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Cited by 61Cellular transport and secretionNeuroscience and Neuropharmacology ResearchExtracellular vesicles in disease

Ral mediates activity‐dependent growth of postsynaptic membranes via recruitment of the exocyst

Open access
Teodoro, Rita O, Pekkurnaz, Gulçin, Nasser, Abdullah, Higashi‐Kovtun, Misao E, Balakireva, Maria, McLachlan, Ian G, Camonis, Jacques, Schwarz, Thomas L·2013·The EMBO Journal
DOI 10.1038/emboj.2013.147PMC PMC3715865
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Abstract

Remodelling neuronal connections by synaptic activity requires membrane trafficking. We present evidence for a signalling pathway by which synaptic activity and its consequent Ca(2+) influx activate the small GTPase Ral and thereby recruit exocyst proteins to postsynaptic zones. In accord with the ability of the exocyst to direct delivery of post-Golgi vesicles, constitutively active Ral expressed in Drosophila muscle causes the exocyst to be concentrated in the region surrounding synaptic boutons and consequently enlarges the membrane folds of the postsynaptic plasma membrane (the subsynaptic reticulum, SSR). SSR growth requires Ral and the exocyst component Sec5 and Ral-induced enlargement of these membrane folds does not occur in sec5(-/-) muscles. Chronic changes in synaptic activity influence the plastic growth of this membrane in a manner consistent with activity-dependent activation of Ral. Thus, Ral regulation of the exocyst represents a control point for postsynaptic plasticity. This pathway may also function in mammals as expression of activated RalA in hippocampal neurons increases dendritic spine density in an exocyst-dependent manner and increases Sec5 in spines.

AI snapshot

Written from the paper’s full text.

TL;DR

Synaptic activity and Ca²⁺ influx activate the small GTPase Ral, which recruits exocyst components to the Drosophila neuromuscular junction postsynapse and drives activity-dependent growth of the subsynaptic reticulum (SSR). Activated RalA also increases dendritic spine density in cultured hippocampal neurons in an exocyst-dependent manner, suggesting a conserved mechanism for postsynaptic membrane plasticity.

Key findings

  • Muscle expression of constitutively active Ral (Ral^CA) recruited Sec5, Sec15, Sec3, and Sec8 to the synaptic region; Ral^inact did not recruit Sec5, and neuronal Ral^CA did not alter Sec5 distribution.
  • Ral^CA enlarged the SSR in third-instar muscles from 695 ± 27 nm (n = 58) to 1088 ± 50 nm (n = 51), a 56% increase; this expansion did not occur in sec5 mutants (sec5^−/−; Ral^CA = 151 ± 11 nm vs sec5^−/− = 149 ± 9 nm).
  • SSR development required Ral and Sec5: at 60 h, control SSR was 580 ± 33 nm, sec5^−/− was 149 ± 9 nm, ral^PG89 was 240 ± 20 nm, and ral^G0501 was 260 ± 27 nm.
  • Chronic activity changes altered SSR width: with temperature cycling, Shi^ts expression reduced SSR to 586 ± 33 nm vs control 862 ± 57 nm, while dTrpA1 expression increased it to 1157 ± 56 nm. In hippocampal neurons, RalA^CA increased spine density 32% (0.30 ± 0.016 to 0.39 ± 0.023 spines/μm) and PSD95-positive spines by 50%; RalA^CADExo did not increase spine density.

Methods

  • Drosophila larval NMJ: muscle- or neuron-specific Gal4 drivers expressed Ral WT/CA/inact, Ras^CA, Rab11^CA, Sec5, Sec5-IR, dTrpA1, or shi^ts; immunocytochemistry quantified synaptic Sec5 and other markers; TEM measured SSR width around type Ib boutons.
  • Stimulation protocols included calcimycin with/without Ca²⁺, 30 mM glutamate, dTrpA1 activation at 37°C, nerve stimulation at 10 Hz, and chronic temperature shifts/cycling to manipulate activity.
  • Mammalian hippocampal cultures from E18 rat were transfected at DIV19 and analyzed at DIV21 for spine density, PSD95-positive spines, and HA-Sec5 distribution after RalA constructs or 60 mM KCl depolarization; statistics used t-tests or ANOVA with post hoc Bonferroni.

Limitations

  • The paper does not include a dedicated limitations section. It describes the mammalian hippocampal experiments as “a preliminary inquiry” and states that further studies may establish whether the same pathway functions at mammalian synapses.
  • The authors note that chronic TrpA1/shi^ts manipulations produced weaker effects than constant Ral^CA expression, probably because those manipulations are less potent.
  • Evident from the text: the function of the SSR is “poorly understood,” and the mammalian spine findings are largely correlative/overexpression-based, with no direct loss-of-function test of Ral/exocyst in hippocampal spine plasticity.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/emboj.2013.147

Cites

105 of 105 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

40 of 75 cited sources checked · 18 supported · 2 flagged · accuracy not yet measured

  • Premisecitation

    RalA binds directly to the exocyst members Sec5 and Exo84.

    “RalA binds directly to the exocyst members Sec5 and Exo84”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Moskalenko et al, 2002 source
    • cites Moskalenko et al, 2003 source
    • cites Sugihara et al, 2002 source
  • Premisecitation

    Exocytic trafficking from recycling compartments contributes to dendritic spine growth in response to activity.

    “Exocytic trafficking from recycling compartments contributes to dendritic spine growth in response to activity (Park et al, 2006;Kennedy et al, 2010)”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Park et al, 2006 source
    • cites Kennedy et al, 2010 source
  • Interpretationcitation

    The presence of normal EPSPs despite smaller mEPSPs can likely be attributed to a homeostatic increase in quantal content.

    “The presence of normal EPSPs despite smaller mEPSPs can likely be attributed to a homeostatic increase in quantal content (Davis and Bezprozvanny, 2001;Heckscher et al, 2007).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Davis and Bezprozvanny, 2001 source
    • cites Heckscher et al, 2007 source
  • Premisecitation

    Remodelling neuronal connections by synaptic activity requires membrane trafficking.

    “Remodelling neuronal connections by synaptic activity requires membrane trafficking.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Steiner et al, 2002 source
    • cites Lalli and Hall, 2005 source

      The Introduction says neurite branching likely involves membrane delivery, and the Discussion proposes membrane delivery as part of a mechanism promoting branching. Neither passage establishes that remodelling driven by synaptic activity requires membrane trafficking; the paper discusses developmental branching and other cues instead. Page numbers are not available in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    The subcellular localization of the exocyst in these activated muscles is consistent with the known function of the exocyst in directing post-Golgi vesicles to target membranes for localized membrane addition.

    “The subcellular localization of the exocyst in these activated muscles is consistent with the known function of the exocyst in directing post-Golgi vesicles to target membranes for localized membrane addition”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites Zhang et al, 2001 source
    • cites Dupraz et al, 2009 source
    • cites He and Guo, 2009 source
  • Classified as probably not a substantial claim. 32
  • May combine two claims. 60
  • The quote only partly states this claim. 3

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ral is inactivated by PKC phosphorylation of the effector Sec5.

    “and it is inactivated by PKC phosphorylation of the effector Sec5 (Chen et al, 2011).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Chen et al, 2011 source

      The Introduction distinguishes RalA inactivation from modification of its exocyst partner Sec5 and reports that PKC phosphorylation causes Sec5 to dissociate from active RalA. The Results section on allosteric phosphorylation further shows that the released RalA can bind fresh, unphosphorylated Sec5. The paper therefore supports disruption of the RalA–Sec5 interaction, not inactivation of RalA. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ral also has exocyst-independent neuronal functions, including regulation of NMDA receptor endocytosis in response to LTD, via RalBP1.

    “Conversely, Ral also has exocyst-independent neuronal functions, including regulation of NMDA receptor endocytosis in response to LTD, via RalBP1”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Han et al, 2009 source

      The Introduction and Discussion describe RalA–RalBP1 regulation of AMPA receptor endocytosis during NMDA receptor-dependent LTD, not NMDA receptor endocytosis. The Introduction distinguishes this endocytic pathway from RalA’s exocyst-associated exocytic pathway, but the citing sentence identifies the wrong receptor as the endocytosis target. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Syndapin is a peripheral membrane protein located in the SSR and its F-bar domain may contribute to the folding and tubulation of this postsynaptic membrane network.

    “Syndapin is a peripheral membrane protein located in the SSR and its F-bar domain may contribute to the folding and tubulation of this postsynaptic membrane network (Kumar et al, 2009;Wang et al, 2009;Rao et al, 2010).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Kumar et al, 2009 source
    • cites Wang et al, 2009 source
    • cites Rao et al, 2010 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Both Ca2+ and Ras can regulate synaptic development and plasticity.

    “Both Ca 2 þ and Ras can regulate synaptic development and plasticity (Tada and Sheng, 2006;Alvarez and Sabatini, 2007;Harvey et al, 2008).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Tada and Sheng, 2006 source
    • cites Alvarez and Sabatini, 2007 source
    • cites Harvey et al, 2008 source

      The discussion on p. 2 links Ca2+ influx and Ras signaling to long-term potentiation, and the p. 3 synaptic-crosstalk experiment shows that Ras-dependent signaling is necessary for local plasticity. These passages support the plasticity part of the claim, but our copy does not establish that both regulate synaptic development. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    The exocyst is a protein complex that can govern the polarized cell-surface delivery of membrane and membrane proteins.

    “The exocyst is a protein complex that can govern the polarized cell-surface delivery of membrane and membrane proteins (Munson and Novick, 2006;Wu et al, 2008;He and Guo, 2009;Jin et al, 2011).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Munson and Novick, 2006 source
    • cites Wu et al, 2008

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites He and Guo, 2009 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The SSR develops during the second half of larval life and is also present at the NMJs of other arthropods.

    “This structure develops during the second half of larval life and is also present at the NMJs of other arthropods (Jahromi and Atwood, 1974;Rheuben, 1985;Feeney et al, 1998).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Jahromi and Atwood, 1974 source

      The discussion of crayfish synapse growth describes axon branches developing at an unspecified stage of life, not SSR development during the second half of larval life. The paper also notes similar postsynaptic specializations at insect neuromuscular synapses (p. 601), but does not identify them as SSR. Searches for “subsynaptic,” “reticulum,” and “larval” found no passage establishing either part of the claim. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites Rheuben, 1985 source

      The Materials and Methods describe adult Manduca muscles, while the section on plaque and postsynaptic membrane specializations mentions early-fifth-instar larval junctions without stating when the SSR develops. Searches for subsynaptic reticulum, larval development, and its presence at other arthropod NMJs found no passage addressing either part of the claim. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites Feeney et al, 1998

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The exocyst is not required for the exocytosis of synaptic vesicles.

    “although not required for the exocytosis of synaptic vesicles (Murthy et al, 2003)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Murthy et al, 2003 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The distribution of the exocyst within a cell can be highly dynamic, consistent with its role in directing membrane fusion to specific domains.

    “The distribution of the exocyst within a cell can be highly dynamic, consistent with its role in directing membrane fusion to specific domains (Boyd et al, 2004;Beronja et al, 2005;Mehta et al, 2005;Zhang et al, 2008;Murthy et al, 2010).”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites Boyd et al, 2004 source
    • cites Beronja et al, 2005 source
    • cites Mehta et al, 2005 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Membrane addition is critical for permitting neurite outgrowth and branching.

    “Membrane addition is critical for permitting neurite outgrowth and branching (Steiner et al, 2002;Lalli and Hall, 2005)”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Steiner et al, 2002 source
    • cites Lalli and Hall, 2005 source

      The Introduction says neurite branching likely requires membrane delivery, and the Discussion proposes that exocyst-mediated membrane delivery could promote branching. The paper also reports that disrupting exocyst components prevents neurite extension, but it does not establish that membrane addition itself is critical for both outgrowth and branching; its branching mechanism is presented as a possibility. Our copy provides section labels but no page numbers. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The exocyst is required for neurite outgrowth and the addition of neuronal membrane proteins, including the insertion of glutamate receptors, and the maturation of photoreceptors.

    “It is required for neurite outgrowth and the addition of neuronal membrane proteins, including the insertion of glutamate receptors, and the maturation of photoreceptors (Brymora et al, 2001;Vega and Hsu, 2001;Murthy et al, 2003;Sans et al, 2003;Beronja et al, 2005;Liebl et al, 2005;Mehta et al, 2005;Gerges et al, 2006).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Brymora et al, 2001 source

      Our copy states that the exocyst is required for neurite outgrowth (passage [2], labeled Abstract; page unavailable). It does not address the addition of neuronal membrane proteins, glutamate-receptor insertion, or photoreceptor maturation; searches for those terms returned no supporting passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Vega and Hsu, 2001 source

      The section on neurite outgrowth reports that disrupting the exocyst blocks NGF-induced neurite outgrowth in PC12 cells; the Discussion also links vesicle targeting to membrane addition. Our copy does not establish addition of specific neuronal membrane proteins, glutamate-receptor insertion, or photoreceptor maturation (searched for glutamate receptors and photoreceptor maturation). Page numbers are not supplied for the deciding passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Murthy et al, 2003 source

      The Results (page unavailable) conclude that Sec5 is required for neurite outgrowth and show that loss of Sec5 impairs insertion of the mCD8 membrane-protein reporter into neuronal surfaces. Our copy does not establish that this paper supports glutamate-receptor insertion or photoreceptor maturation, the other examples in the multiply cited sentence. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Regulatory interactions of exocyst proteins with several small GTPases, including RalA and RalB, have been identified.

    “have identified regulatory interactions of exocyst proteins with several small GTPases (Lipschutz and Mostov, 2002;Wu et al, 2008), including the two mammalian Ral isoforms, RalA and RalB.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Lipschutz and Mostov, 2002 source

      The indexed text (labeled Abstract; page unavailable) describes exocyst interactions with several small GTPases and explicitly identifies RalA. It does not identify RalB as an interacting isoform, so that part of the claim is not supported by this paper. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Wu et al, 2008

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The interaction of RalA with Sec5 and Exo84 is thought to promote complex assembly, vesicle exocytosis, and membrane addition.

    “this interaction is thought to promote complex assembly, vesicle exocytosis, and membrane addition (Moskalenko et al, 2002(Moskalenko et al, , 2003;;Sugihara et al, 2002;Fukai et al, 2003;Mott et al, 2003;Wang et al, 2004;Jin et al, 2005;Hase et al, 2009).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Moskalenko et al, 2002 source
    • cites Fukai et al, 2003 source

      The ‘Implications of the Sec5–Ral interaction’ section proposes that RalA–Sec5 contact may regulate sec6/8 complex assembly and initiate vesicle tethering before membrane fusion. The Introduction links the exocyst to exocytosis and sites of plasma-membrane expansion. The paper identifies Exo84 as an exocyst component, but does not say that RalA interacts with Exo84 or that the RalA interaction itself promotes membrane addition. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Mott et al, 2003 source

      The Abstract (page not provided) says Ral binds Sec5 and that this interaction is thought necessary to anchor the exocyst to secretory vesicles; it also links Ral to regulation of exocytosis. The concluding passage (page not provided) proposes studying the interaction’s role in exocyst assembly, but does not establish that it promotes assembly. Our copy lists Exo84 as an exocyst component without saying it binds RalA, and does not attribute membrane addition to the RalA–Sec5 interaction. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ral is expressed in the nervous system.

    “Like the exocyst, Ral is expressed in the nervous system (Ngsee et al, 1991;Huber et al, 1994;Peng et al, 2004;Han et al, 2009).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Ngsee et al, 1991 source
    • cites Huber et al, 1994 source
    • cites Peng et al, 2004 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Overexpression of Syndapin promotes the growth of the SSR.

    “Overexpression of Syndapin promotes the growth of the SSR (Kumar et al, 2009)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Kumar et al, 2009 source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Ral can be activated either by Ras indirectly via a Ral-GEF or by Ca2+/calmodulin binding.

    “Ral can be activated either by Ras indirectly via a Ral-GEF (Guanine nucleotide Exchange Factor) or by Ca 2 þ /calmodulin binding (Hofer et al, 1994(Hofer et al, , 1998;;Kikuchi et al, 1994;Wolthuis et al, 1998;Wang and Roufogalis, 1999;Wolthuis and Bos, 1999;Harvey et al, 2008)”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Hofer et al, 1994 source
    • cites Kikuchi et al, 1994 source
    • cites Hofer et al, 1998 source

      The Results and discussion (page not provided) show that Ras can activate Ral and that RalGDS can mediate Ral activation. The Abstract (page not provided) identifies a Ca2+-dependent, Ras-independent pathway in Rat-2 cells, but presents direct Ca2+–calmodulin binding to Ral only as a possible mechanism, not an established route of activation. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Spine growth is likely to be a multifaceted event involving regulated changes in cytoskeletal elements, membrane addition, and receptor trafficking under the control of multiple small GTPases.

    “Indeed spine growth is likely to be a multifaceted event involving regulated changes in cytoskeletal elements, membrane addition, and receptor trafficking under the control of multiple small GTPases”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Park et al, 2004 source
    • cites Murakoshi et al, 2011 source

      The indexed Abstract (page not provided) links spine enlargement to RhoA and Cdc42 signalling and describes Rho GTPases as regulators of the actin cytoskeleton. It does not describe membrane addition or receptor trafficking as components of spine growth, so it supports only part of the attributed claim. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Murakoshi and Yasuda, 2012 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    RalA and the exocyst are known to regulate early stages of neuronal development, including neurite formation.

    “RalA and the exocyst are known to regulate early stages of neuronal development, including neurite formation (Lalli and Hall, 2005).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Lalli and Hall, 2005 source

      The Abstract reports that RalA promotes neurite branching through the exocyst complex, supporting a role in neuronal development. But the Discussion distinguishes branching from neurite formation: neurites still grow when Ral is inhibited, and the authors say Ral is not necessary for neurite extension per se. The claim's reference to neurite formation therefore goes beyond this paper's finding about RalA. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    LTP increases spine volume, a process that requires the addition of membranes whose source remains largely unknown.

    “In particular, LTP increases spine volume, a process that requires the addition of membranes whose source remains largely unknown”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Cooney et al, 2002 source

      The Discussion (page unavailable) addresses the possible sources of synaptic receptors inserted during LTP, not membrane added as spines enlarge. Searches for LTP-related spine volume, enlargement, and membrane addition found no passage in our copy stating that LTP increases spine volume or that the source of membrane for such growth is largely unknown. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
    • cites Park et al, 2006 source
    • cites Bourne and Harris, 2007 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    These findings raise the possibility that the mechanism by which the postsynaptic membrane expands with spine growth in response to glutamate receptor activation may resemble the Ral/exocyst pathway by which the glutamatergic synapse at the fly NMJ expands its postsynaptic membrane.

    “These findings raise the possibility that the mechanism by which the postsynaptic membrane expands with spine growth in response to glutamate receptor activation (Kwon and Sabatini, 2011) may resemble the Ral/exocyst pathway by which the glutamatergic synapse at the fly NMJ expands its postsynaptic membrane.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Kwon and Sabatini, 2011 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Exocyst-mediated AMPA receptor targeting, insertion, and recycling are thought to be independent of Ral.

    “exocystmediated AMPA receptor targeting, insertion, and recycling (Sans et al, 2003;Gerges et al, 2006;Mao et al, 2010) are thought to be independent of Ral.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Sans et al, 2003 source
    • cites Gerges et al, 2006 source

      The Introduction and Discussion describe exocyst-mediated AMPA receptor targeting and insertion, and the section on Sec8 describes its role in constitutive synaptic cycling. Neither those passages nor the searches for Ral and Ral GTPase establish that these processes are independent of Ral. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Mao et al, 2010 source

      The Discussion reports exocyst involvement in AMPA receptor transport, synaptic accumulation, and recycling, supporting that part of the attribution. It does not address Ral or say that these processes are independent of Ral (searched: Ral, Ral-independent exocyst trafficking). Page numbers are unavailable in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Ca2+ entry is a consequence of excitatory transmission in the mammalian brain and mammalian neurons contain both Ral and the exocyst.

    “Ca 2 þ entry is a consequence of excitatory transmission in the mammalian brain and mammalian neurons contain both Ral and the exocyst”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Brymora et al, 2001 source

      The Abstract reports that RalA is enriched in brain and that rat exocyst proteins bind active RalA in nerve terminals, supporting the statement that mammalian neurons contain both. It also says nerve-terminal exocytosis and endocytosis are Ca2+-dependent, but does not say that Ca2+ entry is a consequence of excitatory transmission in the mammalian brain. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    While RalA activity promotes the outgrowth and branching of neurites in newly dissociated neurons in a Sec5-dependent manner, at later stages, it may regulate aspects of dendritic spine dynamics.

    “While RalA activity promotes the outgrowth and branching of neurites in newly dissociated neurons in a Sec5-dependent manner (Lalli and Hall, 2005), at later stages, it may regulate aspects of dendritic spine dynamics.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Lalli and Hall, 2005 source

      The Results (‘Active Ral increases neurite branching’; ‘RalA and -B regulate branching through different effectors’, page unavailable) show that active RalA increases branching in recently plated neurons and that preventing its interaction with the Sec5-containing exocyst impairs this effect. They also report increased axon length with active RalA, but do not establish that this outgrowth effect is Sec5-dependent. The later-stage dendritic-spine suggestion follows the citation and is not attributed to this paper. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Ral, via the exocyst, promotes neurite branching and participates in establishing neuronal polarity.

    “Ral, via the exocyst, promotes neurite branching and participates in establishing neuronal polarity”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Lalli and Hall, 2005 source

      The Introduction and Discussion support the claim that RalA promotes neurite branching through the exocyst complex. But the Discussion says Ral activity is dispensable for establishing neuronal polarity, contrary to the claim’s second part. Page numbers were not provided in our copy. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites Lalli, 2009 source

  • May combine two claims.

    Findingown data

    The increase in the SSR was apparent in each Ral CA larva examined and frequently changed the overall appearance of the SSR.

    “The increase in the SSR was apparent in each Ral CA larva examined and frequently changed the overall appearance of the SSR.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both CD8-GFP fluorescence intensity surrounding boutons and its thickness were increased at the synapse when Ral CA was expressed.

    “We found that both parameters were increased at the synapse when Ral CA was expressed (Supplementary Figure S2D-I), as expected from the EM.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ral CA expression selectively evoked membrane overgrowth of the SSR; total muscle surface area was not changed, invaginations of the membrane were not encountered elsewhere along the muscle surface and other synaptic parameters quantified by TEM were also unchanged.

    “Ral CA expression selectively evoked membrane overgrowth of the SSR; total muscle surface area was not changed, invaginations of the membrane were not encountered elsewhere along the muscle surface and other synaptic parameters quantified by TEM were also unchanged (Supplementary Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SSR growth requires Ral and the exocyst component Sec5.

    “SSR growth requires Ral and the exocyst component Sec5”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of Ral WT in the muscle showed a Ral CA phenotype, though less pronounced, likely due to partial activation of Ral WT.

    “Expression of Ral wild-type (Ral WT ) in the muscle showed a Ral CA phenotype, though less pronounced, likely due to partial activation of Ral WT (Figure 1H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only minor electrophysiological differences were observed between muscles expressing Ral CA and controls: the amplitude of evoked EPSPs was unchanged and the spontaneous mEPSP amplitude and frequency were mildly reduced.

    “Only minor electrophysiological differences were observed between muscles expressing Ral CA and controls: the amplitude of evoked excitatory postsynaptic potentials (EPSPs) was unchanged and the spontaneous miniature EPSP (mEPSP) amplitude and frequency were mildly reduced (Supplementary Figure S2A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Sec15 immunoreactivity and epitope-tagged Sec3-HA and Sec8-HA also became concentrated in the vicinity of the boutons in muscles expressing Ral CA.

    “When we examined other exocyst components, Sec15 immunoreactivity (Figure 1I-J) and the epitope-tagged transgenes Sec3-HA and Sec8-HA (Supplementary Figure S1B-E) also became concentrated in the vicinity of the boutons in muscles expressing Ral CA .”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The SSR of muscles expressing Ral CA was larger in electron micrographs than that of controls, increasing in width by 56% from 695±27 nm (n=58) to 1088±50 nm (n=51).

    “The SSR of muscles expressing Ral CA was obviously larger in electron micrographs than that of controls (Figure 2D and E), increasing in width by 56% from 695±27 nm (n ¼ 58) to 1088±50 nm (n ¼ 51) (Figure 2D and G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GluRIIB receptor and Par-1 levels were increased by 22 and 35%, respectively, and Dlg was reduced by 20% by Ral CA expression.

    “GluRIIB receptor and Par-1 levels were increased by 22 and 35%, respectively, and Dlg was reduced by 20% (Figure 2A; Supplementary Figure S2B and C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Whereas CD8-GFP is always abundant surrounding boutons due to the concentration of membrane in that region, Sec5 moves to the SSR only in response to the activation of Ral.

    “Second, whereas CD8-GFP is always abundant surrounding boutons due to the concentration of membrane in that region, Sec5 moves to the SSR only in response to the activation of Ral.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ral CA surrounded the boutons of both control and sec5-/- muscles even though there was little or no SSR in the sec5-/- muscles.

    “Ral CA surrounded the boutons of both genotypes even though there was little or no SSR in the sec5 À / À muscles (Figure 3K-L).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The growth of the SSR does not occur in sec5 mutants or when Sec5 is selectively knocked down in the muscle by RNAi.

    “First, the growth of the SSR does not occur in sec5 mutants or when Sec5 is selectively knocked down in the muscle by RNAi.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Calcimycin exposure increases synaptic Sec5 enrichment in wild-type larvae in both Ca2+-containing and Ca2+-free saline.

    “Sec5 immunoreactivity (grey) was enriched at synapses (anti-HRP labelled; magenta) of second-instar larvae (60 h) upon calcimycin exposure in Ca 2 þ -containing (E) or Ca 2 þ -free (D) saline.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Calcimycin treatment elevates intracellular Ca2+ in larval muscles.

  • The quote only partly states this claim.

    Findingown data

    Suction electrode stimulation of the nerve for 5 min at 10 Hz in 1 mM Ca2+ significantly increased Sec5 immunoreactivity at the stimulated synapses relative to the unstimulated side.

    “Sec5 immunoreactivity was significantly increased at the stimulated synapses relative to the unstimulated side (Supplementary Figure S7).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Ral localization to the NMJ was independent of Sec5 and of the nucleotide-bound state of Ral.

    “However, Ral localization to the NMJ was independent of Sec5 and of the nucleotide-bound state of Ral.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In sec5 mutants, only one or two SSR folds were visible around most boutons; among 34 boutons examined from three sec5 larvae, only one bouton had a nearly normal thickness of SSR, but it extended only around half the bouton.

    “In these mutants, only one or two SSR folds were visible around most boutons (Figure 3B); among 34 boutons examined from three sec5 larvae, only one bouton had a nearly normal thickness of SSR, but it extended only around half the bouton, with the other half lacking SSR (Supplementary Figure S3E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SSR size and complexity were more variable in ral mutants (ral PG89 = 240 ± 20 nm and ral G0501 = 260 ± 27 nm) than in sec5 mutants, but their SSR was significantly smaller than control larvae.

    “SSR size and complexity were more variable in ral mutants (ral PG89 ¼ 240 ± 20 nm and ral G0501 ¼ 260 ± 27 nm) than in sec5 mutants, but their SSR was significantly smaller than control larvae (Figure 3C, D, and J; Supplementary Figure S3F and G).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Enhancing neuronal activity by expressing dTrpA1 recruits Sec5 to the NMJ.

    “(G, H) Neuronal activity was enhanced by expression of the temperature-activated dTrpA1 (OK6-Gal4/UAS-dTrpA1, G) and compared to control larvae subjected to the same temperature regimen but lacking the channel (OK6-Gal4/ þ , H).”
    • Also stated in: § Hippocampal culture, transfection, and immunostaining · plain
      “(K) of nerve stimulation by dTrpA1 activation (as in G and H).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Expression of dTrpA1 increases neuronal activity.

  • May combine two claims.

    Findingown data

    Although spine density changed, average neck length and head diameter did not change.

    “Although spine density changed, their average neck length and head diameter did not”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    ral G0501 third-instar larvae had a very small SSR compared to control and the SSR at this stage was not significantly different from ral G0501 mutants (237±27) at 60 h.

    “ral G0501 third-instar larvae had a very small SSR compared to control and indeed the SSR at this stage was not significantly different from ral G0501 mutants (237±27) at 60 h.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Ca2+/Ral/exocyst pathway altered postsynaptic morphology independently of other properties of the NMJ: presynaptic properties including bouton number and size were unaffected when SSR growth was promoted and glutamate receptors were largely unaltered.

    “Notably, the Ca 2 þ /Ral/exocyst pathway altered postsynaptic morphology independently of other properties of the NMJ: presynaptic properties including bouton number and size were unaffected when SSR growth was promoted and glutamate receptors were largely unaltered.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RalA CA expression alters spine density without changing spine shape (length and width).

    “(E, F) Cumulative distribution histograms of spine length and width indicate that RalA alters spine density without changing spine shape.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In 60 h larval muscles exposed to 30 mM glutamate, Sec5 was recruited to the NMJ, but only if Ca2+ was present in the medium and only in wild-type but not in ral PG89 mutant larvae.

    “In 60 h, larval muscles exposed to 30 mM glutamate, Sec5 was recruited to the NMJ, but only if Ca 2 þ was present in the medium and only in wild-type but not in ral PG89 mutant larvae (Figure 5A-D and I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The SSR is a plastic structure whose size is regulated by levels of synaptic activity.

    “Finally, we established that the SSR is indeed a plastic structure whose size is regulated by levels of synaptic activity”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither RalA inact nor RalA CADExo altered spine density relative to controls.

    “In contrast, neither RalA inact nor RalA CADExo altered spine density (0.27±0.023 and 0.29±0.022spine/mm) relative to controls.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RalA CA expression increased levels of Sec5 in spines, whereas RalA inact and Ral CADExo expression did not.

    “RalA CA expression increased levels of Sec5 in spines, whereas RalA inact and Ral CADExo expression did not”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the absence of Ca2+ influx, ral PG89 larvae were identical to controls; Ral is therefore not necessary for the expression and resting distribution of Sec5.

    “In the absence of Ca 2 þ influx, ral PG89 larvae were identical to controls; Ral is therefore not necessary for the expression and resting distribution of Sec5.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Ca2+-dependent exocyst recruitment could be accomplished with an ionophore, by applying glutamate, or by nerve stimulation either via direct electrical excitation or activation of dTrpA1 channels.

    “Ca 2 þ -dependent exocyst recruitment could be accomplished with an ionophore, by applying glutamate, or by nerve stimulation either via direct electrical excitation or activation of dTrpA1 channels.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    sec5 mutant muscles had only a rudimentary SSR, considerably thinner (149±9 nm in sec5-/-) than that of controls (580 ± 33 nm).

    “sec5 mutant muscles had only a rudimentary SSR, considerably thinner (149±9 nm in sec5 À / À ) than that of controls (580 ± 33 nm) (Figure 3A, B, and J).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Postsynaptic activation of Ral, but not presynaptic, recruited Sec5, Sec15, Sec3, and Sec8 to the NMJ.

    “We show that postsynaptic activation of Ral, but not presynaptic, recruited to the NMJ Sec5, Sec15, Sec3, and Sec8”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of Sec5 or Ral WT back into the mutant larvae fully rescued SSR development, confirming that the observed defects are specific to these genes.

    “Expression of Sec5 or Ral WT back into the mutant larvae fully rescued SSR development (Supplementary Figure S4C-E), confirming that the observed defects are specific to these genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Even though ral mutants have reduced synaptic arbors, NMJs were present in ral-/- embryos and the ral G0501 allele survives to pupation though no Ral protein is detectable.

    “even though ral mutants have reduced synaptic arbors, NMJs were present in ral À / À embryos and the ral G0501 allele survives to pupation though no Ral protein is detectable.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both glutamate receptor activation and Ca2+ influx through calcimycin promoted Sec5 recruitment selectively to the vicinity of the synapse.

    “Both glutamate receptor activation and Ca 2 þ influx through calcimycin promoted Sec5 recruitment selectively to the vicinity of the synapse”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The six-fold increase in Sec5 intensity caused by Ral CA was far greater than the 1.5-fold change in CD8-GFP; thus, the exocyst was actively recruited to the postsynaptic region and not a passive consequence of the presence of more membrane.

    “The six-fold increase in Sec5 intensity caused by Ral CA was far greater than the 1.5-fold change in CD8-GFP; thus, the exocyst was actively recruited to the postsynaptic region and not a passive consequence of the presence of more membrane.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In control animals, expression of Ral CA induced Sec5 recruitment starting at 48 h and Sec5 levels increased as the SSR expanded at 60 h.

    “In control animals, expression of Ral CA induced Sec5 recruitment starting at 48 h and Sec5 levels increased as the SSR expanded at 60 h (Figure 3E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only constitutively active RalA (RalA CA) increases spine density; inactive RalA and RalA CADexo do not.

    “Only RalA CA increased spine density.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RalA CADexo blocks binding to exocyst members Sec5 and Exo84.

  • May combine two claims.

    Findingown data

    We found that spine density was increased by activated RalA and that both activated RalA and neuronal depolarization increase Sec5 in dendritic spines.

    “We found that spine density was increased by activated RalA and that both activated RalA and neuronal depolarization increase Sec5 in dendritic spines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Although loss of function mutations in ral prevented exocyst-dependent SSR growth, other exocyst-catalysed processes persisted.

    “Although loss of function mutations in ral prevented exocyst-dependent SSR growth, other exocyst-catalysed processes persisted.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The synaptic localization of Ral is independent of its nucleotide state, but the recruitment of the exocyst occurs only in the GTP-bound state.

    “it indicates that the synaptic localization of Ral is independent of its nucleotide state, but the recruitment of the exocyst occurs only in the GTP-bound state.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Both membrane addition and membrane internalization are required for the growth and retraction of spines.

    “both membrane addition and membrane internalization are required for the growth and retraction of spines (Holtmaat and Svoboda, 2009;Newpher and Ehlers, 2009;Kelly et al, 2011).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Holtmaat and Svoboda, 2009 source
    • cites Newpher and Ehlers, 2009 source
    • cites Kelly et al, 2011 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that has proven to be a good model system to study synaptic development and plasticity.

    “The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that has proven to be a good model system to study synaptic development and plasticity (Collins and DiAntonio, 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Collins and DiAntonio, 2007 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In response to changes in synaptic activity, neurons can alter both pre- and postsynaptic elements of the synapse, including the number, size, and shape of dendritic spines.

    “In response to changes in synaptic activity, neurons can alter both pre-and postsynaptic elements of the synapse, including the number, size, and shape of dendritic spines (Kennedy and Ehlers, 2006;Alvarez and Sabatini, 2007;Bourne and Harris, 2007;Hanus and Ehlers, 2008;Newpher and Ehlers, 2009;Kasai et al, 2010).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Kennedy and Ehlers, 2006 source
    • cites Alvarez and Sabatini, 2007 source
    • cites Bourne and Harris, 2007 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    The exocyst may be important in other aspects of synapse growth and plasticity through its involvement in and regulation of the tethering, docking, and fusion of post-Golgi vesicles with the plasma membrane.

    “the exocyst may be important in other aspects of synapse growth and plasticity through its involvement in and regulation of the tethering, docking, and fusion of post-Golgi vesicles with the plasma membrane.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The SSR contains scaffolding proteins and cytoskeletal specializations.

    “The SSR contains scaffolding proteins and cytoskeletal specializations (Rheuben et al, 1999;Ataman et al, 2006)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Rheuben et al, 1999 source
    • cites Ataman et al, 2006 source

  • May combine two claims.

    Interpretationown data

    The SSR is a plastic structure whose size depends on synaptic activity.

    “We here report that the SSR is a plastic structure whose size depends on synaptic activity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The effect of TrpA1 and shi ts manipulations on Syndapin, GluRIIB, and Dlg is weak probably because manipulations with TrpA1 and shi ts are less potent than constant expression of Ral CA.

    “The effect is weak probably because manipulations with TrpA1 and shi ts are less potent than constant expression of Ral CA .”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Ral localization to the synaptic area is upstream of Sec5 activation and does not require Sec5, whereas Sec5 recruitment by Ral CA parallels the growth of the SSR and is required for that growth.

    “Thus, Ral localization to the synaptic area is upstream of Sec5 activation and does not require Sec5, whereas Sec5 recruitment by Ral CA parallels the growth of the SSR and is required for that growth.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The SSR is a plastic structure whose size can be regulated by the level of synaptic activity, and the changes in the SSR width correlated well with the ability of activity to recruit the exocyst to the synapse.

    “Thus, the SSR is a plastic structure whose size can be regulated by the level of synaptic activity, and the changes in the SSR width correlated well with the ability of activity to recruit the exocyst to the synapse.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The increase in the SSR may occur primarily in its deeper folds and this zone is marked by Syndapin (which was increased surrounding boutons by Ral CA expression) but lacks Dlg (which did not increase).

    “The increase in the SSR may occur primarily in its deeper folds and this zone is marked by Syndapin (which was increased surrounding boutons by Ral CA expression) but lacks Dlg (which did not increase).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    It is likely that SSR development entails two components: an unknown developmental signal that initiates transcription and translation of the building blocks and a membrane trafficking mechanism, dependent on Ral and the exocyst, that mediates and modulates the assembly of those components at the synapse.

    “It is likely that SSR development entails two components: an unknown developmental signal that initiates transcription and translation of the building blocks and a membrane trafficking mechanism, dependent on Ral and the exocyst, that mediates and modulates the assembly of those components at the synapse.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The ability of stimulation by either method to recruit Sec5 to the NMJ is consistent with the ability of glutamate receptors to activate the Ca2+- and Ral-dependent pathway and indicates that this recruitment is likely to occur under normal physiological conditions.

    “The ability of stimulation by either method to recruit Sec5 to the NMJ is consistent with the ability of glutamate receptors to activate the Ca 2 þ -and Ral-dependent pathway and indicates that this recruitment is likely to occur under normal physiological conditions.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    When comparing wild-type and ral mutants, 60 h larvae of each genotype were dissected on the same Sylgard-coated slide and processed together to ensure equivalent treatments.

    “When comparing wild-type and ral mutants, 60 h larvae of each genotype were dissected on the same Sylgard-coated slide and processed together to ensure equivalent treatments.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The glutamatergic NMJ of Drosophila has provided insight into molecular pathways of synaptic development and function that are shared with mammalian synapses.

    “The glutamatergic NMJ of Drosophila has provided insight into molecular pathways of synaptic development and function (Collins and DiAntonio, 2007) that are shared with mammalian synapses.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Collins and DiAntonio, 2007 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    If measurements of SSR thickness did not clearly fit a normal distribution, a log transformation was applied to convert them to a symmetrical distribution.

    “If our measurements of SSR thickness did not clearly fit a normal distribution (as visualized with a qqplot), and were instead positively skewed, then we applied a log transformation to those data sets to convert them to a symmetrical distribution.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    That Sec5 was recruited selectively to the NMJ and not uniformly across the muscle surface suggests that a synaptic targeting mechanism must be present independent of the site of Ca2+ entry and regardless of whether the Ca2+ enters through glutamate receptors or voltage-dependent channels.

    “That Sec5 was recruited selectively to the NMJ and not uniformly across the muscle surface suggests that a synaptic targeting mechanism must be present independent of the site of Ca 2 þ entry and regardless of whether the Ca 2 þ enters through glutamate receptors or voltage-dependent channels.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Calcimycin permeabilizes the entire muscle membrane to Ca2+.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    As with the growth of the SSR, spine dynamics represent changes in postsynaptic architecture that are not necessarily coupled with the formation of new synaptic contacts.

    “As with the growth of the SSR, spine dynamics represent changes in postsynaptic architecture that are not necessarily coupled with the formation of new synaptic contacts.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Each experiment was repeated at least three independent times and quantified data are always from at least four different larvae, segments A2 and A3, and a minimum of eight NMJs.

    “Each experiment was repeated at least three independent times and quantified data are always from at least four different larvae, segments A2 and A3, and a minimum of eight NMJs.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    A minimum of three larvae were quantified per genotype.

    “A minimum of three larvae were quantified per genotype.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The targeting is determined by the localization of Ral to the membrane surrounding the boutons, which in turn recruits and activates the exocyst.

    “We propose instead that the targeting is determined by the localization of Ral to the membrane surrounding the boutons, which in turn recruits and activates the exocyst.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Development of the SSR is dependent on both Ral and Sec5 and the extent of its growth can be influenced by activated Ral in a Sec5-dependent manner.

    “Thus, development of the SSR is dependent on both Ral and Sec5 and the extent of its growth can be influenced by activated Ral in a Sec5-dependent manner.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationnone given

    Neuronal activity triggers Ca2+ entry into the muscle through activation of postsynaptic glutamate receptors and depolarization of the muscle.

    “Neuronal activity triggers Ca 2 þ entry into the muscle through activation of postsynaptic glutamate receptors and depolarization of the muscle.”
    Quote in the paperNot a substantial claim
84 of 84

  • The paper relies on this without support.

    The main source of membrane responsible for activity-dependent dendritic spine growth and the signals that control membrane addition remain elusive.

    “but the main source of membrane responsible for this growth and the signals that control membrane addition remain elusive.”

  • The paper relies on this without support.

    The significance of the SSR is poorly understood.

    “but the significance of the SSR is poorly understood.”

  • The paper relies on this without support.

    PSD95-positive spines are likely to be part of functional synapses.

    “i.e., those that are likely to be part of functional synapses”

  • The paper relies on this without support.

    Ral promotes the assembly of the exocyst complex.

    “Because Ral is known to promote the assembly of the exocyst complex”

  • The paper relies on this without support.

    Calcimycin permeabilizes the entire muscle membrane to Ca2+.

    “Calcimycin, however, permeabilizes the entire muscle membrane to Ca 2 þ .”

  • The paper relies on this without support.

    The RalA CADExo mutation prevents the interaction of RalA with Sec5.

    “a mutation that prevents the interaction of RalA with Sec5”

  • The paper relies on this without support.

    SSR width measured by electron microscopy reflects the amount of postsynaptic membrane.

    “Quantification of SSR width (nm) in the genotypes shown in (A-I).”
    “The addition of this membrane expands the folds of the SSR around each activated bouton.”

  • The paper relies on this without support.

    Fluorescence intensity at synapses reflects the level of the indicated proteins.

    “The effect of expressing Ral CA in larval muscles on the fluorescent intensity at synapses of the indicated proteins.”
    “Fluorescence intensity (mean±s.e.m.) was normalized to that in control larvae”

  • The paper relies on this without support.

    Calcimycin treatment elevates intracellular Ca2+ in larval muscles.

    “Wild-type (w 1118 ) third-instar larvae were treated with calcimycin in either Ca 2 þ -free (A) or 1 mM Ca 2 þ (B) containing saline”
    “Elevated intracellular Ca 2 þ recruits Sec5 to the NMJ, in a ral-dependent manner.”

  • The paper relies on this without support.

    Expression of dTrpA1 increases neuronal activity.

    “Neuronal activity was enhanced by expression of the temperature-activated dTrpA1”

  • The paper relies on this without support.

    Expression of Shi ts blocks transmitter release.

    “expressing in neurons transgenes encoding dTrpA1 to excite neurons or Shi ts to block transmitter release.”

  • The paper relies on this without support.

    RalA CADexo blocks binding to exocyst members Sec5 and Exo84.

    “constitutively active RalA with a point mutation that blocks binding to the exocyst (abbreviated exo) members Sec5 and Exo84 (RalA CADexo )”
12 of 12

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Neurite outgrowth depends on exocytosis to deliver membrane materials to specific surface regions, a process requiring RalA and exocyst.

    “Neurite outgrowth depends on exocytosis to deliver membrane materials to specific surface regions, a process requiring RalA and exocyst (53–55).”

    From Regulation of cargo exocytosis by a Reps1-Ralbp1-RalA module (2023) · cites this paper as [55]

    The Introduction states that membrane addition permits neurite outgrowth and that the exocyst directs membrane delivery to specific surface regions and is required for neurite outgrowth. The Discussion says Ral promotes neurite branching via the exocyst, but also says some exocyst functions do not require Ral. It does not establish that RalA is required for neurite outgrowth. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Different bouton counts for the same SSR measurements

    Minor

    The Results report 58 control and 51 Ral CA boutons for the third-instar SSR measurements, while the Figure 2 legend reports 59 and 52, respectively, for the same measurements.

  • Different SSR widths reported for sec5 mutants expressing Ral CA

    Minor

    The Results give an SSR thickness of 158 ± 11 nm for sec5 mutants expressing Ral CA, but the Figure 3 legend gives 151 ± 11 nm for that genotype.

  • 60-hour ral mutant SSR width conflicts with figure legend

    Minor

    The Results assign 237 ± 27 nm to ral G0501 mutants at 60 hours. The Figure 3 legend instead assigns 260 ± 27 nm to the 60-hour mutants and 237 ± 27 nm to third-instar mutants.

3 of 3

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Authors
Teodoro, R. O., Pekkurnaz, G., Nasser, A., Higashi‐Kovtun, M. E., Balakireva, M., McLachlan, I. G., Camonis, J., & Schwarz, T. L.
Affiliations
  • Boston Children's Hospital (US)
  • Inserm (FR)
Corresponding author
  • Teodoro, Rita O
  • Schwarz, Thomas L
ORCID
Venue
The EMBO Journal
Publisher
Springer Science and Business Media LLC
Published
2013-06-28
Volume
32
Issue
14
Pages
2039-2055
ISSN
  • 0261-4189 (ISSN-L)
  • 1460-2075
DOI
10.1038/emboj.2013.147
PMC
PMC3715865
OpenAlex
W2005984686
Cited by
61
References
105
Citations by year
2026: 4 · 2025: 4 · 2024: 1 · 2023: 2 · 2022: 1 · 2021: 3 · 2020: 10 · 2019: 8 · 2018: 9 · 2017: 5 · 2016: 5 · 2015: 3 · 2014: 3 · 2013: 3
FWCI
2.71
Type
journal article
Language
en
Update notices
erratum (2013-08-28)
Open access
Licence
Locations
  • The EMBO Journal (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health
  • Intellectual and Developmental Disabilities Research Center (P30HD18655)
  • National Institute of Mental Health (MH075058, R01MH075058)
  • National Institute of Neurological Disorders and Stroke (R01NS041062)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (P30HD018655)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
Keywords
Ral GTPase, exocyst, Sec5, membrane trafficking, calcium influx, synaptic boutons, dendritic spines, hippocampal neurons, Drosophila muscle
MeSH terms
Animals, Drosophila melanogaster, Exocytosis, Membrane Proteins, Neuromuscular Junction, Neurons, Synaptic Membranes, Signal Transduction, Genes, Insect, Calcium Signaling, Monomeric GTP-Binding Proteins, ral GTP-Binding Proteins, Protein Transport, Drosophila Proteins, Animals, Genetically Modified, Dendritic Spines, Mutant Proteins, Rats

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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