Hierarchical Sorting And Trafficking Of β - 2 Adrenergic Receptors Via Endosomal Microdomains

A defining feature of the eukaryotic cell is the abundance and diversity of membranous organelles. This allowed for a single cell to compartmentalize various functions and laid a foundation for the specialization of cell types in multicellular organisms. Greater complexity, however, requires more work to maintain, thus cells evolved machinery for directing the trafficking of proteins to their designated membranes. Early studies on membrane trafficking focused on understanding the biosynthetic pathway that takes proteins from their initial synthesis to be excreted or inserted in the plasma membrane. Transport of transmembrane proteins is not, however, a one way street. Many proteins, as a cycle of nutrient retrieval or signal activation, recycle from the surface through membrane compartments. Endosomes have risen as vital in the recycling and sorting process, but much of the mechanisms used remains unknown. Research into endosomal function has progressed in part by focusing on stereotypical receptors which can typify a class of cargo. Chief among these have been the non-recycling G-protein coupled receptor (GPCR) the δ opioid receptor (DOR), the bulk recycling transferrin receptor (TfR) and the sequence dependent recycling GPCR the β-2 adrenergic receptor (β2AR). The work in this document utilizes the β2AR to explore the intersection of membrane trafficking via early endosomes and intracellular signaling. We initially discovered that β2AR is selectively sorted to tubule microdomains associated with a dynamic actin cytoskeleton. This makes the recycling of active β2AR dependent on actin while bulk cargo can recycle independent of actin. We next connect β2AR actin dependence with the regulatory action of Protein Kinase A (PKA). Phosphorylation of β2AR via PKA in the cytoplasmic tail is necessary for restriction of β2AR to actin dependent tubules. Loss of phosohorylation by PKA led to defects in desensitization and recycling rate. Targeting actin to recycling tubules provides a powerful point of signaling regulation under control of signaling down-stream of β2AR itself. We wondered if this domain allowed for more than homeostatic control and shifted focus to endosomal actin as a potential regulator of sequence dependent recycling. We found the actin modifier cortactin, when phosphorylated by Src family kinases, promotes recycling of phosphorylated β2AR. Restriction of β2AR to and actin dependent pathway provides a link between β 2AR trafficking and the signaling cascades regulating actin. Having established the potential importance of interacting partners to β2AR trafficking we next began a search for the effector proteins which mediate the restriction of β2AR to actin dependent tubules. We concluded that such a binding partner would have to be localized to the body of the early endosome and selectively interact with the phosphorylated receptor. To capture such interacting proteins we optimized conditions for co-immunoprecipication and identified proteins enriched on internalized β2AR using stable isotope labeling of amino acids in cell culture (SILAC) with quantitative mass spectrometry. Bioinformatic analysis revealed proteins and complexes enriched in endosomes. While several known binding partners were detected we also detected a large number of proteins with little known function in mammalian cells. These novel interactions both suggest function for understudied proteins and allude to new regulatory mechanisms for endosomal sorting of β2AR.</p

Src Regulates Sequence-Dependent Beta-2 Adrenergic Receptor Recycling via Cortactin Phosphorylation.

<p>The recycling of internalized signaling receptors, which has direct functional consequences, is subject to multiple sequence and biochemical requirements. Why signaling receptors recycle via a specialized pathway, unlike many other proteins that recycle by bulk, is a fundamental unanswered question. Here, we show that these specialized pathways allow selective control of signaling receptor recycling by heterologous signaling. Using assays to visualize receptor recycling in living cells, we show that the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, is regulated by Src family kinases. The target of Src is cortactin, an essential factor for B2AR sorting into specialized recycling microdomains on the endosome. Phosphorylation of a single cortactin residue, Y466, regulates the rate of fission of B2AR recycling vesicles from these microdomains and, therefore, the rate of delivery of B2AR to the cell surface. Together, our results indicate that actin-stabilized microdomains that mediate signaling receptor recycling can serve as a functional point of convergence for crosstalk between signaling pathways.</p

Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch.

<p>The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling.</p

The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky-Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα.

<p>Vesicle biogenesis machinery components such as coat proteins can interact with the actin cytoskeleton for cargo sorting into multiple pathways. It is unknown, however, whether these interactions are a general requirement for the diverse endosome traffic routes. In this study, we identify actin cytoskeleton regulators as previously unrecognized interactors of complexes associated with the Hermansky-Pudlak syndrome. Two complexes mutated in the Hermansky-Pudlak syndrome, adaptor protein complex-3 and biogenesis of lysosome-related organelles complex-1 (BLOC-1), interact with and are regulated by the lipid kinase phosphatidylinositol-4-kinase type IIα (PI4KIIα). We therefore hypothesized that PI4KIIα interacts with novel regulators of these complexes. To test this hypothesis, we immunoaffinity purified PI4KIIα from isotope-labeled cell lysates to quantitatively identify interactors. Strikingly, PI4KIIα isolation preferentially coenriched proteins that regulate the actin cytoskeleton, including guanine exchange factors for Rho family GTPases such as RhoGEF1 and several subunits of the WASH complex. We biochemically confirmed several of these PI4KIIα interactions. Of importance, BLOC-1 complex, WASH complex, RhoGEF1, or PI4KIIα depletions altered the content and/or subcellular distribution of the BLOC-1-sensitive cargoes PI4KIIα, ATP7A, and VAMP7. We conclude that the Hermansky-Pudlak syndrome complex BLOC-1 and its cargo PI4KIIα interact with regulators of the actin cytoskeleton.</p

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains.

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.</p

Antibody Feedback Limits the Expansion of B Cell Responses to Malaria Vaccination but Drives Diversification of the Humoral Response

Generating sufficient antibody to block infection is a key challenge for vaccines against malaria. Here, we show that antibody titers to a key target, the repeat region of the Plasmodium falciparum circumsporozoite protein (PfCSP), plateaued after two immunizations in a clinical trial of the radiation-attenuated sporozoite vaccine. To understand the mechanisms limiting vaccine responsiveness, we developed immunoglobulin (Ig)-knockin mice with elevated numbers of PfCSP-binding B cells. We determined that recall responses were inhibited by antibody feedback, potentially via epitope masking of the immunodominant PfCSP repeat region. Importantly, the amount of antibody that prevents boosting is below the amount of antibody required for protection. Finally, while antibody feedback limited responses to the PfCSP repeat region in vaccinated volunteers, potentially protective subdominant responses to PfCSP C-terminal regions expanded with subsequent boosts. These data suggest that antibody feedback drives the diversification of immune responses and that vaccination for malaria will require targeting multiple antigens.Production and characterization of PfSPZ Vaccine were supported in part by National Insitute of Allergy and Infectious Diseases Small Business Innovation Research grants 5R44AI055229-11 (to S.L.H.), 5R44AI058499-08 (to S.L.H.), and 5R44AI058375-08 (to S.L.H.)

The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo.

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond

A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver

International audienceDiscovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria

Sequence-Dependent Sorting of Recycling Proteins by Actin-Stabilized Endosomal Microdomains

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway. © 2010 Elsevier Inc