Polarized Axonal Surface Expression of Neuronal KCNQ Potassium Channels Is Regulated by Calmodulin Interaction with KCNQ2 Subunit

KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.     

Sar1 GTPase Activity Is Regulated by Membrane Curvature

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5′-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.     

Axonal Pruning Is Actively Regulated by the Microtubule-Destabilizing Protein Kinesin Superfamily Protein 2A

1. Download : Download full-size image

Highlights? During axonal pruning, microtubule disassembly is regulated by KIF2A ? Degradation of Tau precedes microtubule disassembly during axonal pruning ? Kif2a ablation causes skin hyperinnervation by sensory axons during development     

Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation

The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.     

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca^2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.     

Degradation of Connexins from the Plasma Membrane Is Regulated by Inhibitors of Protein Synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways

Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.     

Lipocalin 2 Expression and Secretion Is Highly Regulated by Metabolic Stress,Cytokines, and Nutrients in Adipocytes

Lipocalin 2 (Lcn2) has been recently characterized as a new adipokine having a role in innate immunity and energy metabolism. Nonetheless, the metabolic regulation of Lcn2 production in adipocytes has not been comprehensively studied. To better understand the Lcn2 biology, we investigated the regulation of Lcn2 expression in adipose tissue in response to metabolic stress in mice as well as the control of Lcn2 expression and secretion by cytokines and nutrients in 3T3-L1 adipocytes. Our results showed that the mRNA expression of Lcn2 was upregulated in white and brown adipose tissues as well as liver during fasting and cold stress in mice. Among pro-inflammatory cytokines TNFα, IL-1β, and IL-6, IL-1β showed most profound effect on Lcn2 expression and secretion in 3T3-L1 adipocytes. Insulin stimulated Lcn2 expression and secretion in a dose-dependent manner; this insulin effect was significantly abolished in the presence of low concentration of glucose. Moreover, insulin-stimulated Lcn2 expression and secretion was also attenuated when glucose was replaced by 3-O-methyl-d-glucose or by blocking NFκB pathway activation. Additionally, we showed that palmitate and oleate induced Lcn2 expression and secretion more significantly than EPA, while phytanic acid reduced Lcn2 production. Our results demonstrated that Lcn2 production in adipocytes is highly responsive to metabolic stress, cytokines, and nutrient signals, suggesting an important role of Lcn2 in adipocyte metabolism and inflammation.     

Tetherin Restricts Herpes Simplex Virus 1 and Is Antagonized by Glycoprotein M

Tetherin is a broadly active antiviral effector that works by tethering nascent enveloped virions to a host cell membrane, thus preventing their release. In this study, we demonstrate that herpes simplex virus 1 (HSV-1) is targeted by tetherin. We identify the viral envelope glycoprotein M (gM) as having moderate anti-tetherin activity. We show that gM but not gB or gD efficiently removes tetherin from the plasma membrane and can functionally substitute for the human immunodeficiency virus type 1 (HIV-1) Vpu protein, the prototypic viral tetherin antagonist, in rescuing HIV-1 release from tetherin-expressing cells. Our data emphasize that tetherin is a broadly active antiviral effector and contribute to the emerging hypothesis that viruses must suppress or evade an array of host cell countermeasures in order to establish a productive infection.     

PTEN Hopping on the Cell Membrane Is Regulated via a Positively-Charged C2 Domain

PTEN, a tumor suppressor that is frequently mutated in a wide spectrum of cancers, exerts PI(3,4,5)P₃ phosphatase activities that are regulated by its dynamic shuttling between the membrane and cytoplasm. Direct observation of PTEN in the interfacial environment can offer quantitative information about the shuttling dynamics, but remains elusive. Here we show that positively charged residues located in the cα2 helix of the C2 domain are necessary for the membrane localization of PTEN via stable electrostatic interactions in Dictyostelium discoideum. Single-molecule imaging analyses revealed that PTEN molecules moved distances much larger than expected had they been caused by lateral diffusion, a phenomenon we call “hopping.” Our novel single-particle tracking analysis method found that the cα2 helix aids in regulating the hopping and stable-binding states. The dynamically established membrane localization of PTEN was revealed to be essential for developmental processes and clarified a fundamental regulation mechanism of the protein quantity and activity on the plasma membrane.     

Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction

As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca²⁺ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca²⁺-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca²⁺-ATPase activity was related to an increase in the apparent affinity for Ca²⁺ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca²⁺ homeostasis.