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T F Martin

Publications and source records attributed to T F Martin.

At least 19 recordsLinked to original sources
PubMed2003

PC12 cells as a model for studies of regulated secretion in neuronal and endocrine cells.

Pheochromocytoma-derived cell lines such as PC12 cells maintain a differentiated neuroendocrine phenotype and have been widely used as a convenient model system for a wide variety of cell biological studies on neurotrophin action, monoamine biogenesis, protein trafficking, and secretory vesicle dynamics. This chapter reviews a number of methods that are useful for studies of the regulated dense core vesicle secretory pathway. This includes protocols for maintaining cells and preserving their phenotype. A variety of assays are discussed for monitoring secretion in intact or permeable cells and in transfected cells. Specific methods for immunocytochemical studies in permeable cells are discussed. Finally, protocols for high-efficiency PC12 cell transfections and the isolation of stably transfected cell lines are provided.

Animals↗
PubMed2001

Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles.

In the exocytosis of neurotransmitter, fusion pore opening represents the first instant of fluid contact between the vesicle lumen and extracellular space. The existence of the fusion pore has been established by electrical measurements, but its molecular composition is unknown. The possibility that synaptotagmin regulates fusion pores was investigated with amperometry to monitor exocytosis of single dense-core vesicles. Overexpression of synaptotagmin I prolonged the time from fusion pore opening to dilation, whereas synaptotagmin IV shortened this time. Both synaptotagmin isoforms reduced norepinephrine flux through open fusion pores. Thus, synaptotagmin interacts with fusion pores, possibly by associating with a core complex of membrane proteins and/or lipid.

Animals↗
PubMed2001

PIP kinase Igamma is the major PI(4,5)P(2) synthesizing enzyme at the synapse.

Disruption of the presynaptically enriched polyphosphoinositide phosphatase synaptojanin 1 leads to an increase of clathrin-coated intermediates and of polymerized actin at endocytic zones of nerve terminals. These changes correlate with elevated levels of PI(4,5)P(2) in neurons. We report that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), a major brain PI(4)P 5-kinase, is concentrated at synapses. Synaptojanin 1 and PIPKIgamma antagonize each other in the recruitment of clathrin coats to lipid membranes. Like synaptojanin 1 and other proteins involved in endocytosis, PIPKIgamma undergoes stimulation-dependent dephosphorylation. These results implicate PIPKIgamma in the synthesis of a PI(4,5)P(2) pool that acts as a positive regulator of clathrin coat recruitment and actin function at the synapse.

Actins↗
PubMed2001

Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion.

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.

Amino Acid Sequence↗
PubMed2001

G protein betagamma subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry.

The nervous system can modulate neurotransmitter release by neurotransmitter activation of heterotrimeric GTP-binding protein (G protein)-coupled receptors. We found that microinjection of G protein betagamma subunits (Gbetagamma) mimics serotonin's inhibitory effect on neurotransmission. Release of free Gbetagamma was critical for this effect because a Gbetagamma scavenger blocked serotonin's effect. Gbetagamma had no effect on fast, action potential-evoked intracellular Ca2+ release that triggered neurotransmission. Inhibition of neurotransmitter release by serotonin was still seen after blockade of all classical Gbetagamma effector pathways. Thus, Gbetagamma blocked neurotransmitter release downstream of Ca2+ entry and may directly target the exocytotic fusion machinery at the presynaptic terminal.

Action Potentials↗
PubMed2001

PI(4,5)P(2) regulation of surface membrane traffic.

Phosphatidylinositol 4,5-biphosphate (PI[4,5]P(2)) has emerged as an important signaling molecule in the membrane for regulating vesicle exo- and endocytosis and the accompanying actin cytoskeletal rearrangements. Localization studies with GFP-tagged binding domains and antibodies provide new views of the non-uniform, dynamic distribution of PI(4,5)P(2) in membranes and its organization in raft-like domains. The targeting of phosphoinositide kinases by GTPases can coordinate the reactions of membrane fusion and fission with cytoskeletal assembly, providing a basis for membrane movement.

Animals↗
PubMed2000

The C2B domain of synaptotagmin is a Ca(2+)-sensing module essential for exocytosis.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca(2+) ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca(2+)-sensing module. Here, we report that Ca(2+) drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca(2)+ are mediated by a set of conserved acidic Ca(2)+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca(2)+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

Amino Acid Sequence↗
PubMed2000

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein.

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca(2+)-dependent activator protein for secretion), a protein required for Ca(2+)-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca(2+) elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca(2+) dependencies. A threshold of approximately 10 microM Ca(2+) was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca(2+) activity < 10 microM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca(2+) and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Animals↗
PubMed2000

The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes.

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln(197)-Arg(198), is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca(2+)-dependent interactions between synaptotagmin and SNARE protein complexes.

Animals↗
PubMed2000

Priming in exocytosis: attaining fusion-competence after vesicle docking.

Membrane contact established by tethering or docking mechanisms is not a sufficient condition for membrane fusion. In neural and neuroendocrine cells, only a small fraction of secretory vesicles docked at the plasma membrane are fusion-competent and undergo rapid ATP-independent fusion in response to Ca(2+) elevations. Additional biochemical events termed 'priming' are essential to render vesicles competent for Ca(2+)-triggered fusion. The priming of vesicles is ATP-dependent and a number of ATP-dependent priming reactions have been characterized in permeable neuroendocrine cells. These involve NSF-mediated priming of SNARE protein complexes, the ATP-dependent synthesis of phosphoinositides, and protein kinase-mediated protein phosphorylation. In addition, munc13 is an important protein involved in priming synaptic vesicles. An emphasis in this review is on recent work indicating that priming events identified in the pathways of regulated exocytosis share many features with pre-fusion processes characterized in constitutive fusion pathways.

Animals↗
PubMed1999

Ca(2+)-dependent activator protein for secretion is critical for the fusion of dense-core vesicles with the membrane in calf adrenal chromaffin cells.

Calcium-dependent activator protein for secretion (CAPS) is a neural/endocrine cell-specific protein that has been shown to function at the Ca(2+)-dependent triggering step of dense-core vesicle (DCV) exocytosis in permeabilized PC12 cells. To evaluate the function of CAPS under physiological conditions, we introduced affinity-purified anti-CAPS IgGs into calf adrenal chromaffin (AC) cells via a patch pipette and tested the kinetics of catecholamine secretion using both amperometric and membrane capacitance techniques. The antibodies reacted with a single major approximately 145 kDa protein in AC cells based on immunoblot analysis. AC cells stimulated with sequential trains of action potentials at 7 Hz resulted in successive secretory episodes of equivalent magnitude. When either of two different anti-CAPS IgGs or their Fab fragments were present, a rapid and progressive inhibition of catecholamine release ensued to a maximum of >80%. The effect was specific because preabsorption of IgGs with the respective antigens ablated the inhibitory effect, and the IgGs had no effect on Ca currents. CAPS immunoneutralization not only reduced the number of amperometric spikes but markedly altered the kinetic characteristics of the residual events. The remaining spikes were much smaller (by 85%) and broader (by approximately 3.5-fold) than those in control cells, suggesting that CAPS plays a role in determining release of vesicle contents via the fusion pore. Anti-CAPS IgGs also slowed the rate of the initial exocytotic capacitance burst, representing the docked-and-primed vesicle pool, by approximately 90% but had no effect on the kinetics of rapid endocytosis. These results suggest that CAPS is a key component regulating the fusion of DCVs to the plasma membrane, and possibly fusion pore dilation, in catecholamine secretion from AC cells.

Action Potentials↗
PubMed1999

Yeast Sec14p deficient in phosphatidylinositol transfer activity is functional in vivo.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity. As expected, Sec14pK66,239A fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo. However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1ts and sec14 null mutations. This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs. These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.

Binding Sites↗
PubMed1998

Specific binding of phosphatidylinositol 4,5-bisphosphate to calcium-dependent activator protein for secretion (CAPS), a potential phosphoinositide effector protein for regulated exocytosis.

The calcium-dependent activator protein for secretion (CAPS) is a novel neural/endocrine-specific cytosolic and peripheral membrane protein required for the Ca2+-regulated exocytosis of secretory vesicles. CAPS acts at a stage in exocytosis that follows ATP-dependent priming, which involves the essential synthesis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In the present studies, CAPS is shown to bind liposomes that contain acidic phospholipids and binding was markedly enhanced by inclusion of PtdIns(4,5)P2 but not other phosphoinositides in the absence of Ca2+. PtdIns(4,5)P2, but not other phosphoinositides including PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, altered the susceptibility of CAPS to proteolysis by trypsin and proteinase K, suggesting that phosphoinositide binding promoted a conformational change. Photoaffinity labeling studies with a photoactivatable benzoylcinnimidyl acyl chain derivative of PtdIns(4,5)P2 confirmed the phosphoinositide-binding properties of CAPS and suggested a hydrophobic aspect of the interaction. CAPS, as one of very few characterized proteins with a binding specificity for 4-, 5-phosphorylated inositides over 3-phosphorylated inositides, may function in regulated exocytosis as an effector of PtdIns(4,5)P2.

Animals↗
PubMed1998

Thy-1 is a component common to multiple populations of synaptic vesicles.

Thy-1, a glycosylphosphatidylinositol-linked integral membrane protein of the immunoglobulin superfamily, is a component of both large dense-core and small clear vesicles in PC12 cells. A majority of this protein, formerly recognized only on the plasma membrane of neurons, is localized to regulated secretory vesicles. Thy-1 is also present in synaptic vesicles in rat central nervous system. Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel. These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

Animals↗
PubMed1998

Large dense-core vesicle exocytosis in PC12 cells.

A current major challenge in the study of regulated exocytosis is the identification of essential proteins that mediate the transit of secretory vesicles through trafficking stages such as recruitment, docking, and fusion. Defining the physiological roles and mechanisms of action of these essential proteins is paramount. The reconstitution of stages of regulated exocytosis in cell-free systems provides the opportunity to identify required proteins and establish their stage-specific mechanisms of action. PC12 cells, clonal cell lines of adrenal medullary origin, possess large dense-core vesicles that retain their competence for regulated exocytosis in a variety of permeable cell and isolated membrane preparations. We describe several cell-free systems for studies of regulated exocytosis derived from PC12 cells.

Adrenal Medulla↗
PubMed1998

CAPS (mammalian UNC-31) protein localizes to membranes involved in dense-core vesicle exocytosis.

CAPS is a neural/endocrine-specific protein discovered as a cytosolic factor required for Ca2+-activated dense-core vesicle (DCV) exocytosis in permeable neuroendocrine cells. We report that CAPS is also a membrane-associated, peripherally bound protein in brain homogenates that localizes Selectively to plasma membranes and to DCVs but not to small clear synaptic vesicles (SVs). CAPS exhibits high affinity and saturable binding to DCVs by interaction with bilayer phospholipids. Specific CAPS antibodies inhibit Ca2+-activated norepinephrine release from lysed synaptosomes that contain membrane-associated CAPS, indicating that membrane-bound CAPS is essential for neural DCV exocytosis. CAPS is a functional component of the exocytotic machinery that localizes selectively to DCVs, and it may confer distinct regulatory features on neuropeptide and biogenic amine transmitter secretion.

Animals↗