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1.
J Vis Exp ; (85)2014 Mar 03.
Article in English | MEDLINE | ID: mdl-24638074

ABSTRACT

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.


Subject(s)
Botulinum Toxins/isolation & purification , Complex Mixtures/analysis , Animals , High-Throughput Screening Assays/methods , Mice
2.
Appl Environ Microbiol ; 78(21): 7687-97, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22923410

ABSTRACT

Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-µl samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices.


Subject(s)
Antibodies/immunology , Botulinum Toxins, Type A/analysis , Botulinum Toxins/analysis , High-Throughput Screening Assays , Animals , Biological Assay , Botulinum Toxins/immunology , Botulinum Toxins/metabolism , Botulinum Toxins, Type A/immunology , Botulinum Toxins, Type A/metabolism , Clostridium botulinum , Daucus carota , Humans , Immunomagnetic Separation , Infant , Infant Formula , Limit of Detection , Magnetite Nanoparticles , Milk , Sensitivity and Specificity , Serum
3.
Appl Environ Microbiol ; 77(21): 7815-22, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21908624

ABSTRACT

Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by Förster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n = 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity.


Subject(s)
Bird Diseases/diagnosis , Blood Chemical Analysis/methods , Botulinum Toxins/blood , Botulism/veterinary , Chemistry Techniques, Analytical/methods , Animals , Birds , Botulism/diagnosis , Fluorescence Resonance Energy Transfer/methods , Great Lakes Region , Immunoprecipitation/methods , Sensitivity and Specificity , Time Factors
4.
Anal Biochem ; 411(2): 200-9, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21216216

ABSTRACT

Methods that do not require animal sacrifice to detect botulinum neurotoxins (BoNTs) are critical for BoNT antagonist discovery and the advancement of quantitative assays for biodefense and pharmaceutical applications. Here we describe the development and optimization of fluorogenic reporters that detect the proteolytic activity of BoNT/A, B, D, E, F, and G serotypes in real time with femtomolar to picomolar sensitivity. Notably, the reporters can detect femtomolar concentrations of BoNT/A in 4h and BoNT/E in 20h, sensitivity that equals that of animal-based methods. The reporters can be used to determine the specific activity of BoNT preparations with intra- and inter-assay coefficients of variation of approximately 10%. Finally, we find that the greater sensitivity of our reporters compared with those used in other commercially available assays makes the former attractive candidates for high-throughput screening of BoNT antagonists.


Subject(s)
Botulinum Toxins/analysis , Spectrometry, Fluorescence/methods , Botulinum Toxins/classification , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Genes, Reporter , Kinetics , Serotyping , Temperature
5.
Biochemistry ; 46(9): 2411-8, 2007 Mar 06.
Article in English | MEDLINE | ID: mdl-17288458

ABSTRACT

Two highly conserved amino acid residues near the C-terminus within the gamma subunit of the mitochondrial ATP synthase form a "catch" with an anionic loop on one of the three beta subunits within the catalytic alphabeta hexamer of the F1 segment [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. Forming the catch is considered to be an essential step in cooperative nucleotide binding leading to gamma subunit rotation. The analogous residues, Arg304 and Gln305, in the chloroplast F1 gamma subunit were changed to leucine and alanine, respectively. Each mutant gamma was assembled together with alpha and beta subunits from Rhodospirillum rubrum F1 into a hybrid photosynthetic F1 that carries out both MgATPase and CaATPase activities and ATP-dependent gamma rotation [Tucker, W. C., Schwarcz, A., Levine, T., Du, Z., Gromet-Elhanan, Z., Richter, M. L. and Haran, G. (2004) J. Biol. Chem. 279, 47415-47418]. Surprisingly, changing Arg304 to leucine resulted in a more than 2-fold increase in the kcat for MgATP hydrolysis. In contrast, changing Gln305 to alanine had little effect on the kcat but completely abolished the well-known stimulatory effect of the oxyanion sulfite on MgATP hydrolysis. The MgATPase activities of combined mutants with both residues substituted were strongly inhibited, whereas the CaATPase activities were inhibited, but to a lesser extent. The results indicate that the C-terminus of the photosynthetic F1 gamma subunit, like its mitochondrial counterpart, forms a catch with the alpha and beta subunits that modulates the nucleotide binding properties of the catalytic site(s). The catch is likely to be part of an activation mechanism, overcoming inhibition by free mg2+ ions, but is not essential for cooperative nucleotide exchange.


Subject(s)
Adenosine Triphosphate/metabolism , Photosynthesis , Proton-Translocating ATPases/metabolism , Anions , Catalysis , Hydrolysis , Models, Molecular , Mutagenesis , Protein Conformation
6.
Nat Struct Mol Biol ; 13(4): 323-30, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16565726

ABSTRACT

In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca(2+) and synaptotagmin-1 (syt). Ca(2+) promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we have used a defined reconstituted fusion assay to determine directly whether syt-t-SNARE interactions couple Ca(2+) to membrane fusion by comparing the effects of Ca(2+)-syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca(2+)-syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca(2+)-syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca(2+)-syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca(2+)-syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca(2+)-syt alters t-SNARE structure.


Subject(s)
Calcium/metabolism , Membrane Fusion/physiology , SNARE Proteins/metabolism , Synaptotagmin I/metabolism , Animals , Exocytosis , Fungal Proteins/genetics , Fungal Proteins/metabolism , In Vitro Techniques , Liposomes , Models, Biological , Mutagenesis, Site-Directed , Nerve Endings/metabolism , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SNARE Proteins/genetics , Synaptosomal-Associated Protein 25/genetics , Synaptosomal-Associated Protein 25/metabolism , Synaptotagmin I/genetics
7.
Biophys J ; 89(4): 2458-72, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16055544

ABSTRACT

Docking and fusion of single proteoliposomes reconstituted with full-length v-SNAREs (synaptobrevin) into planar lipid bilayers containing binary t-SNAREs (anchored syntaxin associated with SNAP25) was observed in real time by wide-field fluorescence microscopy. This enabled separate measurement of the docking rate k(dock) and the unimolecular fusion rate k(fus). On low t-SNARE-density bilayers at 37 degrees C, docking is efficient: k(dock) = 2.2 x 10(7) M(-1) s(-1), approximately 40% of the estimated diffusion limited rate. Full vesicle fusion is observed as a prompt increase in fluorescence intensity from labeled lipids, immediately followed by outward radial diffusion (D(lipid) = 0.6 microm2 s(-1)); approximately 80% of the docked vesicles fuse promptly as a homogeneous subpopulation with k(fus) = 40 +/- 15 s(-1) (tau(fus) = 25 ms). This is 10(3)-10(4) times faster than previous in vitro fusion assays. Complete lipid mixing occurs in <15 ms. Both the v-SNARE and the t-SNARE are necessary for efficient docking and fast fusion, but Ca2+ is not. Docking and fusion were quantitatively similar on syntaxin-only bilayers lacking SNAP25. At present, in vitro fusion driven by SNARE complexes alone remains approximately 40 times slower than the fastest, submillisecond presynaptic vesicle population response.


Subject(s)
Lipid Bilayers/chemistry , Liposomes/chemistry , Membrane Fusion , Microscopy, Fluorescence/methods , Image Enhancement/methods , Membrane Fluidity , Proteolipids/chemistry , Time Factors
8.
Mol Biol Cell ; 16(10): 4755-64, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16093350

ABSTRACT

Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was approximately 400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt.target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+.syt action.


Subject(s)
Barium/pharmacology , Calcium/metabolism , Membrane Fusion/physiology , Strontium/pharmacology , Synaptotagmin I/physiology , Animals , Calcium/pharmacology , Cations, Divalent , Ethylmaleimide/chemistry , Humans , In Vitro Techniques , Liposomes/metabolism , Membrane Fusion/drug effects , Phosphatidylserines/metabolism , Protein Binding , Protein Isoforms/physiology , Rats , SNARE Proteins/metabolism
9.
J Biol Chem ; 280(2): 1652-60, 2005 Jan 14.
Article in English | MEDLINE | ID: mdl-15536080

ABSTRACT

Membrane resealing in mammalian cells after injury depends on Ca(2+)-dependent fusion of intracellular vesicles with the plasma membrane. When cells are wounded twice, the subsequent resealing is generally faster. Physiological and biochemical studies have shown the initiation of two different repair signaling pathways, which are termed facilitated and potentiated responses. The facilitated response is dependent on the generation and recruitment of new vesicles, whereas the potentiated response is not. Here, we report that the two responses can be differentially defined molecularly. Using recombinant fragments of synaptobrevin-2 and synaptotagmin C2 domains we were able to dissociate the molecular requirements of vesicle exocytosis for initial membrane resealing and the facilitated and potentiated responses. The initial resealing response was blocked by fragments of synaptobrevin-2 and the C2B domain of synaptotagmin VII. Both the facilitated and potentiated responses were also blocked by the C2B domain of synaptotagmin VII. Although the initial resealing response was not blocked by the C2AB domain of synaptotagmin I or the C2A domain of synaptotagmin VII, recruitment of new vesicles for the facilitated response was inhibited. We also used Ca2+ binding mutant studies to show that the effects of synaptotagmins on membrane resealing are Ca(2+)-dependent. The pattern of inhibition by synaptotagmin C2 fragments that we observed cannot be used to specify a vesicle compartment, such as lysosomes, in membrane repair.


Subject(s)
Cell Membrane/metabolism , Cell Membrane/pathology , Fibroblasts/cytology , Membrane Fusion , Animals , Calcium/metabolism , Calcium/pharmacology , Calcium-Binding Proteins/antagonists & inhibitors , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/pharmacology , Cell Membrane/drug effects , Exocytosis/drug effects , Kinetics , Membrane Fusion/drug effects , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/pharmacology , Membrane Proteins/chemistry , Membrane Proteins/pharmacology , Mice , NIH 3T3 Cells , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , R-SNARE Proteins , Swiss 3T3 Cells , Synaptotagmin I , Synaptotagmins
10.
J Biol Chem ; 279(46): 47415-8, 2004 Nov 12.
Article in English | MEDLINE | ID: mdl-15377671

ABSTRACT

ATP hydrolysis and synthesis by the F(0)F(1)-ATP synthase are coupled to proton translocation across the membrane in the presence of magnesium. Calcium is known, however, to disrupt this coupling in the photosynthetic enzyme in a unique way: it does not support ATP synthesis, and CaATP hydrolysis is decoupled from any proton translocation, but the membrane does not become leaky to protons. Understanding the molecular basis of these calcium-dependent effects can shed light on the as yet unclear mechanism of coupling between proton transport and rotational catalysis. We show here, using an actin filament gamma-rotation assay, that CaATP is capable of sustaining rotational motion in a highly active hybrid photosynthetic F(1)-ATPase consisting of alpha and beta subunits from Rhodospirillum rubrum and gamma subunit from spinach chloroplasts (alpha(R)(3)beta(R)(3)gamma(C)). The rotation was found to be similar to that induced by MgATP in Escherichia coli F(1)-ATPase molecules. Our results suggest a possible long range pathway that enables the bound CaATP to induce full rotational motion of gamma but might block transmission of this rotational motion into proton translocation by the F(0) part of the ATP synthase.


Subject(s)
Calcium/metabolism , Photosynthesis/physiology , Protein Subunits/metabolism , Proton-Translocating ATPases/metabolism , Recombinant Proteins/metabolism , Actins/metabolism , Adenosine Triphosphate/metabolism , Macromolecular Substances , Protein Subunits/genetics , Proton-Translocating ATPases/genetics , Recombinant Proteins/genetics , Rhodospirillum rubrum/enzymology , Rotation , Spinacia oleracea/enzymology
11.
Science ; 304(5669): 435-8, 2004 Apr 16.
Article in English | MEDLINE | ID: mdl-15044754

ABSTRACT

We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca2+, the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca2+-binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca2+-triggered exocytosis.


Subject(s)
Calcium-Binding Proteins , Calcium/metabolism , Membrane Fusion , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Animals , Binding Sites , Exocytosis , Fluorescence Resonance Energy Transfer , Lipid Bilayers , Lipids/analysis , Liposomes/chemistry , Liposomes/metabolism , Membrane Glycoproteins/chemistry , Membrane Proteins/chemistry , Mice , Mutation , Nerve Tissue Proteins/chemistry , Protein Structure, Tertiary , Qa-SNARE Proteins , R-SNARE Proteins , Rats , Synaptic Vesicles/chemistry , Synaptic Vesicles/metabolism , Synaptosomal-Associated Protein 25 , Synaptotagmin I , Synaptotagmins
12.
Am J Physiol Renal Physiol ; 286(6): F1100-6, 2004 Jun.
Article in English | MEDLINE | ID: mdl-14996668

ABSTRACT

Amiloride-sensitive epithelial Na(+) channels (ENaCs) are subject to modulation by many factors. Recent data have also linked the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery to this regulation of ENaC, but the molecular mechanisms that underlie this modulation are poorly understood. In this study, we demonstrate that syntaxin 1A physically interacts with ENaC and functionally regulates ENaC activity. Syntaxin 1A was able to coimmunoprecipitate in vitro-translated gamma-ENaC, but not alpha- or beta-ENaC. Also, using antibodies raised against alpha-, beta-, or gamma-ENaC, we detected syntaxin 1A in immunoprecipitates from Madin-Darby canine kidney cells stably transfected with alphabetagamma-ENaC. In bilayers, syntaxin 1A inhibited ENaC, and this syntaxin 1A modulation of ENaC activity was eliminated by truncations of cytoplasmic domains of the ENaC subunits. Our findings provide evidence for a direct physical interaction between ENaC and syntaxin 1A and suggest involvement of ENaC's cytoplasmic domains in functional modulation of ENaC activity by syntaxin 1A.


Subject(s)
Antigens, Surface/physiology , Nerve Tissue Proteins/physiology , Sodium Channel Blockers , Sodium Channels/drug effects , Sodium Channels/metabolism , Animals , Blotting, Western , Cell Line , Cytoplasm/drug effects , Cytoplasm/metabolism , Dogs , Electrophoresis, Polyacrylamide Gel , Electrophysiology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Sodium Channels , Immunohistochemistry , Kidney/drug effects , Kidney/metabolism , Lipid Bilayers , Precipitin Tests , Protein Biosynthesis/genetics , RNA, Complementary/biosynthesis , RNA, Complementary/genetics , Sodium Channels/genetics , Syntaxin 1 , Transcription, Genetic , Transfection
13.
Nat Struct Mol Biol ; 11(1): 36-44, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14718921

ABSTRACT

Synaptotagmin-1 (syt), the putative Ca2+ sensor for exocytosis, is anchored to the membrane of secretory organelles. Its cytoplasmic domain is composed of two Ca2+-sensing modules, C2A and C2B. Syt binds phosphatidylinositol 4,5-bisphosphate (PIP2), a plasma membrane lipid with an essential role in exocytosis and endocytosis. We resolved two modes of PIP2 binding that are mediated by distinct surfaces on the C2B domain of syt. A novel Ca2+-independent mode of binding predisposes syt to penetrate PIP2-harboring target membranes in response to Ca2+ with submillisecond kinetics. Thus, PIP2 increases the speed of response of syt and steers its membrane-penetration activity toward the plasma membrane. We propose that syt-PIP2 interactions are involved in exocytosis by facilitating the close apposition of the vesicle and target membrane on rapid time scales in response to Ca2+.


Subject(s)
Calcium-Binding Proteins , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Animals , Calcium Signaling , Exocytosis , In Vitro Techniques , Kinetics , Membrane Glycoproteins/chemistry , Membrane Lipids/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/chemistry , Protein Binding , Protein Structure, Tertiary , Proteolipids/metabolism , R-SNARE Proteins , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Synaptotagmin I , Synaptotagmins , Thermodynamics
14.
J Cell Biol ; 162(2): 199-209, 2003 Jul 21.
Article in English | MEDLINE | ID: mdl-12860971

ABSTRACT

The synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin-SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)-binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.


Subject(s)
Calcium-Binding Proteins , Membrane Proteins/metabolism , Animals , Calcium/metabolism , Catecholamines/metabolism , Exocytosis , Kinetics , Membrane Fusion , Membrane Glycoproteins , Membrane Proteins/chemistry , Nerve Tissue Proteins , Neurons/metabolism , PC12 Cells , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Tertiary , Rats , Recombinant Proteins/metabolism , Synaptotagmins
15.
Proc Natl Acad Sci U S A ; 100(4): 2082-7, 2003 Feb 18.
Article in English | MEDLINE | ID: mdl-12578982

ABSTRACT

Neuronal exocytosis is mediated by Ca(2+)-triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca(2+)-sensing protein proposed to regulate fusion pore dynamics via Ca(2+)-promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca(2+)-triggered oligomerization of syt is a critical step in excitation-secretion coupling; however, this activity has recently been called into question. Here, we show that Ca(2+) does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca(2+).C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are approximately 11 nm long and approximately 11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca(2+) first drives C2A-C2B.membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca(2+)-mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.


Subject(s)
Biopolymers/metabolism , Calcium-Binding Proteins , Lipid Metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Animals , Calcium/metabolism , Rats , Recombinant Proteins/metabolism , Synaptotagmin I , Synaptotagmins
16.
Biochem J ; 366(Pt 1): 1-13, 2002 Aug 15.
Article in English | MEDLINE | ID: mdl-12047220

ABSTRACT

The Ca(2+)-binding synaptic-vesicle protein synaptotagmin I has attracted considerable interest as a potential Ca(2+) sensor that regulates exocytosis from neurons and neuroendocrine cells. Recent studies have shed new light on the structure, biochemical/biophysical properties and function of synaptotagmin, and the emerging view is that it plays an important role in both exocytosis and endocytosis. At least a dozen additional isoforms exist, some of which are expressed outside of the nervous system, suggesting that synaptotagmins might regulate membrane traffic in a variety of cell types. Here we provide an overview of the members of this gene family, with particular emphasis on the question of whether and how synaptotagmin I functions during the final stages of membrane fusion: does it regulate the Ca(2+)-triggered opening and dilation of fusion pores?


Subject(s)
Calcium-Binding Proteins , Calcium/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/physiology , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/physiology , Vesicular Transport Proteins , Animals , Cell Membrane/metabolism , Endocytosis , Exocytosis , Humans , Kinetics , Membrane Fusion , Membrane Proteins/metabolism , Models, Biological , Nervous System/metabolism , Protein Isoforms , Protein Structure, Tertiary , SNARE Proteins , Synaptotagmin I , Synaptotagmins
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