The Experts below are selected from a list of 576 Experts worldwide ranked by ideXlab platform
Phillip J Robinson - One of the best experts on this subject based on the ideXlab platform.
-
pyrImIdIne based InhIbItors of DynamIn I gtpase actIvIty competItIve InhIbItIon at the pleckstrIn homology domaIn
Journal of Medicinal Chemistry, 2017Co-Authors: Luke R Odell, Ngoc Chau, James A Daniel, Timothy A Hill, Mohammed K Abdelhamid, Kelly A Young, Fiona M Deane, Jennette A Sakoff, Sofia Andersson, Phillip J RobinsonAbstract:The large GTPase DynamIn medIates membrane fIssIon durIng clathrIn-medIated endocytosIs (CME). The amInopyrImIdIne compounds were reported to dIsrupt DynamIn localIzatIon to the plasma membrane vIa the PH domaIn and ImplIcate thIs mechanIsm In the InhIbItIon of CME. We have used a computatIonal approach of bIndIng sIte IdentIfIcatIon, dockIng, and InteractIon energy calculatIons to desIgn and synthesIze a new lIbrary of amInopyrImIdIne analogues targetIng sIte-2 of the pleckstrIn homology (PH) domaIn. The optImIzed analogues showed low mIcromolar InhIbItIon agaInst both DynamIn I (IC50 = 10.6 ± 1.3 to 1.6 ± 0.3 μM) and CME (IC50(CME) = 65.9 ± 7.7 to 3.7 ± 1.1 mM), whIch makes thIs serIes among the more potent InhIbItors of DynamIn and CME yet reported. In CME and growth InhIbItIon cell-based assays, the data obtaIned was consIstent wIth DynamIn InhIbItIon. CEREP ExpresS profIlIng IdentIfIed off-target effects at the cholecystokInIn, dopamIne D2, hIstamIne H1 and H2, melanocortIn, melatonIn, muscarInIc M1 ...
-
sh3 domaIns dIfferentIally stImulate dIstInct DynamIn I assembly modes and g domaIn actIvIty
PLOS ONE, 2015Co-Authors: Sai Krishnan, Michael Collett, Phillip J RobinsonAbstract:DynamIn I Is a hIghly regulated GTPase enzyme enrIched In nerve termInals whIch medIates vesIcle fIssIon durIng synaptIc vesIcle endocytosIs. One regulatory mechanIsm Involves Its InteractIons wIth proteIns contaInIng Src homology 3 (SH3) domaIns. At least 30 SH3 domaIn-contaInIng proteIns bInd DynamIn at Its prolIne-rIch domaIn (PRD). Those that stImulate DynamIn actIvIty act by promotIng Its olIgomerIsatIon. We undertook a systematIc parallel screenIng of 13 glutathIone-S-transferase (GST)-tagged endocytosIs-related SH3 domaIns on DynamIn bIndIng, GTPase actIvIty and olIgomerIsatIon. No correlatIon was found between DynamIn bIndIng and theIr potency to stImulate GTPase actIvIty. There was lImIted correlatIon between the extent of theIr abIlIty to stImulate DynamIn actIvIty and the level of olIgomerIsatIon, IndIcatIng an as yet uncharacterIsed allosterIc couplIng of the PRD and G domaIn. We examIned the two varIants, DynamIn Iab and Ibb, whIch dIffer In the alternately splIce mIddle domaIn α2 helIx. They responded dIfferently to the panel of SH3s, wIth the extent of stImulatIon between the splIce varIants varyIng greatly between the SH3s. ThIs study reveals that SH3 bIndIng can act as a heterotropIc allosterIc regulator of the G domaIn vIa the mIddle domaIn α2 helIx, suggestIng an Involvement of thIs helIx In communIcatIng the PRD-medIated allostery. ThIs IndIcates that SH3 bIndIng both stabIlIses multIple conformatIons of the tetramerIc buIldIng block of DynamIn, and promotes assembly of DynamIn-SH3 complexes wIth dIstInct rates of GTP hydrolysIs.
-
1 8 naphthalImIde derIvatIves new leads agaInst DynamIn I gtpase actIvIty
Organic and Biomolecular Chemistry, 2015Co-Authors: Mohammed K Abdelhamid, Phillip J Robinson, Luke R Odell, Ngoc Chau, Anna Mariana, Ainslie Whiting, Kylie A Macgregor, Adam MccluskeyAbstract:Fragment-based In sIlIco screenIng agaInst DynamIn I (dynI) GTPase actIvIty IdentIfIed the 1,8-naphthalImIde framework as a potentIal scaffold for the desIgn of new InhIbItors targetIng the GTP bIndIng pocket of dynI. Structure-based desIgn, synthesIs and subsequent optImIzatIon resulted In the development of a lIbrary of 1,8-naphthalImIde derIvatIves, called the Naphthaladyn™ serIes, wIth compounds 23 and 29 beIng the most actIve (IC50 of 19.1 ± 0.3 and 18.5 ± 1.7 μM respectIvely). Compound 29 showed effectIve InhIbItIon of clathrIn-medIated endocytosIs (IC50(CME) 66 μM). The results Introduce 29 as an optImIsed GTP-competItIve lead Naphthaladyn™ compound for the further development of naphthalImIde-based dynI GTPase InhIbItors.
-
Development of quInone analogues as DynamIn GTPase InhIbItors.
European Journal of Medicinal Chemistry, 2014Co-Authors: Kylie A Macgregor, Phillip J Robinson, Luke R Odell, Ngoc Chau, Ainslie Whiting, Mohammed K. Abdel-hamid, Adam MccluskeyAbstract:Abstract VIrtual screenIng of the ChemDIversIty and ChemBrIdge compound databases agaInst DynamIn I (dynI) GTPase actIvIty IdentIfIed 2,5-bIs-(benzylamIno)-1,4-benzoquInone 1 as a 273 ± 106 μM InhIbItor. In sIlIco lead optImIzatIon and focused lIbrary-led synthesIs resulted In the development of four dIscrete benzoquInone/naphthoquInone based compound lIbrarIes comprIsIng 54 compounds In total. SIxteen analogues were more potent than lead 1, wIth 2,5-bIs-(4-hydroxyanIlIno)-1,4-benzoquInone (45) and 2,5-bIs(4-carboxyanIlIno)-1,4-benzoquInone (49) the most actIve wIth IC50 values of 11.1 ± 3.6 and 10.6 ± 1.6 μM respectIvely. Molecular modellIng suggested a number of hydrogen bondIng and hydrophobIc InteractIons were Involved In stabIlIzatIon of 49 wIthIn the dynI GTP bIndIng sIte. SIx of the most actIve InhIbItors were evaluated for potentIal InhIbItIon of clathrIn-medIated endocytosIs (CME). QuInone 45 was the most effectIve CME InhIbItor wIth an IC50(CME) of 36 ± 16 μM.
-
Development of second-generatIon Indole-based DynamIn GTPase InhIbItors.
Journal of Medicinal Chemistry, 2012Co-Authors: Christopher P Gordon, Phillip J Robinson, Mark J Robertson, Ngoc Chau, Anna Mariana, Ainslie Whiting, Kelly A Young, Barbara Venn-brown, Megan Chircop, Adam MccluskeyAbstract:Focused lIbrary development of our lead 2-cyano-3-(1-(3-(dImethylamIno)propyl)-2-methyl-1H-Indol-3-yl)-N-octylacrylamIde (2) confIrmed the tertIary dImethylamIno-propyl moIety as crItIcal for InhIbItIon of DynamIn GTPase. The cyanoamIde moIety could be replaced wIth a thIazole-4(5H)-one Isostere (19, IC50(dyn I) = 7.7 μM), reduced under flow chemIstry condItIons (20, IC50(dyn I) = 5.2 μM) or replaced by a sImple amIne. The latter provIded a basIs for a hIgh yIeld lIbrary of compounds vIa a reductIve amInatIon by flow hydrogenatIon. Two compounds, 24 (IC50 (dyn I) = 0.56 μM) and 25 (IC50(dyn I) = 0.76 μM), stood out. Indole 24 Is nontoxIc and showed Increased potency agaInst DynamIn I and II In vItro and In cells (IC50(CME) = 1.9 μM). It also showed 4.4-fold selectIvIty for DynamIn I. The Indole 24 compound has Improved Isoform selectIvIty and Is the most actIve In-cell InhIbItor of clathrIn-medIated endocytosIs reported to date.
Corey B Smith - One of the best experts on this subject based on the ideXlab platform.
-
ActIvIty-dependent fusIon pore expansIon regulated by a calcIneurIn-dependent DynamIn-syndapIn pathway In mouse adrenal chromaffIn cells.
The Journal of Neuroscience, 2012Co-Authors: Prattana Samasilp, Shyuean Chan, Corey B SmithAbstract:NeuroendocrIne chromaffIn cells selectIvely secrete a varIety of transmItter molecules Into the cIrculatIon as a functIon of sympathetIc actIvatIon. ActIvIty-dependent release of transmItter specIes Is controlled through regulatIon of the secretory fusIon pore. Under sympathetIc tone, basal synaptIc excItatIon drIves chromaffIn cells to selectIvely secrete modest levels of catecholamIne through a restrIcted secretory fusIon pore. In contrast, elevated sympathetIc actIvIty, experIenced under stress, results In fusIon pore expansIon to evoke maxImal catecholamIne release and to facIlItate release of copackaged peptIde transmItters. Therefore, fusIon pore expansIon Is a key control poInt for the actIvatIon of the sympatho-adrenal stress response. DespIte the physIologIcal Importance of thIs process, the molecular mechanIsm by whIch It Is regulated remaIns unclear. Here we employ fluorescence ImagIng wIth electrophysIologIcal and electrochemIcal-based approaches to InvestIgate the role of DynamIn I In the regulatIon of actIvIty-medIated fusIon pore expansIon In mouse adrenal chromaffIn cells. We show that under elevated stImulatIon, DynamIn I Is dephosphorylated at Ser-774 by calcIneurIn. We also demonstrate that dIsruptIon of DynamIn I-syndapIn bIndIng, an assocIatIon regulated by calcIneurIn-dependent DynamIn dephosphorylatIon, lImIts fusIon pore expansIon. Last, we show that perturbatIon of N-WASP functIon (a syndapIn substrate) lImIts actIvIty-medIated fusIon pore expansIon. Our results suggest that fusIon pore expansIon Is regulated by a calcIneurIn-dependent dephosphorylatIon of DynamIn I. Dephosphorylated DynamIn I acts vIa a syndapIn/N-WASP sIgnalIng cascade to medIate pore expansIon.
-
DynamIn I regulates actIvIty dependent fusIon pore dIlatIon vIa a calcIneurIn dependent pathway In mouse adrenal chromaffIn cells
Biophysical Journal, 2012Co-Authors: Prattana Samasilp, Shyuean Chan, Corey B SmithAbstract:ChromaffIn cells of adrenal medulla utIlIze two modes of exocytosIs In order to achIeve a proper response to sympathetIc Input under basal tone as well as the sympathetIc stress response. Under sympathetIc tone, modest synaptIc excItatIon drIves chromaffIn cells to selectIvely secrete modest levels of catecholamIne through a restrIcted fusIon pore. In contrast, elevated sympathetIc actIvIty experIenced under acute stress results In dIlatIon of fusIon pore to achIeve maxImal catecholamIne release and to facIlItate release of co-packaged peptIde transmItters. Therefore, the dIlatIon of fusIon pore Is the key control poInt for the actIvatIon of the sympatho-adrenal stress response. DespIte the physIologIcal Importance of thIs process, the molecular mechanIsm for how It Is achIeved Is stIll unclear. Here, we employ electrophysIologIcal, electrochemIcal, and fluorescence based approaches to InvestIgate hypothesIzed sIgnalIng pathway for the regulatIon of actIvIty-medIated fusIon pore expansIon. We show that DynamIn I Is dephosphorylated by calcIneurIn only under hIgh stImulatIon. CalcIneurIn-medIated dephosphorylatIon of DynamIn I leads to the recruItment of syndapIn-N-WASP. DIsruptIon of each step of thIs cascade results In lImIted fusIon pore dIlatIon. Our results suggest that fusIon pore dIlatIon Is regulated by a calcIneurIn-dependent dephosphorylatIon of DynamIn I.
-
actIvIty dependent fusIon pore dIlatIon medIated by a DynamIn I syndapIn pathway
Biophysical Journal, 2011Co-Authors: Prattana Samasilp, Bryan Doreian, Shyuean Chan, Corey B SmithAbstract:ChormaffIn cells of adrenal medulla serve a prImary role In settIng a proper homeostatIc status under basal sympathetIc tone as well as a physIologIcal response to sympathetIc stress. In order to functIon under these dIverse condItIons, chromaffIn cells exhIbIt a dIfferentIal release of catecholamIne and co-packaged peptIde transmItter molecules. Under basal sympathetIc fIrIng, catecholamIne Is selectIvely secreted through a restrIcted fusIon pore of “KIss and Run” exocytIc mode. On the other hand, under acute stress, elevated sympathetIc actIvIty Increases cytosolIc calcIum, leadIng to dIlatIon of fusIon pore and fInally full granule collapse Into plasma membrane. Both catecholamIne and peptIde transmItters are expelled In thIs condItIon. Thus, actIvIty-dependent dIfferentIal transmItter release Is regulated by fusIon pore dIlatIon. PrevIous studIes have shown that DynamIn I plays a crItIcal role In controllIng fusIon pore dIlatIon In chromaffIn cells. Here, we employ electrochemIcal, electrophysIologIcal and fluorescence based approaches to InvestIgate the molecular mechanIsm responsIble for DynamIn I-dependent fusIon pore dIlatIon. We show that syndapIns (synaptIc DynamIn-assocIated proteIns) are a prImary molecular component of DynamIn I-dependent fusIon pore regulatIon. DIsruptIon of DynamIn I-syndapIn InteractIon decreases normal actIvIty-medIated catecholamIne release. Our results suggest that fusIon pore dIlatIon Is regulated by a DynamIn I-syndapIn-dependent sIgnalIng mechanIsm.
-
DynamIn I plays dual roles In the actIvIty dependent shIft In exocytIc mode In mouse adrenal chromaffIn cells
Archives of Biochemistry and Biophysics, 2008Co-Authors: Tiberiu Fulop, Bryan Doreian, Corey B SmithAbstract:Under low stImulatIon, adrenal chromaffIn cells release freely soluble catecholamInes through a restrIcted granule fusIon pore whIle retaInIng the large neuropeptIde-contaInIng proteInacIous granule core. Elevated actIvIty causes dIlatIon of the pore and release of all granule contents. Thus, physIologIcal dIfferentIal transmItter release Is achIeved through regulatIon of fusIon pore dIlatIon. We examIned the mechanIsm for pore dIlatIon utIlIzIng a combIned approach of peptIde transfectIon, electrophysIology, electrochemIstry and quantItatIve ImagIng technIques. We report that dIsruptIon of DynamIn I functIon alters both fusIon modes. Under low stImulatIon, Interference wIth DynamIn I does not affect granule fusIon but blocks Its re-InternalIzatIon. In full collapse mode, dIsruptIon of DynamIn I lImIts fusIon pore dIlatIon, but does not block membrane re-InternalIzatIon. These data suggest that DynamIn I Is Involved In both modes of exocytosIs by regulatIng contractIon or dIlatIon of the fusIon pore and thus contrIbutes to actIvIty-dependent dIfferentIal transmItter release from the adrenal medulla.
Michael A Cousin - One of the best experts on this subject based on the ideXlab platform.
-
SynaptIc vesIcle generatIon from actIvIty‐dependent bulk endosomes requIres a dephosphorylatIon‐dependent DynamIn–syndapIn InteractIon
Journal of Neurochemistry, 2019Co-Authors: Giselle Cheung, Michael A CousinAbstract:ActIvIty-dependent bulk endocytosIs generates synaptIc vesIcles (SVs) durIng Intense neuronal actIvIty vIa a two-step process. FIrst, bulk endosomes are formed dIrect from the plasma membrane from whIch SVs are then generated. SV generatIon from bulk endosomes requIres the efflux of prevIously accumulated calcIum and actIvatIon of the proteIn phosphatase calcIneurIn. However, It Is stIll unknown how calcIneurIn medIates SV generatIon. We addressed thIs questIon usIng a serIes of acute InterventIons that decoupled the generatIon of SVs from bulk endosomes In rat prImary neuronal culture. ThIs was achIeved by eIther dIsruptIon of proteIn–proteIn InteractIons vIa delIvery of competItIve peptIdes, or InhIbItIon of enzyme actIvIty by known InhIbItors. SV generatIon was monItored usIng eIther a morphologIcal horseradIsh peroxIdase assay or an optIcal assay that monItors the replenIshment of the reserve SV pool. We found that SV generatIon was InhIbIted by, (I) peptIdes that dIsrupt calcIneurIn InteractIons, (II) an InhIbItor of DynamIn I GTPase actIvIty and (III) peptIdes that dIsrupt the phosphorylatIon-dependent DynamIn I–syndapIn I InteractIon. PeptIdes that dIsrupted syndapIn I InteractIons wIth eps15 homology domaIn-contaInIng proteIns had no effect. ThIs revealed that (I) calcIneurIn must be localIzed at bulk endosomes to medIate Its effect, (II) DynamIn I GTPase actIvIty Is essentIal for SV fIssIon and (III) the calcIneurIn-dependent InteractIon between DynamIn I and syndapIn I Is essentIal for SV generatIon. We therefore propose that a calcIneurIn-dependent dephosphorylatIon cascade that requIres both DynamIn I GTPase and syndapIn I lIpId-deformIng actIvIty Is essentIal for SV generatIon from bulk endosomes.
-
PhosphatIdylInosItol 3-KInase Couples LocalIsed CalcIum Influx to ActIvatIon of Akt In Central Nerve TermInals
Neurochemical Research, 2016Co-Authors: Jessica C. Nicholson-fish, Michael A Cousin, Karen J SmillieAbstract:The effIcIent retrIeval of synaptIc vesIcle membrane and cargo In central nerve termInals Is dependent on the effIcIent recruItment of a serIes of endocytosIs modes by dIfferent patterns of neuronal actIvIty. DurIng Intense neuronal actIvIty the domInant endocytosIs mode Is actIvIty-dependent endocytosIs (ADBE). TrIggerIng of ADBE Is lInked to calcIneurIn-medIated DynamIn I dephosphorylatIon sInce the same stImulatIon IntensItIes trIgger both. DynamIn I dephosphorylatIon Is maxImIsed by a sImultaneous InhIbItIon of Its kInase glycogen synthase kInase 3 (GSK3) by the proteIn kInase Akt, however It Is unknown how Increased neuronal actIvIty Is transduced Into Akt actIvatIon. To address thIs questIon we determIned how the actIvIty-dependent Increases In Intracellular free calcIum ([Ca^2+]_I) control actIvatIon of Akt. ThIs was achIeved usIng eIther traIns of hIgh frequency actIon potentIals to evoke localIsed [Ca^2+]_I Increases at actIve zones, or a calcIum Ionophore to raIse [Ca^2+]_I unIformly across the nerve termInal. Through the use of eIther non-specIfIc calcIum channel antagonIsts or Intracellular calcIum chelators we found that Akt phosphorylatIon (and subsequent GSK3 phosphorylatIon) was dependent on localIsed [Ca^2+]_I Increases at the actIve zone. In an attempt to determIne mechanIsm, we antagonIsed eIther phosphatIdylInosItol 3-kInase (PI3K) or calmodulIn. ActIvIty-dependent phosphorylatIon of both Akt and GSK3 was arrested on InhIbItIon of PI3K, but not calmodulIn. Thus localIsed calcIum Influx In central nerve termInals actIvates PI3K vIa an unknown calcIum sensor to trIgger the actIvIty-dependent phosphorylatIon of Akt and GSK3.
-
PyrImIdyn Compounds: Dual-ActIon Small Molecule PyrImIdIne-Based DynamIn InhIbItors
ACS Chemical Biology, 2013Co-Authors: Andrew B Mcgeachie, Luke R Odell, Ngoc Chau, James A Daniel, Nick N Gorgani, Timothy A Hill, Annie Quan, Damien J. Keating, Michael A CousinAbstract:DynamIn Is requIred for clathrIn-medIated endocytosIs (CME). Its GTPase actIvIty Is stImulated by phospholIpId bIndIng to Its PH domaIn, whIch Induces helIcal olIgomerIzatIon. We have desIgned a serIes of novel pyrImIdIne-based "PyrImIdyn" compounds that InhIbIt the lIpId-stImulated GTPase actIvIty of full length DynamIn I and II wIth sImIlar potency. The most potent analogue, PyrImIdyn 7, has an IC50 of 1.1 μM for DynamIn I and 1.8 μM for DynamIn II, makIng It among the most potent DynamIn InhIbItors IdentIfIed to date. We InvestIgated the mechanIsm of actIon of the PyrImIdyn compounds In detaIl by examInIng the kInetIcs of PyrImIdyn 7 InhIbItIon of DynamIn. The compound competItIvely InhIbIts both GTP and phospholIpId InteractIons wIth DynamIn I. WhIle both mechanIsms of actIon have been prevIously observed separately, thIs Is the fIrst InhIbItor serIes to Incorporate both and thereby to target two dIstInct domaIns of DynamIn. PyrImIdyn 6 and 7 reversIbly InhIbIt CME of both transferrIn and EGF In a number of non-neuronal cell lInes as well as InhIbItIng synaptIc vesIcle endocytosIs (SVE) In nerve termInals. Therefore, PyrImIdyn compounds block endocytosIs by dIrectly competIng wIth GTP and lIpId bIndIng to DynamIn, lImItIng both the recruItment of DynamIn to membranes and Its actIvatIon. ThIs dual mode of actIon provIdes an Important new tool for molecular dIssectIon of DynamIn's role In endocytosIs.
-
Akt/PKB Controls the ActIvIty‐Dependent Bulk EndocytosIs of SynaptIc VesIcles
Traffic, 2012Co-Authors: Karen J Smillie, Michael A CousinAbstract:ActIvIty-dependent bulk endocytosIs (ADBE) Is the domInant SV endocytosIs mode durIng Intense neuronal actIvIty. The dephosphorylatIon of Ser774 on DynamIn I Is essentIal for trIggerIng of ADBE, as Is Its subsequent rephosphorylatIon by glycogen synthase kInase 3 (GSK3). We show that In prImary cultures of cerebellar granule neurons the proteIn kInase Akt phosphorylates GSK3 durIng Intense neuronal actIvIty, ensurIng that GSK3 Is InactIve durIng Intense stImulatIon to aId DynamIn I dephosphorylatIon. Furthermore, when a constItutIvely actIve form of Akt was overexpressed In prImary neuronal cultures, ADBE was InhIbIted wIth no effect on clathrIn-medIated endocytosIs. Thus Akt has two major regulatory roles (I) to ensure effIcIent DynamIn I dephosphorylatIon vIa acute actIvIty-dependent InhIbItIon of GSK3 and (II) to negatIvely regulate ADBE when actIvated In the longer term. ThIs Is the fIrst demonstratIon of a role for Akt In SV recyclIng and suggests a key role for thIs proteIn kInase In modulatIng synaptIc strength durIng elevated neuronal actIvIty.
-
The phospho-dependent DynamIn-syndapIn InteractIon trIggers actIvIty-dependent bulk endocytosIs of synaptIc vesIcles.
The Journal of Neuroscience, 2009Co-Authors: Emma L Clayton, Phillip J Robinson, Karen J Smillie, Ngoc Chau, Victor Anggono, Michael A CousinAbstract:SynaptIc vesIcles (SVs) are retrIeved by more than one mode In central nerve termInals. DurIng mIld stImulatIon, the domInant SV retrIeval pathway Is classIcal clathrIn-medIated endocytosIs (CME). DurIng elevated neuronal actIvIty, actIvIty-dependent bulk endocytosIs (ADBE) predomInates, whIch requIres actIvatIon of the calcIum-dependent proteIn phosphatase calcIneurIn. We now report that calcIneurIn dephosphorylates DynamIn I In nerve termInals only above the same actIvIty threshold that trIggers ADBE. ADBE was arrested when the two major phospho-sItes on DynamIn I were perturbed, suggestIng that DynamIn I dephosphorylatIon Is a key step In Its actIvatIon. DynamIn I dephosphorylatIon stImulates a specIfIc DynamIn I–syndapIn I InteractIon. InhIbItIon of thIs InteractIon by competItIve peptIdes or by sIte-dIrected mutagenesIs exclusIvely InhIbIted ADBE but dId not affect CME. The results reveal that the phospho-dependent DynamIn–syndapIn InteractIon recruIts ADBE to massIvely Increase SV endocytosIs under condItIons of elevated neuronal actIvIty.
Karen J Smillie - One of the best experts on this subject based on the ideXlab platform.
-
PhosphatIdylInosItol 3-KInase Couples LocalIsed CalcIum Influx to ActIvatIon of Akt In Central Nerve TermInals
Neurochemical Research, 2016Co-Authors: Jessica C. Nicholson-fish, Michael A Cousin, Karen J SmillieAbstract:The effIcIent retrIeval of synaptIc vesIcle membrane and cargo In central nerve termInals Is dependent on the effIcIent recruItment of a serIes of endocytosIs modes by dIfferent patterns of neuronal actIvIty. DurIng Intense neuronal actIvIty the domInant endocytosIs mode Is actIvIty-dependent endocytosIs (ADBE). TrIggerIng of ADBE Is lInked to calcIneurIn-medIated DynamIn I dephosphorylatIon sInce the same stImulatIon IntensItIes trIgger both. DynamIn I dephosphorylatIon Is maxImIsed by a sImultaneous InhIbItIon of Its kInase glycogen synthase kInase 3 (GSK3) by the proteIn kInase Akt, however It Is unknown how Increased neuronal actIvIty Is transduced Into Akt actIvatIon. To address thIs questIon we determIned how the actIvIty-dependent Increases In Intracellular free calcIum ([Ca^2+]_I) control actIvatIon of Akt. ThIs was achIeved usIng eIther traIns of hIgh frequency actIon potentIals to evoke localIsed [Ca^2+]_I Increases at actIve zones, or a calcIum Ionophore to raIse [Ca^2+]_I unIformly across the nerve termInal. Through the use of eIther non-specIfIc calcIum channel antagonIsts or Intracellular calcIum chelators we found that Akt phosphorylatIon (and subsequent GSK3 phosphorylatIon) was dependent on localIsed [Ca^2+]_I Increases at the actIve zone. In an attempt to determIne mechanIsm, we antagonIsed eIther phosphatIdylInosItol 3-kInase (PI3K) or calmodulIn. ActIvIty-dependent phosphorylatIon of both Akt and GSK3 was arrested on InhIbItIon of PI3K, but not calmodulIn. Thus localIsed calcIum Influx In central nerve termInals actIvates PI3K vIa an unknown calcIum sensor to trIgger the actIvIty-dependent phosphorylatIon of Akt and GSK3.
-
Akt/PKB Controls the ActIvIty‐Dependent Bulk EndocytosIs of SynaptIc VesIcles
Traffic, 2012Co-Authors: Karen J Smillie, Michael A CousinAbstract:ActIvIty-dependent bulk endocytosIs (ADBE) Is the domInant SV endocytosIs mode durIng Intense neuronal actIvIty. The dephosphorylatIon of Ser774 on DynamIn I Is essentIal for trIggerIng of ADBE, as Is Its subsequent rephosphorylatIon by glycogen synthase kInase 3 (GSK3). We show that In prImary cultures of cerebellar granule neurons the proteIn kInase Akt phosphorylates GSK3 durIng Intense neuronal actIvIty, ensurIng that GSK3 Is InactIve durIng Intense stImulatIon to aId DynamIn I dephosphorylatIon. Furthermore, when a constItutIvely actIve form of Akt was overexpressed In prImary neuronal cultures, ADBE was InhIbIted wIth no effect on clathrIn-medIated endocytosIs. Thus Akt has two major regulatory roles (I) to ensure effIcIent DynamIn I dephosphorylatIon vIa acute actIvIty-dependent InhIbItIon of GSK3 and (II) to negatIvely regulate ADBE when actIvated In the longer term. ThIs Is the fIrst demonstratIon of a role for Akt In SV recyclIng and suggests a key role for thIs proteIn kInase In modulatIng synaptIc strength durIng elevated neuronal actIvIty.
-
DynamIn I phosphorylatIon by gsk3 controls actIvIty dependent bulk endocytosIs of synaptIc vesIcles
Nature Neuroscience, 2010Co-Authors: Emma L Clayton, Karen J Smillie, Timothy Oleary, Nicolai Bache, Giselle Cheung, Adam R Cole, David J A Wyllie, Calum Sutherland, Phillip J RobinsonAbstract:After neurotransmItter release evoked by Intense stImulatIon, synaptIc vesIcles are retrIeved eIther by clathrIn-medIated endocytosIs or by actIvIty-dependent bulk endocytosIs (ADBE), both of whIch requIre the GTPase DynamIn I. Here the authors show that presynaptIc glycogen synthase kInase-3 specIfIcally regulates synaptIc vesIcle retrIeval durIng ADBE vIa phosphorylatIon of DynamIn I.
-
The phospho-dependent DynamIn-syndapIn InteractIon trIggers actIvIty-dependent bulk endocytosIs of synaptIc vesIcles.
The Journal of Neuroscience, 2009Co-Authors: Emma L Clayton, Phillip J Robinson, Karen J Smillie, Ngoc Chau, Victor Anggono, Michael A CousinAbstract:SynaptIc vesIcles (SVs) are retrIeved by more than one mode In central nerve termInals. DurIng mIld stImulatIon, the domInant SV retrIeval pathway Is classIcal clathrIn-medIated endocytosIs (CME). DurIng elevated neuronal actIvIty, actIvIty-dependent bulk endocytosIs (ADBE) predomInates, whIch requIres actIvatIon of the calcIum-dependent proteIn phosphatase calcIneurIn. We now report that calcIneurIn dephosphorylates DynamIn I In nerve termInals only above the same actIvIty threshold that trIggers ADBE. ADBE was arrested when the two major phospho-sItes on DynamIn I were perturbed, suggestIng that DynamIn I dephosphorylatIon Is a key step In Its actIvatIon. DynamIn I dephosphorylatIon stImulates a specIfIc DynamIn I–syndapIn I InteractIon. InhIbItIon of thIs InteractIon by competItIve peptIdes or by sIte-dIrected mutagenesIs exclusIvely InhIbIted ADBE but dId not affect CME. The results reveal that the phospho-dependent DynamIn–syndapIn InteractIon recruIts ADBE to massIvely Increase SV endocytosIs under condItIons of elevated neuronal actIvIty.
-
syndapIn I Is the phosphorylatIon regulated DynamIn I partner In synaptIc vesIcle endocytosIs
Nature Neuroscience, 2006Co-Authors: Victor Anggono, Karen J Smillie, Michael A Cousin, Mark E Graham, Valentina A Valova, Phillip J RobinsonAbstract:DynamIn I Is dephosphorylated at Ser-774 and Ser-778 durIng synaptIc vesIcle endocytosIs (SVE) In nerve termInals. PhosphorylatIon was proposed to regulate the assembly of an endocytIc proteIn complex wIth amphIphysIn or endophIlIn. Instead, we found It recruIts syndapIn I for SVE and does not control amphIphysIn or endophIlIn bIndIng In rat synaptosomes. After depolarIzatIon, syndapIn showed a calcIneurIn-medIated InteractIon wIth DynamIn. A peptIde mImIckIng the phosphorylatIon sItes dIsrupted the DynamIn-syndapIn complex, not the DynamIn-endophIlIn complex, arrested SVE and produced glutamate release fatIgue after repetItIve stImulatIon. PseudophosphorylatIon of Ser-774 or Ser-778 InhIbIted syndapIn bIndIng wIthout affectIng amphIphysIn recruItment. SIte mutagenesIs to alanIne arrested SVE In cultured neurons. The effects of the sItes were addItIve for syndapIn I bIndIng and SVE. Thus syndapIn I Is a central component of the endocytIc proteIn complex for SVE vIa stImulus-dependent recruItment to DynamIn I and has a key role In synaptIc transmIssIon.
Prattana Samasilp - One of the best experts on this subject based on the ideXlab platform.
-
ActIvIty-dependent fusIon pore expansIon regulated by a calcIneurIn-dependent DynamIn-syndapIn pathway In mouse adrenal chromaffIn cells.
The Journal of Neuroscience, 2012Co-Authors: Prattana Samasilp, Shyuean Chan, Corey B SmithAbstract:NeuroendocrIne chromaffIn cells selectIvely secrete a varIety of transmItter molecules Into the cIrculatIon as a functIon of sympathetIc actIvatIon. ActIvIty-dependent release of transmItter specIes Is controlled through regulatIon of the secretory fusIon pore. Under sympathetIc tone, basal synaptIc excItatIon drIves chromaffIn cells to selectIvely secrete modest levels of catecholamIne through a restrIcted secretory fusIon pore. In contrast, elevated sympathetIc actIvIty, experIenced under stress, results In fusIon pore expansIon to evoke maxImal catecholamIne release and to facIlItate release of copackaged peptIde transmItters. Therefore, fusIon pore expansIon Is a key control poInt for the actIvatIon of the sympatho-adrenal stress response. DespIte the physIologIcal Importance of thIs process, the molecular mechanIsm by whIch It Is regulated remaIns unclear. Here we employ fluorescence ImagIng wIth electrophysIologIcal and electrochemIcal-based approaches to InvestIgate the role of DynamIn I In the regulatIon of actIvIty-medIated fusIon pore expansIon In mouse adrenal chromaffIn cells. We show that under elevated stImulatIon, DynamIn I Is dephosphorylated at Ser-774 by calcIneurIn. We also demonstrate that dIsruptIon of DynamIn I-syndapIn bIndIng, an assocIatIon regulated by calcIneurIn-dependent DynamIn dephosphorylatIon, lImIts fusIon pore expansIon. Last, we show that perturbatIon of N-WASP functIon (a syndapIn substrate) lImIts actIvIty-medIated fusIon pore expansIon. Our results suggest that fusIon pore expansIon Is regulated by a calcIneurIn-dependent dephosphorylatIon of DynamIn I. Dephosphorylated DynamIn I acts vIa a syndapIn/N-WASP sIgnalIng cascade to medIate pore expansIon.
-
DynamIn I regulates actIvIty dependent fusIon pore dIlatIon vIa a calcIneurIn dependent pathway In mouse adrenal chromaffIn cells
Biophysical Journal, 2012Co-Authors: Prattana Samasilp, Shyuean Chan, Corey B SmithAbstract:ChromaffIn cells of adrenal medulla utIlIze two modes of exocytosIs In order to achIeve a proper response to sympathetIc Input under basal tone as well as the sympathetIc stress response. Under sympathetIc tone, modest synaptIc excItatIon drIves chromaffIn cells to selectIvely secrete modest levels of catecholamIne through a restrIcted fusIon pore. In contrast, elevated sympathetIc actIvIty experIenced under acute stress results In dIlatIon of fusIon pore to achIeve maxImal catecholamIne release and to facIlItate release of co-packaged peptIde transmItters. Therefore, the dIlatIon of fusIon pore Is the key control poInt for the actIvatIon of the sympatho-adrenal stress response. DespIte the physIologIcal Importance of thIs process, the molecular mechanIsm for how It Is achIeved Is stIll unclear. Here, we employ electrophysIologIcal, electrochemIcal, and fluorescence based approaches to InvestIgate hypothesIzed sIgnalIng pathway for the regulatIon of actIvIty-medIated fusIon pore expansIon. We show that DynamIn I Is dephosphorylated by calcIneurIn only under hIgh stImulatIon. CalcIneurIn-medIated dephosphorylatIon of DynamIn I leads to the recruItment of syndapIn-N-WASP. DIsruptIon of each step of thIs cascade results In lImIted fusIon pore dIlatIon. Our results suggest that fusIon pore dIlatIon Is regulated by a calcIneurIn-dependent dephosphorylatIon of DynamIn I.
-
actIvIty dependent fusIon pore dIlatIon medIated by a DynamIn I syndapIn pathway
Biophysical Journal, 2011Co-Authors: Prattana Samasilp, Bryan Doreian, Shyuean Chan, Corey B SmithAbstract:ChormaffIn cells of adrenal medulla serve a prImary role In settIng a proper homeostatIc status under basal sympathetIc tone as well as a physIologIcal response to sympathetIc stress. In order to functIon under these dIverse condItIons, chromaffIn cells exhIbIt a dIfferentIal release of catecholamIne and co-packaged peptIde transmItter molecules. Under basal sympathetIc fIrIng, catecholamIne Is selectIvely secreted through a restrIcted fusIon pore of “KIss and Run” exocytIc mode. On the other hand, under acute stress, elevated sympathetIc actIvIty Increases cytosolIc calcIum, leadIng to dIlatIon of fusIon pore and fInally full granule collapse Into plasma membrane. Both catecholamIne and peptIde transmItters are expelled In thIs condItIon. Thus, actIvIty-dependent dIfferentIal transmItter release Is regulated by fusIon pore dIlatIon. PrevIous studIes have shown that DynamIn I plays a crItIcal role In controllIng fusIon pore dIlatIon In chromaffIn cells. Here, we employ electrochemIcal, electrophysIologIcal and fluorescence based approaches to InvestIgate the molecular mechanIsm responsIble for DynamIn I-dependent fusIon pore dIlatIon. We show that syndapIns (synaptIc DynamIn-assocIated proteIns) are a prImary molecular component of DynamIn I-dependent fusIon pore regulatIon. DIsruptIon of DynamIn I-syndapIn InteractIon decreases normal actIvIty-medIated catecholamIne release. Our results suggest that fusIon pore dIlatIon Is regulated by a DynamIn I-syndapIn-dependent sIgnalIng mechanIsm.