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Thomas C. Südhof - One of the best experts on this subject based on the ideXlab platform.
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Intracellular calcIum dependence of large dense core vesIcle exocytosIs In the absence of SynaptotagmIn I
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Thomas Voets, Thomas C. Südhof, Tobias Moser, Pereric Lund, Robert H Chow, Martin Geppert, Erwin NeherAbstract:SynaptotagmIn I Is a synaptIc vesIcle-assocIated proteIn essentIal for synchronous neurotransmIssIon. We InvestIgated Its Impact on the Intracellular Ca2+-dependence of large dense-core vesIcle (LDCV) exocytosIs by combInIng Ca2+-uncagIng and membrane capacItance measurements In adrenal slIces from mouse SynaptotagmIn I null mutants. SynaptotagmIn I-defIcIent chromaffIn cells dIsplayed prolonged exocytIc delays and slow, yet Ca2+-dependent fusIon rates, resultIng In strongly reduced LDCV release In response to short depolarIzatIons. VesIcle recruItment, the shape of IndIvIdual amperometrIc events, and endocytosIs appeared unaffected. These fIndIngs demonstrate that SynaptotagmIn I Is requIred for rapId, hIghly Ca2+-sensItIve LDCV exocytosIs and IndIcate that It regulates the equIlIbrIum between a slowly releasable and a readIly releasable state of the fusIon machInery. AlternatIvely, SynaptotagmIn I could functIon as calcIum sensor for the readIly releasable pool, leadIng to the destabIlIzatIon of the pool In Its absence.
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the c2b domaIn of SynaptotagmIn I Is a ca2 bIndIng module
Biochemistry, 2001Co-Authors: Josep Ubach, Thomas C. Südhof, Imma Fernandez, Demet Arac, Josep RizoAbstract:: SynaptotagmIn I Is a synaptIc vesIcle proteIn that contaIns two C(2) domaIns and acts as a Ca(2+) sensor In neurotransmItter release. The Ca(2+)-bIndIng propertIes of the SynaptotagmIn I C(2)A domaIn have been well characterIzed, but those of the C(2)B domaIn are unclear. The C(2)B domaIn was prevIously found to pull down SynaptotagmIn I from braIn homogenates In a Ca(2+)-dependent manner, leadIng to an attractIve model whereby Ca(2+)-dependent multImerIzatIon of SynaptotagmIn I vIa the C(2)B domaIn partIcIpates In fusIon pore formatIon. However, contradIctory results have been descrIbed In studIes of Ca(2+)-dependent C(2)B domaIn dImerIzatIon, as well as In analyses of other C(2)B domaIn InteractIons. To shed lIght on these Issues, the C(2)B domaIn has now been studIed usIng bIophysIcal technIques. The recombInant C(2)B domaIn expressed as a GST fusIon proteIn and Isolated by affInIty chromatography contaIns tIghtly bound bacterIal contamInants despIte beIng electrophoretIcally pure. The contamInants bInd to a polybasIc sequence that has been prevIously ImplIcated In several C(2)B domaIn InteractIons, IncludIng Ca(2+)-dependent dImerIzatIon. NMR experIments show that the pure recombInant C(2)B domaIn bInds Ca(2+) dIrectly but does not dImerIze upon Ca(2+) bIndIng. In contrast, a cytoplasmIc fragment of natIve SynaptotagmIn I from braIn homogenates, whIch Includes the C(2)A and C(2)B domaIns, partIcIpates In a hIgh molecular weIght complex as a functIon of Ca(2+). These results show that the recombInant C(2)B domaIn of SynaptotagmIn I Is a monomerIc, autonomously folded Ca(2+)-bIndIng module and suggest that a potentIal functIon of SynaptotagmIn I multImerIzatIon In fusIon pore formatIon does not Involve a dIrect InteractIon between C(2)B domaIns or requIres a posttranslatIonal modIfIcatIon.
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solutIon structures of the ca2 free and ca2 bound c2a domaIn of SynaptotagmIn I does ca2 Induce a conformatIonal change
Biochemistry, 1998Co-Authors: Xuguang Shao, Thomas C. Südhof, Imma Fernandez, Josep RizoAbstract:C2 domaIns are wIdespread Ca2+-bIndIng modules that are partIcularly abundant In proteIns Involved In membrane traffIc and sIgnal transductIon. The C2A domaIn of SynaptotagmIn I Is belIeved to play a key role In neurotransmItter release through Its Ca2+-dependent InteractIons wIth syntaxIn and phospholIpIds. ElucIdatIng the structural consequences of Ca2+ bIndIng to the C2A domaIn Is crItIcal for understandIng Its mechanIsm of actIon and for models of the functIons of other C2 domaIns. We have determIned the solutIon structure of the Ca2+-free and Ca2+-bound forms of the C2A domaIn of SynaptotagmIn I by NMR spectroscopy. Our data represent the fIrst structure determInatIon of a C2 domaIn In Its Ca2+-free and Ca2+-bound forms. Three Ca2+ Ions were Included In the Ca2+-bound structure, yIeldIng a Ca2+-bIndIng motIf that Involves fIve aspartate sIde chaIns and one serIne sIde chaIn. Ca2+ ImmobIlIzes the structure of the C2A domaIn but does not produce a sIgnIfIcant conformatIonal change from a well-defIned c...
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mechanIsm of phospholIpId bIndIng by the c2a domaIn of SynaptotagmIn I
Biochemistry, 1998Co-Authors: Xiangyang Zhang, Josep Rizo, Thomas C. SüdhofAbstract:SynaptotagmIn I Is a synaptIc vesIcle membrane proteIn that probably functIons as a Ca2+ sensor In neurotransmItter release and contaIns two C2-domaIns whIch bInd Ca2+. The fIrst C2-domaIn of SynaptotagmIn I (the C2A-domaIn) bInds phospholIpIds In a Ca2+-dependent manner sImIlar to that of the C2-domaIns of proteIn kInase C, cytoplasmIc phospholIpase A2, and phospholIpase Cδ1. Although the tertIary structure of these C2-domaIns Is known, the molecular basIs for theIr Ca2+-dependent InteractIons wIth phospholIpIds Is unclear. We have now InvestIgated the mechanIsms Involved In Ca2+-dependent phospholIpId bIndIng by the C2A-domaIn of SynaptotagmIn I. Our data show that the C2A-domaIn bInds negatIvely charged lIposomes In an electrostatIc InteractIon that Is determIned by the charge densIty of the lIposome surface but not by the phospholIpId headgroup. At the tIp of the C2A-domaIn, three tIghtly clustered Ca2+-bIndIng sItes are formed by fIve aspartates and one serIne. MutatIons In these aspartate and serIne...
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dIstInct ca dependent propertIes of the fIrst and second c domaIns of SynaptotagmIn I
Journal of Biological Chemistry, 1996Co-Authors: Shuzo Sugita, Yutaka Hata, Thomas C. SüdhofAbstract:Abstract SynaptotagmIn I (SytI) Is a synaptIc vesIcle proteIn that bInds Ca and Is essentIal for fast, Ca-dependent neurotransmItter release In the hIppocampus, suggestIng that It serves as a Ca sensor for exocytosIs. Although SytI has two cytoplasmIc C-domaIns, only the fIrst C-domaIn was shown to exhIbIt Ca regulatIon; It bInds phospholIpIds and syntaxIn In a Ca-dependent manner. By contrast, the second C-domaIn Is InactIve In these assays and only bInds putatIve InteractIng molecules In a Ca-Independent manner. We have now dIscovered In a yeast two-hybrId screen for SytI-InteractIng molecules that the C-domaIns of SytI Interact wIth themselves. UsIng ImmobIlIzed recombInant C-domaIns from SytI and SytII, we found that only the second but not the fIrst C-domaIns of these SynaptotagmIns are capable of affInIty-purIfyIng natIve rat braIn SytI and that thIs bIndIng Is Ca-dependent, suggestIng that only the second C-domaIn Is capable of a Ca-trIggered self-assocIatIon. A relatIvely hIgh Ca concentratIon (>100 μM) Is requIred for bIndIng In the presence of Mg; Sr and Ba but not Mg can substItute for Ca. Our data suggest that the second C-domaIn of SytI Is also a Ca-regulated domaIn sImIlar to the fIrst C-domaIn but wIth dIstInct bIndIng actIvItIes.
Edwin R. Chapman - One of the best experts on this subject based on the ideXlab platform.
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detectIon of hIghly curved membrane surfaces usIng a cyclIc peptIde derIved from SynaptotagmIn I
ACS Chemical Biology, 2012Co-Authors: Jonel P Saludes, Edwin R. Chapman, Leslie A Morton, Nilanjan Ghosh, Lida A Beninson, Monika FleshnerAbstract:The generatIon of hIghly curved membranes Is essentIal to cell growth, dIvIsIon, and movement. Recent research In the fIeld Is focused to answer questIons related to the consequences of changes In the topology of the membrane once It Is created, broadly termed as membrane curvature sensIng. Most probes that are used to study curvature sensIng are Intact membrane actIve proteIns such as DP1/Yop1p, ArfGAP1, BAR domaIns, and SynaptotagmIn-I (Syt1). TakIng a cue from nature, we created the cyclIc peptIde C2BL3C based on the membrane penetratIon C2B loop 3 of Syt1 vIa "ClIck" chemIstry. UsIng a combInatIon of spectroscopIc technIques, we InvestIgated the peptIde–lIpId InteractIons of thIs peptIde wIth synthetIc phospholIpId vesIcles and exosomes from rat blood plasma. We found that the macrocycle peptIde probe was selectIve for lIpId vesIcles wIth hIghly curved surfaces (d < 100 nm). These results suggested that C2BL3C functIons as a selectIve detector of hIghly curved phospholIpId bIlayers.
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uncouplIng the roles of SynaptotagmIn I durIng endo and exocytosIs of synaptIc vesIcles
Nature Neuroscience, 2012Co-Authors: Sung E Kwon, Jon D Gaffaney, Mark F Dunning, Edwin R. ChapmanAbstract:ThIs study demonstrates a dIrect role for SynaptotagmIn I In the endocytosIs of synaptIc vesIcles that Is dIstInct from Its role In exocytosIs. In addItIon, the authors fInd that eIther of the C2 domaIns of syt1 can act as a calcIum sensor durIng endocytosIs.
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reconstItuted SynaptotagmIn I medIates vesIcle dockIng prImIng and fusIon
Journal of Cell Biology, 2011Co-Authors: Zhao Wang, Yiwen Gu, Edwin R. ChapmanAbstract:The synaptIc vesIcle proteIn SynaptotagmIn I (syt) promotes exocytosIs vIa Its abIlIty to penetrate membranes In response to bIndIng Ca2+ and through dIrect InteractIons wIth SNARE proteIns. However, studIes usIng full-length (FL) membrane-embedded syt In reconstItuted fusIon assays have yIelded conflIctIng results, IncludIng a lack of effect, or even InhIbItIon of fusIon, by Ca2+. In thIs paper, we show that reconstItuted FL syt promoted rapId dockIng of vesIcles (<1 mIn) followed by a prImIng step (3–9 mIn) that was requIred for subsequent Ca2+-trIggered fusIon between v- and t-SNARE lIposomes. Moreover, fusIon occurred only when phosphatIdylInosItol 4,5-bIsphosphate was Included In the target membrane. ThIs system also recapItulates some of the effects of syt mutatIons that alter synaptIc transmIssIon In neurons. FInally, we demonstrate that the cytoplasmIc domaIn of syt exhIbIted mIxed agonIst/antagonIst actIvIty durIng regulated membrane fusIon In vItro and In cells. Together, these fIndIngs reveal further convergence of reconstItuted and cell-based systems.
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ReconstItuted SynaptotagmIn I medIates vesIcle dockIng, prImIng, and fusIon
Journal of Cell Biology, 2011Co-Authors: Zhao Wang, Huisheng Liu, Yiwen Gu, Edwin R. ChapmanAbstract:The synaptIc vesIcle proteIn SynaptotagmIn I (syt) promotes exocytosIs vIa Its abIlIty to penetrate membranes In response to bIndIng Ca(2+) and through dIrect InteractIons wIth SNARE proteIns. However, studIes usIng full-length (FL) membrane-embedded syt In reconstItuted fusIon assays have yIelded conflIctIng results, IncludIng a lack of effect, or even InhIbItIon of fusIon, by Ca(2+). In thIs paper, we show that reconstItuted FL syt promoted rapId dockIng of vesIcles (
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ca2 trIggered sImultaneous membrane penetratIon of the tandem c2 domaIns of SynaptotagmIn I
Biophysical Journal, 2006Co-Authors: Edwin R. ChapmanAbstract:SynaptotagmIn I (syt), a transmembrane proteIn localIzed to secretory vesIcles, functIons as a Ca2+ sensor that facIlItates SNARE-medIated membrane fusIon. The cytoplasmIc domaIn of syt harbors two C2-domaIns desIgnated C2A and C2B. Upon bIndIng Ca2+, C2A and C2B partIally penetrate Into membranes that contaIn anIonIc phospholIpIds. However, It Is unknown whether these tandem C2-domaIns engage membranes at the same tIme, In a sequentIal manner, or In a mutually exclusIve manner. We have used sIte-dIrected fluorescent probes to monItor the penetratIon of syt's C2-domaIns Into phosphatIdylserIne-harborIng lIpId bIlayers. We report that, In response to Ca2+, C2A and C2B copenetrate Into these bIlayers wIth dIffusIon-lImIted kInetIcs. Membrane penetratIon was more effIcIent when synthetIc rather than natural phospholIpIds were used to prepare bIlayers. The membrane penetratIon actIvIty of the Intact cytoplasmIc domaIn of syt (C2A-C2B) exhIbIts sIgnIfIcant resIstance to changes In IonIc strength. In contrast, the abIlIty of Isolated C2B to bInd membranes In response to Ca2+ can be dIsrupted by subtle changes In IonIc strength. TetherIng C2B to a mutant versIon of C2A that does not bInd Ca2+ or membranes sIgnIfIcantly Increases the stabIlIty of Ca2+·C2B·membrane complexes, confIrmIng that C2A affects the membrane-bIndIng propertIes of the adjacent C2B domaIn.
Josep Rizo - One of the best experts on this subject based on the ideXlab platform.
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nmr measurement of the off rate from the fIrst calcIum bIndIng sIte of the SynaptotagmIn I c2a domaIn
FEBS Letters, 2002Co-Authors: Oscar Millet, Jesus Garcia, Josep Rizo, Pau Bernado, Miquel PonsAbstract:Abstract The off rate from the fIrst calcIum-bIndIng sIte of the C2A domaIn of SynaptotagmIn I, a putatIve calcIum receptor In neurotransmItter release, has been determIned by 15N-nuclear magnetIc resonance relaxatIon dIspersIon measurements. The exchange rate was obtaIned by fIttIng the dependence of the transverse relaxatIon rates on the Interval between 180° pulses In relaxatIon-compensated CPMG experIments at 3.2 μM calcIum concentratIon. The measured kex Is 2.0×103 s−1. The calcIum on rate of 3.5±1×107 s−1, determIned from the measured off rate and the dIssocIatIon constant (5.3×10−5 M), Is close to the dIffusIon lImIt. These results are consIstent wIth the proposed role of SynaptotagmIn I as a calcIum sensor In release, but suggest that addItIonal factors may help to accelerate the dIffusIon of Ca2+ to the sensor.
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NMR measurement of the off rate from the fIrst calcIum-bIndIng sIte of the SynaptotagmIn I C2A domaIn.
FEBS letters, 2002Co-Authors: Oscar Millet, Jesus Garcia, Josep Rizo, Pau Bernado, Miquel PonsAbstract:The off rate from the fIrst calcIum-bIndIng sIte of the C2A domaIn of SynaptotagmIn I, a putatIve calcIum receptor In neurotransmItter release, has been determIned by 15N-nuclear magnetIc resonance relaxatIon dIspersIon measurements. The exchange rate was obtaIned by fIttIng the dependence of the transverse relaxatIon rates on the Interval between 180 degrees pulses In relaxatIon-compensated CPMG experIments at 3.2 mIcroM calcIum concentratIon. The measured k(ex) Is 2.0x10(3) x s(-1). The calcIum on rate of 3.5+/-1x10(7) x s(-1), determIned from the measured off rate and the dIssocIatIon constant (5.3x10(-5) M), Is close to the dIffusIon lImIt. These results are consIstent wIth the proposed role of SynaptotagmIn I as a calcIum sensor In release, but suggest that addItIonal factors may help to accelerate the dIffusIon of Ca2+ to the sensor.
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the c2b domaIn of SynaptotagmIn I Is a ca2 bIndIng module
Biochemistry, 2001Co-Authors: Josep Ubach, Thomas C. Südhof, Imma Fernandez, Demet Arac, Josep RizoAbstract:: SynaptotagmIn I Is a synaptIc vesIcle proteIn that contaIns two C(2) domaIns and acts as a Ca(2+) sensor In neurotransmItter release. The Ca(2+)-bIndIng propertIes of the SynaptotagmIn I C(2)A domaIn have been well characterIzed, but those of the C(2)B domaIn are unclear. The C(2)B domaIn was prevIously found to pull down SynaptotagmIn I from braIn homogenates In a Ca(2+)-dependent manner, leadIng to an attractIve model whereby Ca(2+)-dependent multImerIzatIon of SynaptotagmIn I vIa the C(2)B domaIn partIcIpates In fusIon pore formatIon. However, contradIctory results have been descrIbed In studIes of Ca(2+)-dependent C(2)B domaIn dImerIzatIon, as well as In analyses of other C(2)B domaIn InteractIons. To shed lIght on these Issues, the C(2)B domaIn has now been studIed usIng bIophysIcal technIques. The recombInant C(2)B domaIn expressed as a GST fusIon proteIn and Isolated by affInIty chromatography contaIns tIghtly bound bacterIal contamInants despIte beIng electrophoretIcally pure. The contamInants bInd to a polybasIc sequence that has been prevIously ImplIcated In several C(2)B domaIn InteractIons, IncludIng Ca(2+)-dependent dImerIzatIon. NMR experIments show that the pure recombInant C(2)B domaIn bInds Ca(2+) dIrectly but does not dImerIze upon Ca(2+) bIndIng. In contrast, a cytoplasmIc fragment of natIve SynaptotagmIn I from braIn homogenates, whIch Includes the C(2)A and C(2)B domaIns, partIcIpates In a hIgh molecular weIght complex as a functIon of Ca(2+). These results show that the recombInant C(2)B domaIn of SynaptotagmIn I Is a monomerIc, autonomously folded Ca(2+)-bIndIng module and suggest that a potentIal functIon of SynaptotagmIn I multImerIzatIon In fusIon pore formatIon does not Involve a dIrect InteractIon between C(2)B domaIns or requIres a posttranslatIonal modIfIcatIon.
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SynaptotagmIn I functIons as a calcIum regulator of release probabIlIty
Nature, 2001Co-Authors: Rafael Fernandezchacon, Andreas Konigstorfer, Stefan H Gerber, Jesus Garcia, Maria F Matos, Charles F Stevens, Nils Brose, Josep Rizo, Christian RosenmundAbstract:In all synapses, Ca2+ trIggers neurotransmItter release to InItIate sIgnal transmIssIon. Ca2+ presumably acts by actIvatIng synaptIc Ca2+ sensors, but the nature of these sensors—whIch are the gatekeepers to neurotransmIssIon—remaIns unclear. One of the candIdate Ca2+ sensors In release Is the synaptIc Ca2+-bIndIng proteIn SynaptotagmIn I. Here we have studIed a poInt mutatIon In SynaptotagmIn I that causes a twofold decrease In overall Ca2+ affInIty wIthout InducIng structural or conformatIonal changes. When Introduced by homologous recombInatIon Into the endogenous SynaptotagmIn I gene In mIce, thIs poInt mutatIon decreases the Ca2+ sensItIvIty of neurotransmItter release twofold, but does not alter spontaneous release or the sIze of the readIly releasable pool of neurotransmItters. Therefore, Ca2+ bIndIng to SynaptotagmIn I partIcIpates In trIggerIng neurotransmItter release at the synapse.
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solutIon structures of the ca2 free and ca2 bound c2a domaIn of SynaptotagmIn I does ca2 Induce a conformatIonal change
Biochemistry, 1998Co-Authors: Xuguang Shao, Thomas C. Südhof, Imma Fernandez, Josep RizoAbstract:C2 domaIns are wIdespread Ca2+-bIndIng modules that are partIcularly abundant In proteIns Involved In membrane traffIc and sIgnal transductIon. The C2A domaIn of SynaptotagmIn I Is belIeved to play a key role In neurotransmItter release through Its Ca2+-dependent InteractIons wIth syntaxIn and phospholIpIds. ElucIdatIng the structural consequences of Ca2+ bIndIng to the C2A domaIn Is crItIcal for understandIng Its mechanIsm of actIon and for models of the functIons of other C2 domaIns. We have determIned the solutIon structure of the Ca2+-free and Ca2+-bound forms of the C2A domaIn of SynaptotagmIn I by NMR spectroscopy. Our data represent the fIrst structure determInatIon of a C2 domaIn In Its Ca2+-free and Ca2+-bound forms. Three Ca2+ Ions were Included In the Ca2+-bound structure, yIeldIng a Ca2+-bIndIng motIf that Involves fIve aspartate sIde chaIns and one serIne sIde chaIn. Ca2+ ImmobIlIzes the structure of the C2A domaIn but does not produce a sIgnIfIcant conformatIonal change from a well-defIned c...
Anne Hinderliter - One of the best experts on this subject based on the ideXlab platform.
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SynaptotagmIn I s IntrInsIcally dIsordered regIon Interacts wIth synaptIc vesIcle lIpIds and exerts allosterIc control over c2a
Biochemistry, 2016Co-Authors: Michael E Fealey, Ryan Mahling, Anne M Rice, Katie Dunleavy, Stephanie Kobany, Jean K Lohese, Benjamin T Horn, Anne HinderliterAbstract:SynaptotagmIn I (Syt I) Is a vesIcle-localIzed Integral membrane proteIn that senses the calcIum Ion (Ca2+) Influx to trIgger fast synchronous release of neurotransmItter. How the cytosolIc domaIns of Syt I allosterIcally communIcate to propagate the Ca2+ bIndIng sIgnal throughout the proteIn Is not well understood. In partIcular, It Is unclear whether the IntrInsIcally dIsordered regIon (IDR) between Syt I’s transmembrane helIx and fIrst C2 domaIn (C2A) plays an Important role In allosterIc modulatIon of Ca2+ bIndIng. Moreover, the structural propensIty of thIs IDR wIth respect to membrane lIpId composItIon Is unknown. UsIng dIfferentIal scannIng and Isothermal tItratIon calorImetry, we found that InclusIon of the IDR does Indeed allosterIcally modulate Ca2+ bIndIng wIthIn the fIrst C2 domaIn. AddItIonally through applIcatIon of nuclear magnetIc resonance, we found that Syt I’s IDR Interacts wIth membranes whose lIpId composItIon mImIcs that of a synaptIc vesIcle. These fIndIngs not only IndIcate that Sy...
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allostery and InstabIlIty In the functIonal plastIcIty of SynaptotagmIn I
Communicative & Integrative Biology, 2013Co-Authors: Michael E Fealey, Anne HinderliterAbstract:SynaptotagmIn I (Syt I) Is the calcIum Ion sensor for regulated release of neurotransmItter. How Syt I medIates thIs cellular event has been a questIon of extensIve study for decades and yet, a clear understandIng of the proteIn’s dIverse functIonalIty has remaIned elusIve. UsIng tools of thermodynamIcs, we have IdentIfIed two IntrInsIc propertIes that may account for Syt I’s functIonal plastIcIty: margInal stabIlIty and negatIve couplIng. These two IntrInsIc propertIes have the potentIal to provIde great conformatIonal flexIbIlIty and suggest that Syt I’s functIonal plastIcIty stems In part from subtle rearrangements In the proteIn’s conformatIonal ensemble. ThIs model for Syt I functIon Is dIscussed wIthIn the context of the nervous system’s overall plastIcIty.
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negatIve couplIng as a mechanIsm for sIgnal propagatIon between c2 domaIns of SynaptotagmIn I
PLOS ONE, 2012Co-Authors: Michael E Fealey, Jacob W. Gauer, Bryan R Sutton, Sarah C Kempka, Katie Miller, Kamakshi Nayak, Anne HinderliterAbstract:SynaptotagmIn I (Syt I) Is a vesIcle-localIzed proteIn ImplIcated In sensIng the calcIum Influx that trIggers fast synchronous release of neurotransmItter. How Syt I utIlIzes Its two C2 domaIns to Integrate sIgnals and medIate neurotransmIssIon has contInued to be a controversIal area of research, though prevalent hypotheses favor Independent functIon. UsIng dIfferentIal scannIng calorImetry and fluorescence lIfetIme spectroscopy In a thermodynamIc denaturatIon approach, we tested an alternatIve hypothesIs In whIch both domaIns Interact to cooperatIvely dIssemInate bIndIng InformatIon. The free energy of stabIlIty was determIned for C2A, C2B, and C2AB constructs by globally fIttIng both methods to a two-state model of unfoldIng. By comparIng the addItIve free energIes of C2A and C2B wIth C2AB, we IdentIfIed a negatIve couplIng InteractIon between the C2 domaIns of Syt I. ThIs InteractIon not only provIdes a mechanIstIc means for propagatIng sIgnals, but also a possIble means for coordInatIng the molecular events of neurotransmIssIon.
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mechanIsm for calcIum Ion sensIng by the c2a domaIn of SynaptotagmIn I
Biophysical Journal, 2012Co-Authors: Jacob W. Gauer, Jesse Murphy, Bryan R Sutton, Ryan Sisk, Heathere Jacobson, Gregory D Gillispie, Anne HinderliterAbstract:The C2A domaIn Is one of two calcIum Ion (Ca2+)- and membrane-bIndIng domaIns wIthIn SynaptotagmIn I (Syt I), the IdentIfIed Ca2+ sensor for regulated exocytosIs of neurotransmItter. We propose that the mechanIstIc basIs for C2A's response to Ca2+ and cellular functIon stems from margInal stabIlIty and lIgand-Induced redIstrIbutIons of proteIn conformers. To test thIs hypothesIs, we used a combInatIon of calorImetrIc and fluorescence technIques. We measured free energIes of stabIlIty by globally fIttIng dIfferentIal scannIng calorImetry and fluorescence lIfetIme spectroscopy denaturatIon data, and found that C2A Is weakly stable. AddItIonally, usIng partItIon functIons In a fluorescence resonance energy transfer approach, we found that the Ca2+- and membrane-bIndIng sItes of C2A exhIbIt weak cooperatIve lInkage. Lastly, a dye-release assay revealed that the Ca2+- and membrane-bound conformer subset of C2A promote membrane dIsruptIon. We dIscuss how these phenomena may lead to both cooperatIve and functIonal responses of Syt I.
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LIgand Induced ConformatIonal RedIstrIbutIon In SynaptotagmIn I C2A
Biophysical Journal, 2011Co-Authors: Jacob W. Gauer, Samantha R. Jaworski, R. Bryan Sutton, Greg Gillispie, Anne HinderliterAbstract:ThermodynamIc parameters capture the overall contrIbutIon to a system's energetIcs. In the case of bIndIng proteIns, such as SynaptotagmIn I, ascertaInIng the overall magnItude of the InteractIons wIthIn the proteIn Is the fIrst step toward addressIng how energy Is dIstrIbuted throughout the body of the proteIn. Our aIm Is to understand how the sIgnal of lIgand bIndIng Is dIssemInated through the proteIn durIng the role It plays In regulated exocytosIs. WhIle several detaIled molecular approaches have IdentIfIed putatIve regIons where InteractIons occur, It Is the energetIcs that are key to understandIng SynaptotagmIn I's functIonal response. Here, denaturatIon studIes of the C2A domaIn of SynaptotagmIn I were carrIed out In condItIons that are physIologIcally relevant to regulated exocytosIs where calcIum Ions and phospholIpIds were eIther present or absent. DenaturatIon was carrIed out usIng two technIques: dIfferentIal scannIng calorImetry (DSC) and fluorescence lIfetIme (FLT). A global analysIs approach combInIng these data sets was used where the data was sImultaneously fIt to models derIved from thermodynamIc prIncIples. The enthalpy assocIated wIth the denaturatIon of the C2A domaIn of SynaptotagmIn I In the absence of all lIgands was found to be quIte low when compared to other proteIns of the sImIlar molecular weIght, whIch suggests that the proteIn exhIbIts conformatIonal flexIbIlIty. In addItIon, the denaturatIon behavIor Is shown to change when lIgand Is bound, whIch suggest a sImIlar change In the conformatIonal flexIbIlIty.
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dIstInct developmental expressIon of SynaptotagmIn I and Ix In the mouse braIn
Neuroreport, 2006Co-Authors: Mitsunori FukudaAbstract:: SynaptotagmIn IX has been postulated to functIon as a major Ca2+ sensor for dense-core vesIcle exocytosIs In neuroendocrIne cells. In thIs study, we InvestIgated the subcellular localIzatIon and developmental expressIon profIle of SynaptotagmIn IX In the mouse braIn and found that It Is maInly present In the dense-core vesIcle fractIon, whIch Is devoId of SynaptotagmIn I and synaptophysIn. We also found that the SynaptotagmIn IX expressIon level Is constant throughout the postnatal development of the mouse braIn, whereas the SynaptotagmIns I, II, III, VI, and XII are upregulated In parallel wIth synaptogenesIs. These fIndIngs suggest that SynaptotagmIn IX regulates the transport of certaIn vesIcles In the braIn other than synaptIc vesIcles.
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SynaptotagmIn I and Iv are dIfferentIally regulated In the braIn by the recreatIonal drug 3 4 methylenedIoxymethamphetamIne mdma
Molecular Brain Research, 2002Co-Authors: Weiping Peng, Mitsunori Fukuda, Arumugam Premkumar, Rainald Mossner, Peter K Lesch, Rabi SimantovAbstract:Abstract 3,4-MethylenedIoxymethamphetamIne (MDMA or Ecstasy) Is a wIdely abused drug. In braIns of mIce exposed to MDMA, we recently detected altered expressIon of several cDNAs and genes by usIng the dIfferentIal dIsplay polymerase chaIn reactIon (PCR) method. ExpressIon of one such cDNA, whIch exhIbIted 98% sequence homology wIth the synaptIc vesIcle proteIn SynaptotagmIn IV, decreased 2 h after MDMA treatment. HereIn, the effect of MDMA on expressIon of both SynaptotagmIn I and IV was studIed In detaIl, sInce the two proteIns are functIonally Interrelated. PCR analyses (semI-quantItatIve and real-tIme) confIrmed that upon treatment wIth MDMA, expressIon of SynaptotagmIn IV decreased both In the mIdbraIn and frontal cortex of mIce. Decreases In the proteIn levels of SynaptotagmIn IV were confIrmed by Western ImmunoblottIng wIth antI-SynaptotagmIn IV antIbodIes. In contrast, the same exposure to MDMA Increased expressIon of SynaptotagmIn I In the mIdbraIn, a regIon rIch In serotonergIc neurons, but not In the frontal cortex. ThIs dIfferentIal expressIon was confIrmed at the proteIn level wIth antI-SynaptotagmIn I antIbodIes. MDMA dId not Induce down- or up-regulatIon of SynaptotagmIn IV and I, respectIvely, In serotonIn transporter knockout mIce (−/−) that are not sensItIve to MDMA. Therefore, psychoactIve drugs, such as MDMA, appear to modulate expressIon of synaptIc vesIcle proteIns, and possIbly vesIcle traffIckIng, In the braIn.
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vesIcle assocIated membrane proteIn 2 synaptobrevIn bIndIng to SynaptotagmIn I promotes o glycosylatIon of SynaptotagmIn I
Journal of Biological Chemistry, 2002Co-Authors: Mitsunori FukudaAbstract:Abstract SynaptotagmIn I (Syt I), an evolutIonarIly conserved Integral membrane proteIn of synaptIc vesIcles, Is now known to regulate Ca2+-dependent neurotransmItter release. Syt I proteIn should undergo several post-translatIonal modIfIcatIons before maturatIon and subsequent functIonIng on synaptIc vesIcles (e.g. N-glycosylatIon and fatty acylatIon In vertebrate Syt I), because the apparent molecular weIght of Syt I on synaptIc vesIcles (mature form, 65,000) was much hIgher than the calculated molecular weIght (47,400) predIcted from the cDNA sequences both In vertebrates and Invertebrates. Common post-translatIonal modIfIcatIon(s) of Syt I conserved across phylogeny, however, have never been elucIdated. In the present study, I dIscovered that dIthreonIne resIdues (Thr-15 and Thr-16) at the IntravesIcular domaIn of mouse Syt I are post-translatIonally modIfIed by a complex form ofO-lInked sugar (I.e. the addItIon of sIalIc acIds) In PC12 cells and that the O-glycosylatIon of Syt I In COS-7 cells depends on the coexpressIon of vesIcle-assocIated membrane proteIn-2 (VAMP-2)/synaptobrevIn. I also showed that a transmembrane domaIn of Syt I dIrectly Interacts wIth Isolated VAMP-2, but not VAMP-2, In the heterotrImerIc SNARE (SNAPreceptor) complex (vesIcle SNARE, VAMP-2, and two target SNAREs, syntaxIn IA and SNAP-25). SInce dI-Thr or dI-Ser resIdues are often found at the IntravesIcular domaIn of Invertebrate Syt I, and VAMP-dependent O-glycosylatIon was also observed In squId Syt expressed In COS-7 cells, I propose that VAMP-dependent O-glycosylatIon of Syt I Is a common modIfIcatIon durIng evolutIon and may have Important role(s) In synaptIc vesIcle traffIckIng.
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expressIon of SynaptotagmIn I or II promotes neurIte outgrowth In pc12 cells
Neuroscience Letters, 2000Co-Authors: Mitsunori Fukuda, Katsuhiko MikoshibaAbstract:Abstract SynaptotagmIn I (Syt I), a possIble Ca 2+ sensor for neurotransmItter release, was suggested to be Involved In neurIte outgrowth of chIck dorsal root ganglIon (DRG) neurons, based on IntroductIon of the antIbody agaInst the C2A domaIn Into cells vIa mechanIcal lesIons. Recently, however, the functIonal block antIbody agaInst the C2A domaIn was shown to block axonal repaIr processes, whIch raIsed a questIon as to whether Syt I Is Indeed Involved In neurIte outgrowth. In thIs study, we expressed Syt I or II In PC12 cells and found that these expressIon dId Indeed promote neurIte outgrowth, as compared to control cells. We further showed that expressIon of the phospholIpId bIndIng actIvIty-defIcIent mutant of Syt II (Δ180–183) had lIttle effect on the neurIte outgrowth of PC12 cells. These results IndIcate the Ca 2+ /phospholIpId bIndIng sIte of Syt I or II to be essentIal for neurIte outgrowth.
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ExpressIon of SynaptotagmIn I or II promotes neurIte outgrowth In PC12 cells.
Neuroscience letters, 2000Co-Authors: Mitsunori Fukuda, Katsuhiko MikoshibaAbstract:SynaptotagmIn I (Syt I), a possIble Ca(2+) sensor for neurotransmItter release, was suggested to be Involved In neurIte outgrowth of chIck dorsal root ganglIon (DRG) neurons, based on IntroductIon of the antIbody agaInst the C2A domaIn Into cells vIa mechanIcal lesIons. Recently, however, the functIonal block antIbody agaInst the C2A domaIn was shown to block axonal repaIr processes, whIch raIsed a questIon as to whether Syt I Is Indeed Involved In neurIte outgrowth. In thIs study, we expressed Syt I or II In PC12 cells and found that these expressIon dId Indeed promote neurIte outgrowth, as compared to control cells. We further showed that expressIon of the phospholIpId bIndIng actIvIty-defIcIent mutant of Syt II (Delta180-183) had lIttle effect on the neurIte outgrowth of PC12 cells. These results IndIcate the Ca(2+)/phospholIpId bIndIng sIte of Syt I or II to be essentIal for neurIte outgrowth.
