Biomedical subjects

Alan Morgan

Publications and source records attributed to Alan Morgan.

At least 19 recordsLinked to original sources

PubMed2006

Analysis of the interacting partners of the neuronal calcium-binding proteins L-CaBP1, hippocalcin, NCS-1 and neurocalcin delta.

Intracellular Ca2+ signals are transduced by the binding of Ca2+ to sensor proteins, which subsequently modify the activity of their target proteins. Identification of these target proteins is, therefore, important for an understanding of cellular signalling processes. We have investigated the binding partners of four EF-hand Ca2+-binding proteins. Three proteins of the neuronal calcium sensor (NCS) family, hippocalcin, NCS-1 and neurocalcin delta were prepared as N-terminally tagged GST fusion proteins, and the less closely related protein L-CaBP1 was prepared in both N- and C-terminally tagged forms, the latter requiring generation of a new vector. Immobilised fusion proteins were used to purify binding partners from bovine brain cytosol and membrane extracts in the presence of 1 microM free Ca2+. Bound proteins were eluted with Ca2+-free and high-salt buffers and eluted proteins were identified by MALDI-MS and Western blotting. New protein targets detected included ARF1, Ca2+-dependent activator protein for secretion 1, cyclic nucleotide 3',5'-phosphodiesterase, the vacuolar ATPase, AP1 and AP2 complexes and the type I TGF-beta receptor. While certain of these interactions occurred with more than one of the Ca2+-binding proteins, others were found to be specific targets for particular Ca2+ sensors, and many of these did not overlap with known calmodulin-binding proteins. These findings provide new clues to the functional roles of the neuronal calcium sensor proteins.

Animals↗

PubMed2005

Protein kinase B/Akt is a novel cysteine string protein kinase that regulates exocytosis release kinetics and quantal size.

Protein kinase B/Akt has been implicated in the insulin-dependent exocytosis of GLUT4-containing vesicles, and, more recently, insulin secretion. To determine if Akt also regulates insulin-independent exocytosis, we used adrenal chromaffin cells, a popular neuronal model. Akt1 was the predominant isoform expressed in chromaffin cells, although lower levels of Akt2 and Akt3 were also found. Secretory stimuli in both intact and permeabilized cells induced Akt phosphorylation on serine 473, and the time course of Ca2+-induced Akt phosphorylation was similar to that of exocytosis in permeabilized cells. To determine if Akt modulated exocytosis, we transfected chromaffin cells with Akt constructs and monitored catecholamine release by amperometry. Wild-type Akt had no effect on the overall number of exocytotic events, but slowed the kinetics of catecholamine release from individual vesicles, resulting in an increased quantal size. This effect was due to phosphorylation by Akt, because it was not seen in cells transfected with kinase-dead mutant Akt. As overexpression of cysteine string protein (CSP) results in a similar alteration in release kinetics and quantal size, we determined if CSP was an Akt substrate. In vitro 32P-phosphorylation studies revealed that Akt phosphorylates CSP on serine 10. Using phospho-Ser10-specific antisera, we found that both transfected and endogenous cellular CSP is phosphorylated by Akt on this residue. Taken together, these findings reveal a novel role for Akt phosphorylation in regulating the late stages of exocytosis and suggest that this is achieved via the phosphorylation of CSP on serine 10.

Adrenal Glands↗

PubMed2005

Amisyn regulates exocytosis and fusion pore stability by both syntaxin-dependent and syntaxin-independent mechanisms.

Amisyn and tomosyn are related by the possession of a C-terminal vesicle-associated membrane protein-like domain that allows them to bind to syntaxin 1 and assemble into SNARE complexes. The formation of inactive complexes may sequester syntaxin and allow tomosyn and amisyn to act as inhibitors of exocytosis. We aimed to use adrenal chromaffin and PC12 cells to probe this possible mode of action of amisyn and tomosyn in dense core granule exocytosis. Although tomosyn is expressed by adrenal chromaffin and PC12 cells, amisyn expression could not be detected allowing examination of the effect of introduction of amisyn expression onto a neuronal-like background. Overexpression of m-tomosyn1 and expression of amisyn both inhibited Ca2+-induced exocytosis in transfected PC12 cells. Surprisingly, this inhibition was not removed when amisyn and tomosyn constructs were used in which key residues required for efficient binding to syntaxin1 were mutated. The effect of amisyn was further characterized using carbon fiber amperometry in chromaffin cells. Expression of amisyn had no effect on the basic characteristics of the amperometric spikes but reduced the number of spikes elicited. This inhibitory action on the extent of exocytosis was also seen with the amisyn mutant deficient in syntaxin1 binding. In addition, expression of amisyn resulted in an increase in the lifetime of the prespike foot, and this effect was abolished by the mutations. These results show that tomosyn and amisyn can negatively regulate exocytosis independently of syntaxin and also that amisyn can regulate the stability of the fusion pore.

Amino Acid Sequence↗

PubMed2005

Yeast Nhp6A/B and mammalian Hmgb1 facilitate the maintenance of genome stability.

Saccharomyces cerevisiae Nhp6A and Nhp6B are chromatin architectural factors that belong to the high-mobility group box (HMGB) superfamily and appear to be functionally related to mammalian Hmgb1. They bind to the minor groove of double-stranded DNA in a non-sequence-specific manner and thereby influence chromatin structure. Previous work has implicated these proteins in a variety of nuclear processes, including chromatin remodeling, DNA replication, transcription, and recombination . Here, we show that Nhp6A/B loss leads to increased genomic instability, hypersensitivity to DNA-damaging agents, and shortened yeast cell life span that is associated with elevated levels of extrachromosomal rDNA circles. Furthermore, we show that hypersensitivity toward UV light does not appear to reflect a decreased capacity for DNA repair but instead correlates with higher levels of UV-induced thymine dimer adducts being formed in cells lacking Nhp6A/B. Likewise, we show that mouse fibroblasts lacking Hmgb1 display higher rates of damage after UV irradiation than wild-type controls and also exhibit pronounced chromosomal instability. Taken together, these data indicate that Nhp6A/B and Hmgb1 protect DNA from damaging agents and thus guard against the generation of genomic aberrations.

Animals↗

PubMed2005

Phosphorylation of cysteine string protein in the brain: developmental, regional and synaptic specificity.

Protein phosphorylation modulates regulated exocytosis in most cells, including neurons. Cysteine string protein (CSP) has been implicated in this process because its phosphorylation on Ser10 alters its interactions with syntaxin and synaptotagmin, and because the effect of CSP overexpression on exocytosis kinetics in chromaffin cells requires phosphorylatable Ser10. To characterize CSP phosphorylation in the brain, we raised phosphospecific antibodies to Ser10. Western blotting revealed that the proportion of phosphorylated CSP (P-CSP) varies between distinct brain regions and also exhibits developmental regulation, with P-CSP highest early in development. Immunohistochemical analysis of the cerebellar cortex revealed a novel pool of P-CSP that did not colocalize with synaptic vesicle markers during early development. Strikingly, in the adult cerebellar granular layer P-CSP was highly enriched in a subset of glutamatergic synapses but undetectable in neighbouring GABA-ergic synapses. In view of the functional consequences of CSP phosphorylation, such differences could contribute to the synapse-specific regulation of neurotransmitter release.

Aging↗

PubMed2004

Munc18-1 regulates early and late stages of exocytosis via syntaxin-independent protein interactions.

Sec1/Munc18 (SM) proteins are involved in various intracellular membrane trafficking steps. Many SM proteins bind to appropriate syntaxin homologues involved in these steps, suggesting that SM proteins function as syntaxin chaperones. Organisms with mutations in SM genes, however, exhibit defects in either early (docking) or late (fusion) stages of exocytosis, implying that SM proteins may have multiple functions. To gain insight into the role of SM proteins, we introduced mutations modeled on those identified in Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae into mammalian Munc18-1. As expected, several mutants exhibited reduced binding to syntaxin1A. However, three mutants displayed wild-type syntaxin binding affinities, indicating syntaxin-independent defects. Expression of these mutants in chromaffin cells either increased the rate and extent of exocytosis or altered the kinetics of individual release events. This latter effect was associated with a reduced Mint binding affinity in one mutant, implying a potential mechanism for the observed alteration in release kinetics. Furthermore, this phenotype persisted when the mutation was combined with a second mutation that greatly reduced syntaxin binding affinity. These results clarify the data on the function of SM proteins in mutant organisms and indicate that Munc18-1 controls multiple stages of exocytosis via both syntaxin-dependent and -independent protein interactions.

Adrenal Medulla↗

PubMed2004

Membrane traffic: controlling membrane fusion by modifying NSF.

Recent studies show that NSF, isolated over 15 years ago as a protein required for membrane fusion in vitro, can be reversibly inactivated by both S-nitrosylation and tyrosine phosphorylation. Different cell types use distinct post-translational modifications of NSF for localized regulation of membrane fusion.

Membrane Fusion↗

PubMed2004

A protein-protein binding assay using coated microtitre plates: increased throughput, reproducibility and speed compared to bead-based assays.

Protein-protein interactions, and the factors affecting them, are of fundamental importance to all biological systems. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITR) are powerful methods for assaying such interactions, but are expensive to implement. In contrast, bead-based pull-down assays using affinity tags such as glutathione-S-transferase (GST), require no specialist equipment. As a result, such assays are the most popular method for analysing protein-protein interactions, despite being time-consuming and prone to variability. In respect of these problems, we have modified this form of binding assay, using glutathione-coated 96-well plates rather than glutathione-Sepharose beads to bind the primary bait protein. Quantitation of bound protein utilises ELISA for purified proteins and scintillation counting for in vitro translated proteins, rather than the SDS-PAGE-based detection methods used in traditional bead-based assays. These modifications result in an approximately 10-fold increase in the number of samples that can be assayed daily, and allow results to be obtained within hours as opposed to days. We validate the modified assay by analysing the equilibrium binding of Munc18 and syntaxin, and also demonstrate that association and dissociation kinetics may be measured using this approach. The method we describe is generally applicable to any protein-protein interaction assay based on affinity tags and is amenable to automation, and so should benefit a wide range of biochemical research.

Automation↗

PubMed2004

Regulation of the fusion pore conductance during exocytosis by cyclin-dependent kinase 5.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase involved in synaptogenesis and brain development, and its enzymatic activity is essential for slow forms of synaptic vesicle endocytosis. Recent work also has implicated Cdk5 in exocytosis and synaptic plasticity. Pharmacological inhibition of Cdk5 modifies secretion in neuroendocrine cells, synaptosomes, and brain slices; however, the specific mechanisms involved remain unclear. Here we demonstrate that dominant-negative inhibition of Cdk5 increases quantal size and broadens the kinetics of individual exocytotic events measured by amperometry in adrenal chromaffin cells. Conversely, Cdk5 overexpression narrows the kinetics of fusion, consistent with an increase in the extent of kiss-and-run exocytosis. Cdk5 inhibition also increases the total charge and current of catecholamine released during the amperometric foot, representing a modification of the conductance of the initial fusion pore connecting the granule and plasma membrane. We suggest that these effects are not attributable to an alteration in catecholamine content of secretory granules and therefore represent an effect on the fusion mechanism itself. Finally, mutational silencing of the Cdk5 phosphorylation site in Munc18, an essential protein of the late stages of vesicle fusion, has identical effects on amperometric spikes as dominant-negative Cdk5 but does not affect the amperometric feet. Cells expressing Munc18 T574A have increased quantal size and broader kinetics of fusion. These results suggest that Cdk5 could, in part, control the kinetics of exocytosis through phosphorylation of Munc18, but Cdk5 also must have Munc18-independent effects that modify fusion pore conductance, which may underlie a role of Cdk5 in synaptic plasticity.

Animals↗

PubMed2004

Rural policy development: an NRHA and PACE association collaborative model.

The Program of All-Inclusive Care for the Elderly (PACE) offers a unique model of comprehensive care for frail, elderly people. To date, all of the PACE programs have been located in urban areas. Rural advocates and policymakers, however, believe the program may hold great promise for use in rural areas, which have higher percentages of elderly residents than urban areas. In 2002, the National Rural Health Association and the National PACE Association convened a meeting that brought together PACE experts, policymakers, and rural health care providers to examine PACE and its applicability for rural communities. The meeting participants concluded that there were many rural communities where the PACE model might not only be appropriate but also highly successful in caring for rural, frail, elderly people. This article examines the notion of expanding the PACE model to rural communities, including some of the barriers and some of the possible solutions that might make PACE a viable part of the rural health care delivery system.

Aged↗

PubMed2003

Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis.

Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.

Animals↗

PubMed2003

Suppression of homologous recombination by the Saccharomyces cerevisiae linker histone.

The basic unit of chromatin in eukaryotes is the nucleosome, comprising 146 bp of DNA wound around two copies of each of four core histones. Chromatin is further condensed by association with linker histones. Saccharomyces cerevisiae Hho1p has sequence homology to other known linker histones and interacts with nucleosomes in vitro. However, disruption of HHO1 results in no significant changes in the phenotypes examined thus far. Here, we show that Hho1p is inhibitory to DNA repair by homologous recombination (HR). We find Hho1p is abundant and associated with the genome, consistent with a global role in DNA repair. Furthermore, we establish that Hho1p is required for a full life span and propose that this is mechanistically linked to its role in HR. Finally, we show that Hho1p is inhibitory to the recombination-dependent mechanism of telomere maintenance. The role of linker histones in genome stability, aging, and tumorigenesis is discussed.

Base Sequence↗

PubMed2003

Tying everything together: the multiple roles of cysteine string protein (CSP) in regulated exocytosis.

In addition to the core vesicle fusion machinery, the SNARE proteins, a large number of regulatory proteins have been implicated in the process of Ca2+-dependent exocytosis. How these exocytotic proteins are properly targeted and how their myriad interactions are temporally and spatially coordinated is poorly understood. Cysteine string protein (CSP), a secretory vesicle membrane protein and a member of the dnaJ family of co-chaperones, may assist in performing this function. Through its interaction with the ubiquitous chaperone, Hsc70, it is thought that cysteine string protein targets chaperone complexes to the exocytotic machinery to facilitate the correct folding of polypeptides or to regulate the assembly of protein complexes. Since its discovery, there have been conflicting reports from different systems concerned with whether cysteine string protein exerts its effects on exocytosis either up- or down-stream of Ca2+-influx. In this review, we summarize recent experiments that associate cysteine string protein with the regulation of vesicle filling, vesicle docking, Ca2+-channels and the SNARE proteins themselves, hence supporting a role for cysteine string protein as a multifunctional secretory co-chaperone. In addition, we provide an update on the mammalian isoforms of cysteine string protein following the recent discovery of two novel cysteine string proteins.

Amino Acid Sequence↗

PubMed2003

Physiological regulation of Munc18/nSec1 phosphorylation on serine-313.

Increased protein phosphorylation enhances exocytosis in most secretory cell types, including neurones. However, the molecular mechanisms by which this occurs and the specific protein targets remain unclear. Munc18-1/nSec1 is essential for exocytosis in neurones, and is known to be phosphorylated by protein kinase C (PKC) in vitro at Ser-313. This phosphorylation has been shown to decrease its affinity for syntaxin, and to alter the kinetics of exocytosis in chromaffin cells. However, there are no data on the physiological regulation of Ser-313 phosphorylation. Using phospho-Ser-313-specific antisera, we demonstrate here that Ser-313 is phosphorylated in intact and permeabilized chromaffin cells in response to histamine and Ca2+ respectively. Furthermore, Ser-313 is rapidly and transiently phosphorylated in intact synaptosomes in response to depolarization by KCl treatment or by 4-aminopyridine, and by the metabotropic glutamate receptor agonist dihydroxyphenylglycine. PKC was identified as the kinase, and PP1 and PP2B as the phosphatases responsible for regulating Ser-313 phosphorylation. As phosphorylation of nSec1 on Ser-313 affects the rate of transmitter release in chromaffin cells, the demonstration here that this phosphorylation event occurs in neurones suggests that synaptic neurotransmitter release may be similarly regulated by nSec1 phosphorylation. Furthermore, such changes in release kinetics are associated with long-term potentiation and depression, thus implicating nSec1 phosphorylation as a potential regulatory mechanism underlying presynaptic plasticity.

Animals↗

PubMed2003

Secretory granule exocytosis.

Regulated exocytosis of secretory granules or dense-core granules has been examined in many well-characterized cell types including neurons, neuroendocrine, endocrine, exocrine, and hemopoietic cells and also in other less well-studied cell types. Secretory granule exocytosis occurs through mechanisms with many aspects in common with synaptic vesicle exocytosis and most likely uses the same basic protein components. Despite the widespread expression and conservation of a core exocytotic machinery, many variations occur in the control of secretory granule exocytosis that are related to the specialized physiological role of particular cell types. In this review we describe the wide range of cell types in which regulated secretory granule exocytosis occurs and assess the evidence for the expression of the conserved fusion machinery in these cells. The signals that trigger and regulate exocytosis are reviewed. Aspects of the control of exocytosis that are specific for secretory granules compared with synaptic vesicles or for particular cell types are described and compared to define the range of accessory control mechanisms that exert their effects on the core exocytotic machinery.

Animals↗

PubMed2002

Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I.

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His(6)-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

Animals↗

PubMed2002

Analysis of NSF mutants reveals residues involved in SNAP binding and ATPase stimulation.

N-Ethylmaleimide-sensitive fusion protein (NSF) and its yeast orthologue, Sec18, are cytoplasmic AAA(+) ATPases required for most intracellular membrane fusion events. The primary function of NSF is thought to be the disassembly of cis-SNARE complexes, thus allowing trans-SNARE complex formation and subsequent membrane fusion. The importance of NSF/Sec18 in intracellular membrane traffic in vivo is highlighted by the inhibition of neurotransmission in Drosophila comatose (NSF) mutants and of constitutive secretion in yeast sec18 mutants. However, the underlying biochemical defects in these mutant proteins are largely unknown. Here, we identify the sec18-1 mutation as a G89D substitution in the N domain of Sec18p. This mutation results in an inhibition of the mutant protein's ability to bind to Sec17p (yeast alpha-SNAP). In contrast, engineering the comatose(st53)() mutation (S483L) into mammalian NSF (S491L) has no effect on alpha-SNAP binding. Instead, the stimulation of ATPase activity by alpha-SNAP required for wild-type NSF to disassemble SNARE complexes does not occur in the mutant NSF(st53) protein. This biochemical phenotype predicts a dominant negative effect, which was confirmed by engineering the st53 mutation into Sec18 (A505L), resulting in a dominant lethal phenotype in vivo. These findings suggest a biochemical basis for the block in membrane fusion observed in the mutant organisms. Furthermore, the mutants characterized here define key residues involved in two essential, but mechanistically distinct, biochemical functions of NSF: SNAP binding and SNAP-dependent ATPase stimulation.

Adenosine Triphosphatases↗

PubMed2002

SGS1 is a multicopy suppressor of srs2: functional overlap between DNA helicases.

Sgs1 is a member of the RecQ family of DNA helicases, which have been implicated in genomic stability, cancer and ageing. Srs2 is another DNA helicase that shares several phenotypic features with Sgs1 and double sgs1srs2 mutants have a severe synthetic growth phenotype. This suggests that there may be functional overlap between these two DNA helicases. Consistent with this idea, we found the srs2Delta mutant to have a similar genotoxin sensitivity profile and replicative lifespan to the sgs1Delta mutant. In order to directly test if Sgs1 and Srs2 are functionally interchangeable, the ability of high-copy SGS1 and SRS2 plasmids to complement the srs2Delta and sgs1Delta mutants was assessed. We report here that SGS1 is a multicopy suppressor of the methyl methanesulphonate (MMS) and hydroxyurea sensitivity of the srs2Delta mutant, whereas SRS2 overexpression had no complementing ability in the sgs1Delta mutant. Domains of Sgs1 directly required for processing MMS-induced DNA damage, most notably the helicase domain, are also required for complementation of the srs2Delta mutant. Although SGS1 overexpression was unable to rescue the shortened mean replicative lifespan of the srs2Delta mutant, maximum lifespan was significantly increased by multicopy SGS1. We conclude that Sgs1 is able to partially compensate for the loss of Srs2.

Cell Division↗