 3 of 4 βadp1-treated hearts exhibited spontaneous ventricular tachycardias. Conclusions: These data support a role for β1-mediated adhesion in maintaining close apposition between Na V 1.5-rich perinexal membranes. Importantly, acute inhibition of β1-mediated adhesion resulted in disruption of close membrane apposition within the GJ perinexus and precipitated severe conduction slowing and spontaneous arrhythmias.

Membrane Trafficking: An Endosome Tether Meets a Rab and Collapses

Long-range tethering is a ubiquitous recognition event preceding membrane fusion. A new study shows that Rab GTPase binding causes 'entropic collapse' of the coiled-coil endosome tether EEA1, driving membrane apposition and facilitating short-range interactions required for fusion.

The evolution of the placenta.

The very apt definition of a placenta is coined by Mossman, namely apposition or fusion of the fetal membranes to the uterine mucosa for physiological exchange. As such, it is a specialized organ whose purpose is to provide continuing support to the developing young. By this definition, placentas have evolved within every vertebrate class other than birds. They have evolved on multiple occasions, often within quite narrow taxonomic groups. As the placenta and the maternal system associate more intimately, such that the conceptus relies extensively on maternal support, the relationship leads to increased conflict that drives adaptive changes on both sides. The story of vertebrate placentation, therefore, is one of convergent evolution at both the macromolecular and molecular levels. In this short review, we first describe the emergence of placental-like structures in nonmammalian vertebrates and then transition to mammals themselves. We close the review by discussing the mechanisms that might have favored diversity and hence evolution of the morphology and physiology of the placentas of eutherian mammals.

AKT Signaling Prevailing in Mesenchymal Stromal Cells Modulates the Functionality of Hematopoietic Stem Cells via Intercellular Communication.

The AKT pathway plays an important role in various aspects of stem cell biology. However, the consequences of constitutive activation of AKT in mesenchymal stromal cells (MSCs) on the fate of hematopoietic stem cells (HSCs) were unknown. Here, we show that bone marrow-derived MSCs expressing a constitutively active AKT1 expand HSCs, but severely affect their functionality. Conversely, stromal cells with silenced AKT1 limit HSC proliferation, but boost their functionality. These effects were related to differential modulation of several important regulatory genes, in both, the cocultured HSCs and in the stromal cells themselves. The detrimental effect of stromal cells with constitutively activated AKT1 involved dynamin-dependent endocytosis, whereas the salutary effect of stromal cells devoid of AKT1 was mediated via GAP junctions. Constitutive activation of AKT1 led to deregulated formation of GAP junctions in the stromal cells, which consequently exhibited strikingly increased intercellular transfer of molecular cargo to the HSCs. Conversely, stromal cells with silenced AKT1 exhibited normal intercellular arrangement of GAP junctions at appositional membrane areas, and did not show aberrant intercellular transfer. Micro-vesicles isolated from conditioned media of the stromal cells not only mimicked the effect of these cells, but also showed stronger effects. This is perhaps the first report demonstrating that AKT1 signaling prevailing in the MSCs regulates HSC functionality through various intercellular communication mechanisms. These findings could have important implications in the use of MSCs in regenerative medicine. Stem Cells 2016;34:2354-2367.

Studying cytokinesis and midbody remnants using correlative light/scanning EM.

Cytokinesis is an essential step of cell proliferation leading to the physical separation of the dividing cells. Cytokinesis relies on both large scale and local scale cell shape changes, and terminates with the final abscission cut that requires close apposition of the plasma membrane. While furrow ingression is a prominent feature of the early phase of cytokinesis and is easy to visualize in all models, from dividing eggs to culture cells, the later steps of cytokinesis until abscission can be much more difficult to visualize. One key issue is to combine live-cell imaging over several hours and detailed, structural analysis of the cell shape changes in 3D, in particular at the time of cytokinetic abscission. Here, we describe the methodologies that we recently developed for studying cytokinetic abscission in human culture cells using live-cell phase-contrast microscopy, combined with correlative scanning electron microscopy. This allows us to determine the membrane surface and underlying cytoskeleton of the intercellular bridge with unprecedented precision and to determine the fate of the midbody remnant after abscission.

SNARE zippering.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly conserved set of membrane-associated proteins that mediate intracellular membrane fusion. Cognate SNAREs from two separate membranes zipper to facilitate membrane apposition and fusion. Though the stable post-fusion conformation of SNARE complex has been extensively studied with biochemical and biophysical means, the pathway of SNARE zippering has been elusive. In this review, we describe some recent progress in understanding the pathway of SNARE zippering. We particularly focus on the half-zippered intermediate, which is most likely to serve as the main point of regulation by the auxiliary factors.

Identification and functional analysis of two novel connexin 50 mutations associated with autosome dominant congenital cataracts.

Autosomal dominant congenital cataracts (ADCC) are clinically and genetically heterogeneous diseases. The present study recruited two Chinese families with bilateral nuclear cataract or zonular pulverulent phenotype. Direct sequencing of candidate genes identified two novel missense mutations of Cx50, Cx50P59A (c.175C > G) and Cx50R76H (c.227G > A), both co-segregated well with all affected individuals. Bioinformatics analysis predicted deleterious for both mutations. Functional and cellular behaviors of wild type and mutant Cx50 examined by stably transfecting recombinant systems revealed similar protein expression levels. Protein distribution pattern by fluorescence microscopy showed that Cx50R76H localized at appositional membranes forming gap junctions with enormous cytoplasmic protein accumulation, whereas the Cx50P59A mutation was found inefficient at forming detectable plaques. Cell growth test by MTT assay showed that induction of Cx50P59A decreased cell viability. Our study constitutes the first report that the Cx50P59A and Cx50R76H mutations are associated with ADCC and expands the mutation spectrum of Cx50 in association with congenital cataracts. The genetic, cellular, and functional data suggest that the altered intercellular communication governed by mutated Cx50 proteins may act as the molecular mechanism underlying ADCC, which further confirms the role of Cx50 in the maintenance of human lens transparency.

Novel Storm-Based Quantitative Assessment of Relative Protein Localization Reveals New Role for Sodium Channel β1 Subunit in Cardiac Conduction

We recently demonstrated that cardiac sodium channels (Nav1.5) localized within gap junction (GJ) -adjacent microdomains within the intercalated disc (ID) may enable ephaptic coupling between cardiac myocytes. Further we demonstrated conduction slowing and elevated arrhythmia risk secondary to disruption of close membrane apposition (<10nm) within these microdomains. Confocal micrographs of guinea pig ventricular myocardium revealed expression of β1 (SCN1b), a sodium channel auxiliary subunit and cell adhesion molecule, throughout the interplicate regions of the ID. Therefore, we hypothesized that β1-mediated adhesion may be key to close membrane apposition within Nav1.5-rich ID microdomains and thereby, to ephaptic coupling. In order to assess β1-localization within the ID, we analyzed STORM images of guinea pig ventricular sections immunolabeled for β1 along with Cx43 and N-cadherin (N-Cad) using custom algorithms. Overall, 16±2% of Cx43 clusters overlapped with a β1 cluster while 30±3% had a β1 cluster <200nm away, with a median distance of 55±6nm between Cx43 and β1. In contrast, only 7±3% of N-Cad overlapped with β1 while 16±2% had β1 <200nm away (p<0.05 vs. Cx43-β1), with a median distance of 154±98nm between N-Cad and β1. These data suggest preferential β1 localization adjacent Cx43 GJ relative (interplicate ID regions) to N-cadherin (plicate ID regions). Next we examined β1-mediated adhesion using electric cell-substrate impedance spectroscopy (ECIS): 1610 cells heterologously overexpressing β1 revealed 3-fold higher barrier function relative to native 1610 cells. Further, βadp1, a peptide mimetic of the β1 adhesion domain, inhibited barrier function in dose dependent fashion in β1-overexpressing 1610 cells but not in native 1610 cells. Taken together our results support a role for β1-mediated adhesion in modulating ephaptic coupling in the heart and highlight it as a potential target for novel anti-arrhythmic therapy.

Fluorescence Density Mapping: Extending the Possibilities of TIRFM to Study PM-ER Junctions

Total internal reflection microscopy (TIRFM) is an established method to study protein localization, dynamics and interactions of plasmalemmal as well as sub-plasmalemmal cellular structures by fluorescence microscopy. TIRFM features combine high temporal and spatial resolution and thus, in many applications, exceed the possibilities of confocal microscopy. TIRFM has been established as a versatile method to study vesicle traffic, fusion and docking as well as dynamics of proteins within PM-ER junctions. Increasing experimental evidence demonstrates a pivotal role of PM-ER junctional areas in a variety of cellular processes including lipid- and calcium homeostasis which crucially involve close apposition and physical contact between intracellular compartments like the ER and the PM. Microscopic characterization of properties and architecture of PM-ER junctions is currently limited to the use of fluorescent fusion proteins related to ER resident signaling molecules. These markers bear the potential to interfere with native junctional properties. Here we introduce a simple, novel TIRFM approach, which allows to study basolateral junctional areas of cells based on mapping the optical density of small cytosolic fluorophores within the evanescent field. As optical density of small diffusible fluorophores is reduced in junctional membrane appositions, TIRFM can be employed to visualize ER-PM junctions without overexpression of specific ER protein markers and this method enables unprecendeted insight into the dynamics of trans-compartimental signaling. We provide proof of concept by utilizing this new method for in-depth analysis of the role of PM-ER junctions in mast cell calcium signaling. Our results reveal a surprisingly dense, structurally well defined compartment that provides a dynamic platform for key signaling molecules.

Mechanobiology in Cell-Cell Fusion: Roles of Myosin II and Spectrin in Mechanosensing and Force Generation during Cell-Cell Fusion

Fusion of one cell into another cell engages several biological processes including cell adhesion molecule interaction, signal transduction, cytoskeleton rearrangement and cell membrane fusion. Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers which eventually merge into a single bilayer. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an “attacking” cell drills actin-based finger-like protrusions into the “receiving” cell to promote fusion. In the studies presented here, we revealed that the receiving cell utilizes mechanosensory responses to build up cytoskeletal structures at the site of fusion (fusogenic synapse) in response to invasion by the attacking cell. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusogenic synapse in the receiving cell, and the mechanosensory response of MyoII is amplified by cell adhesion molecule-initiated and GTPase/kinase-mediated chemical signaling. The accumulated MyoII, in turn, increases cortical tension which provides resistance on the receiving cell cortex and promotes fusion pore formation. In a similar way, the receiving cell accumulates spectrin at the fusogenic synapse. Spectrin functions as a molecular barrier which concentrates cell adhesion molecules at the fusogenic synapse. The concentrated cell adhesion molecules, in turn, induce formation of effective invasive protrusions in the attacking cell which exert pushing force into the receiving cell. We propose that the protrusive and resisting forces from two fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome the energy barriers for membrane apposition and drives cell membrane fusion.

Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse.

ABSTRACTMonocyte-derived cells use an extracellular, acidic, lytic compartment (a lysosomal synapse) for initial degradation of large objects or species bound to the extracellular matrix. Akin to osteoclast degradation of bone, extracellular catabolism is used by macrophages to degrade aggregates of low density lipoprotein (LDL) similar to those encountered during atherogenesis. However, unlike osteoclast catabolism, the lysosomal synapse is a highly dynamic and intricate structure. In this study, we use high resolution three dimensional imaging to visualize compartments formed by macrophages to catabolize aggregated LDL. We show that these compartments are topologically complex, have a convoluted structure and contain sub-regions that are acidified. These sub-regions are characterized by a close apposition of the macrophage plasma membrane and aggregates of LDL that are still connected to the extracellular space. Compartment formation is dependent on local actin polymerization. However, once formed, compartments are able to maintain a pH gradient when actin is depolymerized. These observations explain how compartments are able to maintain a proton gradient while remaining outside the boundaries of the plasma membrane.

Abstract 11138: Superresolution Studies of Sodium Channels Within Intercalated Disk Microdomains Suggest Novel Arrhythmia Mechanism

Pore-forming (Nav1.5) and auxiliary (β1; SCN1b) subunits of cardiac sodium channels are enriched at the cardiomyocyte intercalated disk (ID). Mathematical models suggest that this may facilitate conduction via ephaptic mechanisms. We recently demonstrated Nav1.5 enrichment (gSTED superresolution microscopy) and close membrane apposition (&lt;10 nm; electron microscopy) within the perinexus, a microdomain surrounding connexin43 (Cx43) gap junctions (GJ). These data identified the perinexus as a candidate structure for the cardiac ephapse. Further studies using gSTED and STORM superresolution microscopy revealed Nav1.5 and β1 enrichment within ID regions not containing dense clusters of Cx43 and N-Cadherin. Notably, both were identified within the perinexus: Overall, 22% of Nav1.5 &amp; β1 were located within perinexal regions while only 2 and 5% respectively overlapped with Cx43 clusters. Importantly, acute interstitial edema (AIE) increased intermembrane distance at perinexal, but not at non-perinexal sites in adult guinea pig myocardium. Functionally, this correlated with decreased transverse conduction velocity (CV-T; 15.2±0.3 vs. 19.6±0.1cm/s) and increased anisotropic ratio (AR; 3.0±0.2 vs. 2.8±0.1) relative to control, in perfused guinea pig ventricles. Nav1.5 blockade (0.5 μM flecainide) by itself decreased CV (18%) without changing AR. However, Nav1.5 inhibition during AIE preferentially decreased CV-T (13.0±0.6cm/s), increased AR (3.3±0.2) and increased spontaneous arrhythmias (7/9 vs. 4/11) compared to AIE alone. Notably, only a computer model including ephaptic coupling and the ID localization of Nav1.5 could recapitulate these results. Next we investigated the role of β1 in ephaptic coupling: Electrical cell-substrate impedance spectroscopy of 1610 cells heterologously overexpressing β1 revealed 3-fold higher paracellular resistance relative to native 1610 cells. These data along with the known cell adhesion function of β1 in neural tissue suggest that β1-mediated adhesion may facilitate close membrane apposition within the perinexus. Taken together, our results identify β1-mediated adhesion as a novel determinant of anisotropic conduction and potential antiarrhythmic target.

A family of sterol sensors/transporters at membrane contact sites: Regulation of ORP-VAP complexes by sterol ligands

The functions of lipids as signaling compounds and their interorganelle transport are topics that have recently moved to the center stage of cell biological and biomedical research. In this context the concept of membrane contact sites (MCS), sites of close apposition of organelle membranes, is an emerging major theme. An increasing number of studies have revealed crucial roles of such contacts as sites with prominent functions in interorganelle lipid transport and metabolism as well as signaling nodes, and molecular machineries operating at MCSs are being identified (reviewed by ). One of the protein families reported to localize at membrane contacts are the Oxysterol-binding protein/OSBP-related proteins (ORPs), sterol/phospholipid binding proteins implicated in a variety of cellular functions: lipid metabolism and transport, vesicle transport and signaling cascades .

Pannexin-1 channels and their emerging functions in cardiovascular diseases.

Pannexin-1, Pannexin-2, and Pannexin-3 are three members of the Pannexin family of channel-forming glycoprotein. Their primary function is defined by their ability to form single-membrane channels. Pannexin-1 ubiquitously exists in many cells and organs throughout the body and is specially distributed in the circulatory system, while the expressions of Pannexin-2 and Pannexin-3 are mostly restricted to organs and tissues. Pannexin-1 oligomers have been shown to be functional single membrane channels that connect intracellular and extracellular compartments and are not intercellular channels in appositional membranes. The physiological functions of Pannexin-1 are to link to the adenosine triphosphate efflux that acts as a paracrine signal, and regulate cellular inflammasomes in a variety of cell types under physiological and pathophysiological conditions. However, there are still many functions to be explored. This review summarizes recent reports and discusses the role of Pannexin-1 in cardiovascular diseases, including ischemia, arrhythmia, cardiac fibrosis, and hypertension. Pannexin-1 has been suggested as an exciting, clinically relevant target in cardiovascular diseases.

Mechanical Tension Drives Cell Membrane Fusion

Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an "attacking" cell drills finger-like protrusions into the "receiving" cell to promote cell fusion. Here, we show that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its invasive fusion partner. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusion site, and the mechanosensory response of MyoII is amplified by chemical signaling initiated by cell adhesion molecules. The accumulated MyoII, in turn, increases cortical tension and promotes fusion pore formation. We propose that the protrusive and resisting forces from fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome energy barriers for membrane apposition and drives cell membrane fusion.

Sodium channels in the Cx43 gap junction perinexus may constitute a cardiac ephapse: an experimental and modeling study.

It has long been held that electrical excitation spreads from cell-to-cell in the heart via low resistance gap junctions (GJ). However, it has also been proposed that myocytes could interact by non-GJ-mediated “ephaptic” mechanisms, facilitating propagation of action potentials in tandem with direct GJ-mediated coupling. We sought evidence that such mechanisms contribute to cardiac conduction. Using super-resolution microscopy, we demonstrate that Nav1.5 is localized within 200 nm of the GJ plaque (a region termed the perinexus). Electron microscopy revealed close apposition of adjacent cell membranes within perinexi suggesting that perinexal sodium channels could function as an ephapse, enabling ephaptic cell-to-cell transfer of electrical excitation. Acute interstitial edema (AIE) increased intermembrane distance at the perinexus and was associated with preferential transverse conduction slowing and increased spontaneous arrhythmia incidence. Inhibiting sodium channels with 0.5 μM flecainide uniformly slowed conduction, but sodium channel inhibition during AIE slowed conduction anisotropically and increased arrhythmia incidence more than AIE alone. Sodium channel inhibition during GJ uncoupling with 25 μM carbenoxolone slowed conduction anisotropically and was also highly proarrhythmic. A computational model of discretized extracellular microdomains (including ephaptic coupling) revealed that conduction trends associated with altered perinexal width, sodium channel conductance, and GJ coupling can be predicted when sodium channel density in the intercalated disk is relatively high. We provide evidence that cardiac conduction depends on a mathematically predicted ephaptic mode of coupling as well as GJ coupling. These data suggest opportunities for novel anti-arrhythmic therapies targeting noncanonical conduction pathways in the heart.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-014-1675-z) contains supplementary material, which is available to authorized users.

Cytomics of the tumour microenvironment: therapeutic targeting (keynote lecture)

There is a growing appreciation of the influence of both the tumour microenvironment and the dynamic nature of the cellular micro-community in determining tumour progression and therapeutic resistance. Changes in cell heterogeneity, resulting from time-varying changes in the microenvironment, present complex challenges for cancer therapy. For example, the tumour micro-community comprises multiple levels of cellular interactions overlaid with pervading influences such as variation in the levels of oxygenation. Molecular targeting combined with agents that target the microenvironment offer novel approaches. The presentation focused on two examples supported by cytometric techniques. First, dissecting the influences of cellular interactions on tumour progression-related cell behavior. An in vitro, non-adherent small cell lung cancer (SCLC) cell model system enabled the engineering of cellular micro-communities. SCLC cells show surface expression of a potential drug target, polysialylated neural cell adhesion molecule (NCAM; NCAM1 or CD56). The extensive addition of polysialic acid (polySia) to NCAM in SCLC decreases membrane apposition, cell adhesion and enables cell migration. Cytometric studies reveal heterogeneity in the expression of polySia in SCLC cell populations that can be related to proliferation potential both in vitro and in vivo. In constructed 3-D micro-communities, polySia expression was driven in non-expressing variants by the presence of over-expressing subpopulations. This suggests that a degree of homeostasis operates with implications for polySia-NCAM-targeted therapies. The second example addressed the targeting of hypoxia for therapeutic advantage using a novel hypoxia activated prodrug (HAP) technology. Hypoxia in tumour regions, arising from loss of vascularity, generates drug resistant phenotypes. Targeting hypoxic tumour cells using HAPs has been previously attempted using a diverse group of chemicals based mainly on structures with alkylating functionality. Crucially most of these agents are reduced in single electron steps by a number of enzymes including cytochrome P450 reductase. This produces prodrug conversion to species that bind covalently to DNA. Critically, the cytotoxins generated do not persist such that targeted cells upon re-oxygenation may contribute to tumour regrowth. We have developed the concept of a unidirectional hypoxia activated prodrug (uHAP), building upon experience with di-N-oxides of tertiary amine anthraquinones, to develop the novel agent OCT1002. The uHAP represents a distinct class of HAP, reduced to a cytotoxic species through an irreversible two-step process, each involving a two-electron reduction. The bioactive reduction products inhibit DNA processing and topoisomerase II activity, selectively causing cancer cell death. The presentation explored the application of cytometric methods to reveal the mechanisms of action of OCT1002 and to provide an insight into the targeting of hypoxic regions of prostate and pancreatic cancers. The above examples highlight the importance of cell-based approaches in the development of therapeutic strategies that address changing patterns of target presentation in different cellular microenvironments. [PJS acknowledges the contributions of Prof. Rachel Errington (Cardiff University); Prof. Stephanie McKeown (University of Ulster) and Robert Falconer (Bradford University)].

Superresolution Microscopy Reveals Sodium Channel Localization within Intercalated Disk Microdomains: Implications for Ephaptic Coupling

Pore-forming (Nav1.5) and auxiliary (β1; SCN1b) subunits of cardiac sodium channels are enriched at the cardiomyocyte intercalated disk (ID). Mathematical models suggest that this may facilitate conduction via ephaptic mechanisms. We previously demonstrated anisotropic conduction slowing during acute interstitial edema (AIE), possibly due to weakened ephaptic coupling. Here we assessed Nav1.5 and β1 localization to ID microdomains using electron microscopy (EM) and super-resolution microscopy (gSTED, STORM) and used optical mapping and computer modeling to investigate the implications for ephaptic conduction in the heart. gSTED and STORM revealed Nav1.5 and β1 enrichment within ID regions not containing dense clusters of Cx43 and N-Cadherin. Notably, both were identified within the perinexus, a microdomain surrounding Cx43 gap junctions. Overall, 22% of Nav1.5 was located within perinexal regions while only 2% was within Cx43 clusters. EM revealed closer membrane apposition at perinexal ( 10nm) under control conditions. AIE increased intermembrane distance at perinexal, but not at non-perinexal sites. Functionally, this correlated with decreased transverse conduction velocity (CV-T; 15.2±0.3 vs. 19.6±0.1cm/s) and increased anisotropic ratio (AR; 3.0±0.2 vs. 2.8±0.1) relative to control, in perfused guinea pig ventricles. Next, we investigated AIE effects on Nav1.5 function in conduction. Nav1.5 blockade (0.5 µM flecainide) by itself decreased CV (18%) without changing AR. However, Nav1.5 inhibition during AIE preferentially decreased CV-T (13.0±0.6cm/s), increased AR (3.3±0.2) and increased spontaneous arrhythmias (7/9 vs. 4/11) compared to AIE alone. Notably, only a computer model including ephaptic coupling and the ID localization of Nav1.5 could recapitulate these results. In summary, sodium channel complexes localized to ID microdomains such as the perinexus may enable ephaptic conduction in the heart. Further, Nav1.5 functional availability and perinexal membrane spacing emerge as novel determinants of anisotropic conduction.