Response Of Junctions Research Articles

Titanium nanotopography enhances mechano-response of osteocyte three-dimensional network toward osteoblast activation

The bone turnover capability influences the acquisition and maintenance of osseointegration. The architectures of osteocyte three-dimensional (3D) networks determine the direction and activity of bone turnover through osteocyte intercellular crosstalk, which exchanges prostaglandins through gap junctions in response to mechanical loading. Titanium nanosurfaces with anisotropically patterned dense nanospikes promote the development of osteocyte lacunar-canalicular networks. We investigated the effects of titanium nanosurfaces on intercellular network development and regulatory capabilities of bone turnover in osteocytes under cyclic compressive loading. MLO-Y4 mouse osteocyte-like cell lines embedded in type I collagen 3D gels on titanium nanosurfaces promoted the formation of intercellular networks and gap junctions even under static culture conditions, in contrast to the poor intercellular connectivity in machined titanium surfaces. The osteocyte 3D network on the titanium nanosurfaces further enhanced gap junction formation after additional culturing under cyclic compressive loading simulating masticatory loading, beyond the degree observed on machined titanium surfaces. A prostaglandin synthesis inhibitor cancelled the dual effects of titanium nanosurfaces and cyclic compressive loading on the upregulation of gap junction-related genes in the osteocyte 3D culture. Supernatants from osteocyte monolayer culture on titanium nanosurfaces promoted osteocyte maturation and intercellular connections with gap junctions. With cyclic loading, titanium nanosurfaces induced expression of the regulatory factors of bone turnover in osteocyte 3D cultures, toward higher osteoblast activation than that observed on machined surfaces. Titanium nanosurfaces with anisotropically patterned dense nanospikes promoted intercellular 3D network development and regulatory function toward osteoblast activation in osteocytes activated by cyclic compressive loading, through intercellular crosstalk by prostaglandin.

Notch signaling regulates UNC5B to suppress endothelial proliferation, migration, junction activity, and retinal plexus branching

Notch signaling guides vascular development and function by regulating diverse endothelial cell behaviors, including migration, proliferation, vascular density, endothelial junctions, and polarization in response to flow. Notch proteins form transcriptional activation complexes that regulate endothelial gene expression, but few of the downstream effectors that enable these phenotypic changes have been characterized in endothelial cells, limiting our understanding of vascular Notch activities. Using an unbiased screen of translated mRNA rapidly regulated by Notch signaling, we identified novel in vivo targets of Notch signaling in neonatal mouse brain endothelium, including UNC5B, a member of the netrin family of angiogenic-regulatory receptors. Endothelial Notch signaling rapidly upregulates UNC5B in multiple endothelial cell types. Loss or gain of UNC5B recapitulated specific Notch-regulated phenotypes. UNC5B expression inhibited endothelial migration and proliferation and was required for stabilization of endothelial junctions in response to shear stress. Loss of UNC5B partially or wholly blocked the ability of Notch activation to regulate these endothelial cell behaviors. In the developing mouse retina, endothelial-specific loss of UNC5B led to excessive vascularization, including increased vascular outgrowth, density, and branchpoint count. These data indicate that Notch signaling upregulates UNC5B as an effector protein to control specific endothelial cell behaviors and inhibit angiogenic growth.

The ability of the LIMD1 and TRIP6 LIM domains to bind strained f-actin is critical for their tension dependent localization to adherens junctions and association with the Hippo pathway kinase LATS1.

A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitment of the LIM domain proteins TRIP6 and LIMD1 to adherens junctions. Mechanical tension also triggers TRIP6 and LIMD1 to bind and inhibit the Hippo pathway kinase LATS1. How TRIP6 and LIMD1 are recruited to adherens junctions in response to tension is not clear, but previous studies suggested that they could be regulated by the known mechanosensory proteins α-catenin and vinculin at adherens junctions. We found that the three LIM domains of TRIP6 and LIMD1 are necessary and sufficient for tension-dependent localization to adherens junctions. The LIM domains of TRIP6, LIMD1, and certain other LIM domain proteins have been shown to bind to actin networks under strain/tension. Consistent with this, we show that TRIP6 and LIMD1 colocalize with the ends of actin fibers at adherens junctions. Point mutations in a key conserved residue in each LIM domain that are predicted to impair binding to f-actin under strain inhibits TRIP6 and LIMD1 localization to adherens junctions and their ability to bind to and recruit LATS1 to adherens junctions. Together these results show that the ability of TRIP6 and LIMD1 to bind to strained actin underlies their ability to localize to adherens junctions and regulate LATS1 in response to mechanical tension.

Targeting EB3-IP3R3 Interface with Cognate Peptide Protects from Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a lung disease characterized by acute onset of noncardiogenic pulmonary edema, hypoxemia, and respiratory insufficiency. The current treatment for ARDS is mainly supportive in nature, providing a critical need for targeted pharmacological management. We addressed this medical problem by developing a pharmacological treatment for pulmonary vascular leakage, a culprit of alveolar damage and lung inflammation. Our novel therapeutic target is the microtubule accessory factor EB3 (end binding protein 3), which contributes to pulmonary vascular leakage by amplifying pathological calcium signaling in endothelial cells in response to inflammatory stimuli. EB3 interacts with IP3R3 (inositol 1,4,5-trisphosphate receptor 3) and orchestrates calcium release from endoplasmic reticulum stores. Here, we designed and tested the therapeutic benefits of a 14-aa peptide named CIPRI (cognate IP3 receptor inhibitor), which disrupted EB3-IP3R3 interaction invitro and in lungs of mice challenged with endotoxin. Treatment with CIPRI or depletion of IP3R3 in lung microvascular endothelial monolayers mitigated calcium release from endoplasmic reticulum stores and prevented a disassembly of vascular endothelial cadherin junctions in response to the proinflammatory mediator α-thrombin. Furthermore, intravenous administration of CIPRI in mice mitigated inflammation-induced lung injury, blocked pulmonary microvascular leakage, prevented activation of NFAT (nuclear factor of activated T cells) signaling, and reduced production of proinflammatory cytokines in the lung tissue. CIPRI also improved survival of mice from endotoxemia and polymicrobial sepsis. Together, these data demonstrate that targeting EB3-IP3R3 interaction with a cognate peptide is a promising strategy to address hyperpermeability of microvessels in inflammatory lung diseases.

Multi-level Force-dependent Allosteric Enhancement of αE-catenin Binding to F-actin by Vinculin

Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via β-catenin and α-catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in αE-catenin conformation enables it to recruit the actin-binding protein vinculin to cell–cell junctions, which contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell–cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (−) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (−) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization.

Self-Assembly and Growth of Intercellular Junctions in Response to Membrane Tension and Cytoskeletal Contraction

Abstract Mechanical forces are crucial to the regulation of different aspects of cellular phenotype. These forces are transmitted between cells primarily through the bonds of intercellular junctions. A striking feature of cell adhesion junctions is their ability to sense and respond to force. It is known that the size of the junctions correlates with the magnitude of transmitted traction. In this study, we present a physical model for the interaction between two flexible membranes conjugated by a number of mobile bonds. The membranes have finite bending rigidity and are subjected to membrane tension and intracellular contraction. Implementing the first and second laws of thermodynamics, we derived the fundamental equations that control the self-assembly or growth kinetics of the junction. We predict that the junction may grow or shrink in size depending on the magnitude of membrane tension. More importantly, an increase in intracellular contraction always leads to spontaneous growth of the junctions. The balance between the lateral osmotic pressure of aggregated bonds, inter-membrane repulsive pressure, and elastic energy density of deformed membranes controls the equilibrium size of the junction. These passive sensory functions are purely mechanistic, independent from any internal signaling within the cells.

Peripheral Coupling Sites Formed by STIM1 Govern the Contractility of Vascular Smooth Muscle Cells

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). However, the mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal‐interaction molecule 1), a single transmembrane‐domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER‐PM junctions in response to depletion of ER Ca2+ stores and initiates store‐operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR‐PM interacting sites were decreased and SR‐dependent Ca2+ signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen‐inducible, SMC‐specific STIM1‐knockout (Stim1‐smKO) mice. VSMCs from Stim1‐smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+‐release sites on the SR and Ca2+‐activated ion channels on the PM, accompanied by diminished channel activity. Stim1‐smKO mice were hypotensive and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 – independent of SR Ca2+ store depletion – is critically important for stable peripheral coupling in contractile VSMCs.

Functional adaptation of glial cells at neuromuscular junctions in response to injury.

Synaptic elements from neuromuscular junctions (NMJs) undergo massive morphological and functional changes upon nerve injury. While morphological changes of NMJ‐associated glia in response to injury has been investigated, their functional properties remain elusive. Perisynaptic Schwann cells (PSCs), glial cells at the NMJ, are essential for NMJ maintenance and repair, and are involved in synaptic efficacy and plasticity. Importantly, these functions are regulated by PSCs ability to detect synaptic transmission through, notably, muscarinic (mAChRs) and purinergic receptors' activation. Using Ca2+ imaging and electrophysiological recordings of synaptic transmission at the mouse NMJ, we investigated PSC receptors activation following denervation and during reinnervation in adults and at denervated NMJs in an ALS mouse model (SOD1G37R). We observed reduced PSCs mAChR‐mediated Ca2+ responses at denervated and reinnervating NMJs. Importantly, PSC phenotypes during denervation and reinnervation were distinct than the one observed during NMJ maturation. At denervated NMJs, exogenous activation of mAChRs greatly diminished galectin‐3 expression, a glial marker of phagocytosis. PSCs Ca2+ responses at reinnervating NMJs did not correlate with the number of innervating axons or process extensions. Interestingly, we observed an extended period of reduced PSC mAChRs activation after the injury (up to 60 days), suggesting a glial memory of injury. PSCs associated with denervated NMJs in an ALS model (SOD1G37R mice) did not show any muscarinic adaptation, a phenotype incompatible with NMJ repair. Understanding functional mechanisms that underlie this glial response to injury may contribute to favor complete NMJ and motor recovery.

CD44 mediates shear stress mechanotransduction in an in vitro blood-brain barrier model through small GTPases RhoA and Rac1.

Fluid shear stress is an important mediator of vascular permeability, yet the molecular mechanisms underlying the effect of shear on the blood-brain barrier (BBB) have yet to be clarified in cerebral vasculature despite its importance for brain homeostasis. The goal of this study is to probe components of shear mechanotransduction within the BBB to gain a better understanding of pathologies associated with changes in cerebral perfusion including ischemic stroke. Interrogating the effects of shear stress in vivo is complicated by the complexity of factors in the brain parenchyma and the difficulty associated with modulating blood flow regimes. The in vitro model used in this study is compatible with real-time measurement of barrier function using a transendothelial electrical resistance as well as immunocytochemistry and dextran permeability assays. These experiments reveal that there is a threshold level of shear stress required for barrier formation and that the composition of the extracellular matrix, specifically the presence of high molecular weight hyaluronan, dictates the flow response. Gene editing to modulate the expression of CD44, a mechanosensitive receptor for hyaluronan, demonstrates that the receptor is required for the endothelial response to shear stress. Manipulation of small GTPase activity reveals CD44 activates Rac1 while inhibiting RhoA activation. Additionally, adducin-γ localizes to tight junctions in response to shear stress and RhoA inhibition and is required to maintain the barrier. This study identifies specific components of the mechanosensing complex associated with the BBB response to fluid shear stress and, therefore, illuminates potential targets for barrier manipulation in vivo.

STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells.

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). The mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal interaction molecule 1), a single-transmembrane-domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER-PM junctions in response to depletion of ER Ca2+ stores and initiates store-operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR-PM interacting sites were decreased, and SR-dependent Ca2+-signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen-inducible, SMC-specific STIM1-knockout (Stim1-smKO) mice. VSMCs from Stim1-smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+-release sites on the SR and Ca2+-activated ion channels on the PM, accompanied by diminished channel activity. Stim1-smKO mice were hypotensive, and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 - independent of SR Ca2+ store depletion - is critically important for stable peripheral coupling in contractile VSMCs.

A picket fence function for adherens junctions in epithelial cell polarity.

Adherens junctions are a defining feature of all epithelial cells, providing cell-cell adhesion and contractile ring formation that is essential for cell and tissue morphology. In Drosophila, adherens junctions are concentrated between the apical and basolateral plasma membrane domains, defined by aPKC-Par6-Baz and Lgl/Dlg/Scrib, respectively. Whether adherens junctions contribute to apical-basal polarization itself has been unclear because neuroblasts exhibit apical-basal polarization of aPKC-Par6-Baz and Lgl in the absence of adherens junctions. Here we show that, upon disruption of adherens junctions in epithelial cells, apical polarity determinants such as aPKC can still segregate from basolateral Lgl, but lose their sharp boundaries and also overlap with Dlg and Scrib - similar to neuroblasts. In addition, control of apical versus basolateral domain size is lost, along with control of cell shape, in the absence of adherens junctions. Manipulating the levels of apical Par3/Baz or basolateral Lgl polarity determinants in experiments and in computer simulations confirms that adherens junctions provide a 'picket fence' diffusion barrier that restricts the spread of polarity determinants along the membrane to enable precise domain size control. Movement of adherens junctions in response to mechanical forces during morphogenetic change thus enables spontaneous adjustment of apical versus basolateral domain size as an emergent property of the polarising system.

Fluid shear stress promotes embryonic stem cell pluripotency via interplay between β-catenin and vinculin in bioreactor culture.

The expansion of pluripotent stem cells (PSCs) as aggregates in stirred suspension bioreactors is garnering attention as an alternative to adherent culture. However, the hydrodynamic environment in the bioreactor can modulate PSC behavior, pluripotency and differentiation potential in ways that need to be well understood. In this study, we investigated how murine embryonic stem cells (mESCs) sense fluid shear stress and modulate a noncanonical Wnt signaling response to promote pluripotency. mESCs showed higher expression of pluripotency marker genes, Oct4, Sox2, and Nanog in the absence of leukemia inhibitory factor (LIF) in stirred suspension bioreactors compared to adherent culture, a phenomenon we have termed mechanopluripotency. In bioreactor culture, fluid shear promoted the nuclear translocation of the less well-known pluripotency regulator β-catenin and concomitant increase of c-Myc expression, an upstream regulator of Oct4, Sox2, and Nanog. We also observed similar β-catenin nuclear translocation in LIF-free mESCs cultured on E-cadherin substrate under defined fluid shear stress conditions in flow chamber plates. mESCs showed lower shear-induced expression of pluripotency marker genes when β-catenin was inhibited, suggesting that β-catenin signaling is crucial to mESC mechanopluripotency. Key to this process is vinculin, which is known to rearrange and associate more strongly with adherens junctions in response to fluid shear. When the vinculin gene is disrupted, we observe that nuclear β-catenin translocation and mechanopluripotency are abrogated. Our results indicate that mechanotransduction through the adherens junction complex is important for mESC pluripotency maintenance.

A novel role for bone marrow-derived cells to recover damaged keratinocytes from radiation-induced injury

Exposure to moderate doses of ionizing radiation (IR), which is sufficient for causing skin injury, can occur during radiation therapy as well as in radiation accidents. Radiation-induced skin injury occasionally recovers, although its underlying mechanism remains unclear. Moderate-dose IR is frequently utilized for bone marrow transplantation in mice; therefore, this mouse model can help understand the mechanism. We had previously reported that bone marrow-derived cells (BMDCs) migrate to the epidermis-dermis junction in response to IR, although their role remains unknown. Here, we investigated the role of BMDCs in radiation-induced skin injury in BMT mice and observed that BMDCs contributed to skin recovery after IR-induced barrier dysfunction. One of the important mechanisms involved the action of CCL17 secreted by BMDCs on irradiated basal cells, leading to accelerated proliferation and recovery of apoptosis caused by IR. Our findings suggest that BMDCs are key players in IR-induced skin injury recovery.

Par-3 family proteins in cell polarity & adhesion.

The Par‐3/Baz family of polarity determinants is highly conserved across metazoans and includes C. elegans PAR‐3, Drosophila Bazooka (Baz), human Par‐3 (PARD3), and human Par‐3‐like (PARD3B). The C. elegans PAR‐3 protein localises to the anterior pole of asymmetrically dividing zygotes with cell division cycle 42 (CDC42), atypical protein kinase C (aPKC), and PAR‐6. The same C. elegans ‘PAR complex’ can also localise in an apical ring in epithelial cells. Drosophila Baz localises to the apical pole of asymmetrically dividing neuroblasts with Cdc42‐aPKC‐Par6, while in epithelial cells localises both in an apical ring with Cdc42‐aPKC‐Par6 and with E‐cadherin at adherens junctions. These apical and junctional localisations have become separated in human PARD3, which is strictly apical in many epithelia, and human PARD3B, which is strictly junctional in many epithelia. We discuss the molecular basis for this fundamental difference in localisation, as well as the possible functions of Par‐3/Baz family proteins as oligomeric clustering agents at the apical domain or at adherens junctions in epithelial stem cells. The evolution of Par‐3 family proteins into distinct apical PARD3 and junctional PARD3B orthologs coincides with the emergence of stratified squamous epithelia in vertebrates, where PARD3B, but not PARD3, is strongly expressed in basal layer stem cells – which lack a typical apical domain. We speculate that PARD3B may contribute to clustering of E‐cadherin, signalling from adherens junctions via Src family kinases or mitotic spindle orientation by adherens junctions in response to mechanical forces.

Endoplasmic Reticulum-Plasma Membrane Contact Sites: Regulators, Mechanisms, and Physiological Functions.

The endoplasmic reticulum (ER) forms direct membrane contact sites with the plasma membrane (PM) in eukaryotic cells. These ER-PM contact sites play essential roles in lipid homeostasis, ion dynamics, and cell signaling, which are carried out by protein-protein or protein-lipid interactions. Distinct tethering factors dynamically control the architecture of ER-PM junctions in response to intracellular signals or external stimuli. The physiological roles of ER-PM contact sites are dependent on a variety of regulators that individually or cooperatively perform functions in diverse cellular processes. This review focuses on proteins functioning at ER-PM contact sites and highlights the recent progress in their mechanisms and physiological roles.

Snd3 controls nucleus-vacuole junctions in response to glucose signaling

Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites.

STIM2 targets Orai1/STIM1 to the AKAP79 signaling complex and confers coupling of Ca2+ entry with NFAT1 activation

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.

Interactive live fNIRS reveals engagement of the temporoparietal junction in response to social contingency in infants

Live social interaction is the dominant form of human social activity, but it remains unclear if brain processing of live interactive social stimuli differs substantially from processing of non-interactive social stimuli, mainly because of technical difficulties measuring brain activity during natural social interactions. This distinction is particularly important during infancy considering the importance of real-life interactions for various forms of learning. To assess the impact of live social interaction accompanied by ostensive social signals on infant cortical processing, the present study measured the cortical activities of 6–8-month-old and 10−12-month-old infants using functional near-infrared spectroscopy under contingent and non-contingent conditions (appropriately timed versus delayed responsiveness). We found greater activation over the right temporoparietal junction region in response to contingent interactions relative to non-contingent interactions in 6–8-month-old and 10–12-month-old infants. Our study indicates a critical role of contingent responsiveness for differential processing of live interactive social stimuli.

ER stress-linked autophagy stabilizes apoptosis effector PERP and triggers its co-localization with SERCA2b at ER\u2013plasma membrane junctions

Specific molecular interactions that underpin the switch between ER stress-triggered autophagy-mediated cellular repair and cellular death by apoptosis are not characterized. This study reports the unexpected interaction elicited by ER stress between the plasma membrane (PM)-localized apoptosis effector PERP and the ER Ca2+ pump SERCA2b. We show that the p53 effector PERP, which specifically induces apoptosis when expressed above a threshold level, has a heterogeneous distribution across the PM of un-stressed cells and is actively turned over by the lysosome. PERP is upregulated following sustained starvation-induced autophagy, which precedes the onset of apoptosis indicating that PERP protein levels are controlled by a lysosomal pathway that is sensitive to cellular physiological state. Furthermore, ER stress stabilizes PERP at the PM and induces its increasing co-localization with SERCA2b at ER–PM junctions. The findings highlight a novel crosstalk between pro-survival autophagy and pro-death apoptosis pathways and identify, for the first time, accumulation of an apoptosis effector to ER–PM junctions in response to ER stress.

Interactions of Junctophilins and STIM1 with ER Calcium-Releasing Channels

Junctions between the endoplasmic reticulum (ER) and plasma membrane (PM) are found in many cell types, where they mediate local Ca2+ signaling by recruiting a variety of ER and PM channels to the two sides of these junctions. Stim1 and Junctophilins (JPs) are among the major proteins responsible for forming junctions. Stim1 organizes mostly transient junctions in response to ER Ca2+ depletion and is essential for store-operated Ca2+ entry. Junctophilins 1 and 2 coordinate the organization of the calcium releasing units in striated muscle, but the role of the neuronal junctophilins, JP3 and JP4, is less understood. Because studies on double knock-out mice indicated the importance of JP3/JP4 for brain function, we are using expression in tsA201 cells to compare JP3, JP4 and Stim1. Previously, we showed that JP3 and JP4 were both able to recruit the PM channels CaV1.2, CaV2.1 and CaV2.2 to junctions, and that JP3, but not JP4, could recruit the ER channels RyR1 and RyR2. Here we show that RyR3 is recruited to junctions by JP3, and also by JP4 (less efficiently than JP3). RyR1 and RyR3 constructs lacking ER transmembrane domains co-localized with JP3, indicative of an interaction which likely contributes to the ability of JP3 to recruit the full-length proteins to junctions. By contrast, RyR3 lacking transmembrane domains did not co-localize with JP4. Co-localization also did not occur between IP3R1 and either JPs. On the other hand, constitutively active Stim1D76A displayed moderate co-localization with IP3R1 but none with the RyRs. These results suggest that neuronal ER-PM junctions formed by Stim1, JP3 and JP4 likely harbor different sets of channels and that the JP3/JP4 ratio in junctophilin-formed junctions might modulate the composition of such microdomains with possible functional implications.