Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies.

Spatial regulation of exocytosis relies on the exocyst, a hetero-octameric protein complex that tethers vesicles to fusion sites at the plasma membrane. Nevertheless, our understanding of mechanisms regulating exocyst assembly/disassembly, localization, and function are incomplete. Here, we have exploited a panel of anti-Sec6 monoclonal antibodies (mAbs) to probe possible configurational changes accompanying transitions in exocyst function in epithelial MDCK cells. Sec6 is quantitatively associated with Sec8 in high molecular weight complexes, as shown by gel filtration and co-immunoprecipitation studies. We mapped epitopes recognized by more than 20 distinct mAbs to one of six Sec6 segments. Surprisingly, mAbs that bound epitopes in each segment labeled distinct subcellular structures. In general, antibodies to epitopes in N-terminal domains labeled Sec6 in either cytosolic or nuclear pools, whereas those that bound epitopes in C-terminal domains labeled membrane-associated Sec6. In this latter group, we identified antibodies that labeled distinct Sec6 populations at the apical junctional complex, desmosomes, endoplasmic reticulum and vimentin-type intermediate filaments. That each antibody was specific was verified by both Sec6 RNAi and competition with fusion proteins containing each domain. Comparison of non-polarized and polarized cells revealed that many Sec6 epitopes either redistribute or become concealed during epithelial polarization. Transitions in exocyst configurations may be regulated in part by the actions of Ral GTPases, because the exposure of Sec6 C-terminal domain epitopes at the plasma membrane is significantly reduced upon RalA RNAi. To determine whether spatio-temporal changes in epitope accessibility was correlated with differential stability of interactions between Sec6 and other exocyst subunits, we quantified relative amounts of each subunit that co-immunoprecipitated with Sec6 when antibodies to N-terminal or C-terminal epitopes were used. Antibodies to Sec6NT co-precipitated substantially more Sec5, -10, -15, Exo70 and -84 than did those to Sec6CT. In contrast, antibodies to Sec6CT co-precipitated more Sec3 and Sec8 than did those to Sec6NT. These results are consistent with a model in which exocyst activation during periods of rapid membrane expansion is accompanied by molecular rearrangements within the holocomplex or association with accessory proteins, which expose the Sec6 C-terminal domain when the complex is membrane-bound and conceal it when the complex is cytoplasmic.

CREG1 Interacts with Sec8 to Promote Cardiomyogenic Differentiation and Cell-Cell Adhesion.

Understanding the regulation of cell-cell interactions during the formation of compact myocardial structures is important for achieving true cardiac regeneration through enhancing the integration of stem cell-derived cardiomyocytes into the recipient myocardium. In this study, we found that cellular repressor of E1A-stimulated genes 1 (CREG1) is highly expressed in both embryonic and adult hearts. Gain- and loss-of-function analyses demonstrated that CREG1 is required for differentiation of mouse embryonic stem (ES) cell into cardiomyocytes and the formation of cohesive myocardium-like structures in a cell-autonomous fashion. Furthermore, CREG1 directly interacts with Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Site-directed mutagenesis and rescue of CREG1 knockout ES cells showed that CREG1 binding to Sec8 is required for cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8, and N-cadherin colocalize at intercalated discs in vivo and are enriched at cell-cell junctions in cultured cardiomyocytes. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis. Stem Cells 2016;34:2648-2660.

The neural cell adhesion molecule promotes FGFR-dependent phosphorylation and membrane targeting of the exocyst complex to induce exocytosis in growth cones.

The exocyst complex is an essential regulator of polarized exocytosis involved in morphogenesis of neurons. We show that this complex binds to the intracellular domain of the neural cell adhesion molecule (NCAM). NCAM promotes FGF receptor-mediated phosphorylation of two tyrosine residues in the sec8 subunit of the exocyst complex and is required for efficient recruitment of the exocyst complex to growth cones. NCAM at the surface of growth cones induces Ca(2+)-dependent vesicle exocytosis, which is blocked by an inhibitor of L-type voltage-dependent Ca(2+) channels and tetanus toxin. Preferential exocytosis in growth cones underlying neurite outgrowth is inhibited in NCAM-deficient neurons as well as in neurons transfected with phosphorylation-deficient sec8 and dominant-negative peptides derived from the intracellular domain of NCAM. Thus, we reveal a novel role for a cell adhesion molecule in that it regulates addition of the new membrane to the cell surface of growth cones in developing neurons.

A dual role for IQGAP1 in regulating exocytosis.

Polarized secretion is a tightly regulated event generated by conserved, asymmetrically localized multiprotein complexes, and the mechanism(s) underlying its temporal and spatial regulation are only beginning to emerge. Although yeast Iqg1p has been identified as a positional marker linking polarity and exocytosis cues, studies on its mammalian counterpart, IQGAP1, have focused on its role in organizing cytoskeletal architecture, for which the underlying mechanism is unclear. Here, we report that IQGAP1 associates and co-localizes with the exocyst-septin complex, and influences the localization of the exocyst and the organization of septin. We further show that activation of CDC42 GTPase abolishes this association and inhibits secretion in pancreatic beta-cells. Whereas the N-terminus of IQGAP1 binds the exocyst-septin complex, enhances secretion and abrogates the inhibition caused by CDC42 or the depletion of IQGAP1, the C-terminus, which binds CDC42, inhibits secretion. Pulse-chase experiments indicate that IQGAP1 influences protein-synthesis rates, thus regulating exocytosis. We propose and discuss a model in which IQGAP1 serves as a conformational switch to regulate exocytosis.

Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells.

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

Exo70 interacts with the Arp2/3 complex and regulates cell migration.

The exocyst is a multiprotein complex essential for tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. Here, we report that the exocyst component Exo70 interacts with the Arp2/3 complex, a key regulator of actin polymerization. We further show that the exocyst-Arp2/3 interaction is regulated by epidermal growth factor (EGF) signalling. Inhibition of Exo70 by RNA interference (RNAi) or antibody microinjection blocks the formation of actin-based membrane protrusions and affects various aspects of cell motility. We propose that Exo70, in addition to functioning in exocytosis, also regulates actin at the leading edges of migrating cells, therefore coordinating cytoskeleton and membrane traffic during cell migration.

The exocyst complex in polarized exocytosis.

Exocytosis is an essential membrane traffic event mediating the secretion of intracellular protein contents such as hormones and neurotransmitters as well as the incorporation of membrane proteins and lipids to specific domains of the plasma membrane. As a fundamental cell biological process, exocytosis is crucial for cell growth, cell-cell communication, and cell polarity establishment. For most eukaryotic cells exocytosis is polarized. A multiprotein complex, named the exocyst, is required for polarized exocytosis from yeast to mammals. The exocyst consists of eight components: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. They are localized to sites of active exocytosis, where they mediate the targeting and tethering of post-Golgi secretory vesicles for subsequent membrane fusion. Here we review the progress made in the understanding of the exocyst and its role in polarized exocytosis.

The neuronal beta 4 subunit increases the unitary conductance of L-type voltage-gated calcium channels in PC12 cells.

beta subunits of voltage-gated calcium channels influence channel behavior in numerous ways, including enhancing the targeting of alpha1 subunits to the plasma membrane and shifting the voltage dependence of activation and inactivation. Of the four beta subunits that have been identified, beta 4 is of particular interest because mutation of its alpha1 subunit interaction domain produces severe neurological defects. Its differential distribution in the hippocampus prompted us to examine whether this subunit was responsible for the heterogeneity of hippocampal L-type calcium channels. To study the functional effects of the beta 4 subunit on native L-type calcium channels, we transfected beta 4 cDNA subcloned out of embryonic hippocampal neurons into PC12 cells, a cell line that contains the beta 1, beta 2, and beta 3 subunits but not the beta 4 subunit. Cell-attached single-channel recordings of L-type channel activity from untransfected and transfected PC12 cells compared with recordings obtained from hippocampal neurons revealed an effect of the beta 4 subunit on single-channel conductance. L-type channels in untransfected PC12 cells had a significantly smaller conductance (19.8 picosiemens (pS)) than L-type channels in hippocampal neurons (22 pS). After transfection of beta 4, however, L-type single-channel conductance was indistinguishable between the two cell types. Our data suggest that calcium channel beta 4 subunits affect the conductance of L-type calcium channels and that native hippocampal L-type channels contain the beta 4 subunit.

Exocyst complex subunit sec8 binds to postsynaptic density protein-95 (PSD-95): a novel interaction regulated by cypin (cytosolic PSD-95 interactor).

The PDZ domains of postsynaptic density (PSD) protein-95 play a role in the localization of PSD-95 and binding partners to neuronal synapses. The identification of binding partners to these PDZ domains can help us in understanding how signalling complexes are assembled. We observed that one of the subunits in the sec6/8 or exocyst complex, sec8, contains a C-terminal consensus sequence for PDZ binding. Sec8 binds to PDZ1-2 of PSD-95, and this binding can be competed with a peptide that binds to PDZ1 and PDZ2 in the peptide-binding site. In addition, binding of sec8 is dependent on its C-terminal-binding sequence namely Thr-Thr-Val (TTV). Immunoblotting of rat tissue extracts shows that sec8 and PSD-95 are enriched in the same brain regions, and sec8 and PSD-95 have the same subcellular distribution in pheochromocytoma cells, suggesting that these proteins may interact in vivo. Immunoprecipitation studies of sec8 and PSD-95 in brain provide further evidence of a sec8 and PSD-95 interaction. Furthermore, the cytosolic PSD-95 interactor competes with sec8 for interaction with PSD-95. Taken together, our results suggest that the cytosolic PSD-95 interactor may function to regulate the ability of sec8 to bind to PSD-95.

Mammalian septins nomenclature.

There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEPT4_v1.