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1 Centre for Integrative Physiology, George Square, University of Edinburgh, Edinburgh EH8 9XD, Scotland, UK
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Abstract |
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 Top Abstract Multiple synaptic vesicle...Properties of bulk endocytosisThe dephosphins are activity...PerspectivesReferences |
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(Received 29 May 2007;
accepted after revision 18 June 2007;
first published online 21 June 2007)
Corresponding author M. A. Cousin: Centre for Integrative Physiology, George Square, University of Edinburgh, Edinburgh EH8 9XD, Scotland, UK. Email: m.cousin{at}ed.ac.uk
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Multiple synaptic vesicle retrieval pathways in central nerve terminals |
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TopAbstractMultiple synaptic vesicle...Properties of bulk endocytosisThe dephosphins are activity...PerspectivesReferences |
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Properties of bulk endocytosis |
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TopAbstractMultiple synaptic vesicle...Properties of bulk endocytosisThe dephosphins are activity...PerspectivesReferences |
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Bulk endocytosis was first demonstrated in central nerve terminals using electron microscopy (Takei et al. 1996), and this has remained an important tool in observing the process. However, these studies are labour intensive and do not provide real time information on the process. Since the turn of the decade, bulk endocytosis has also been able to be visualized using fluorescent methods, opening up new possibilities to investigate its activation and molecular mechanism. The first studies were performed on the frog neuromuscular junction, where it was shown that the lipid-binding styryl dye FM1-43 could selectively label bulk endocytosis when compared with its more hydrophilic counterpart FM2-10 (Richards et al. 2000). The proposed reason for this disproportionate labelling was that FM2-10 was washed out of the bulk endosomes that were still connected to the plasma membrane, whereas the more hydrophobic FM1-43 could not be removed. The selective labelling of bulk endosomes was confirmed by photoconversion of the dyes and examination of nerve terminals with electron microscopy (Richards et al. 2000). In addition to styryl dyes, large molecular weight dextrans tagged with fluorescent molecules have also been used to demonstrate the presence and kinetics of bulk endocytosis in motor nerve terminals (Holt et al. 2003; Teng et al. 2007). These fluid phase markers are too large to be accumulated inside single SVs, and therefore they selectively label the bulk endocytosis pathway.
These flourescent studies have allowed the traffic of SVs generated by bulk endocytosis to be followed in real time. It transpires that the primary route for these SVs is to replenish the reserve pool of SVs. This conclusion was first drawn in the frog neuromuscular junction, since SVs labelled by FM1-43 during strong stimulation could not immediately undergo exocytosis, whereas those SVs labelled with FM2-10 could (Richards et al. 2000). The FM1-43-labelled SVs could eventually be released, but only after a delay of approximately 10–15 min. The most obvious explanation for this time lag is that this is the time required to generate new SVs from bulk endosomes. Recently our group has shown that SVs derived from bulk endocytosis also replenish the reserve SV pool in central nerve terminals (Evans & Cousin, 2007). In these studies, a sustained component of exocytosis was observed that was attributable to the reserve pool when FM1-43 was loaded into SVs using a strong stimulus, but was absent when FM2-10 was loaded under identical conditions. Thus SVs generated by bulk endocytosis are unable to be immediately used and rejoin the SV recycling pool in the reserve pool.
Since the kinetics of bulk endocytosis and fate of SVs derived from this pathway are now known, it is perhaps surprising that very little is understood about the molecular mechanism of the process itself. While it is thought that clathrin-dependent endocytosis is responsible for SV budding from bulk endosomes (Takei et al. 1996), the molecules that activate and mediate the invagination and fission of the membrane are relatively unknown. Bulk endocytosis has been linked to the process of macropinocytosis in non-neuronal cells where membrane protrusions gather large amounts of the fluid phase in a ‘cell drinking’ mechanism (Holt et al. 2003; Teng et al. 2007). Macropinocytosis is dependent on the activation of Rho family GTPases which stimulate the actin-driven formation of these protrusions (Conner & Schmid, 2003). Interestingly in both the frog neuromuscular junction and in retinal bipolar neurones, disruption of actin function with pharmacological agents resulted in an inhibition of bulk endocytosis (Holt et al. 2003; Richards et al. 2004). In addition, bulk endocytosis was also retarded by inhibition of phosphatidylinositol 3-kinase activity (Holt et al. 2003; Richards et al. 2004), suggesting a link between this signalling cascade and the actin dynamics required for the process. Thus a requirement for some molecules in bulk endocytosis has been identified, with actin dynamics and rearrangement a major factor in the process.
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The dephosphins are activity-dependent triggers for bulk endocytosis |
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TopAbstractMultiple synaptic vesicle...Properties of bulk endocytosisThe dephosphins are activity...PerspectivesReferences |
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(PIPKI). After their stimulus-dependent dephosphorylation, the dephosphins are rephosphorylated by their respective protein kinases, such as cyclin-dependent kinase 5 (cdk5), which rephosphorylates dynamin I, synaptojanin and PIPKI in vivo (Tan et al. 2003; Lee et al. 2005). We have identified both the dephosphorylation of the dephosphins and their subsequent rephosphorylation as essential events in bulk endocytosis (Evans & Cousin, 2007). The evidence for this is as follows: (i) inhibition of either calcineurin or cdk5 by either pharmacological antagonists or overexpression of dominant negative constructs in primary neuronal culture arrested the uptake of FM1-43 but not FM2-10 during strong stimulation; (ii) inhibition of cdk5 blocked the uptake of the fluid-phase marker horseradish peroxidase (HRP) into plasma-membrane-generated endosomes, but not single SVs, during strong stimulation; (iii) inhibition of cdk5 had no effect on either FM1-43 loading or HRP labelling if nerve terminals were challenged with a mild stimulus; and finally (iv) the sustained component of exocytosis observed when SVs were labelled with FM1-43 was abolished when either calcineurin or cdk5 activity was inhibited during the dye-loading phase (Evans & Cousin, 2007).
These findings place some or all of the dephosphins as the key mediators of bulk endocytosis, with their dephosphorylation by calcineurin as the activity-dependent trigger for the process. There is ample evidence that calcineurin can fulfil the temporal requirements of this role, since the dephosphorylation by calcineurin of the dephosphins is synchronous and rapid (<1 s; Robinson et al. 1994). Furthermore the intracellular free Ca2+ increase required to maximally dephosphorylate the dephosphins in nerve terminals (approximately 1 µM; Sihra et al. 1992) correlates well with the predicted increase in intracelluar free Ca2+ during strong stimulation. However, the key test as to whether calcineurin is the activity-dependent Ca2+ sensor for bulk endocytosis is whether it is able to dephosphorylate its substrates only at stimulation frequencies at which bulk endocytosis is observed. Unpublished experiments recently performed in our laboratory confirm that this is the case. In these experiments no dephosphorylation of the calcineurin substrate dynamin I was observed at mild stimulation frequencies, but a robust dephosphorylation was seen with increasing stimulation frequency in our neuronal cultures. Thus we propose that the activity-dependent dephosphorylation of the dephosphins is the trigger for bulk endocytosis, and that some if not all of the dephosphins are involved in the process.
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Perspectives |
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TopAbstractMultiple synaptic vesicle...Properties of bulk endocytosisThe dephosphins are activity...PerspectivesReferences |
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Footnotes |
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References |
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TopAbstractMultiple synaptic vesicle...Properties of bulk endocytosisThe dephosphins are activity...PerspectivesReferences |
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Braun A, Pinyol R, Dahlhaus R, Koch D, Fonarev P, Grant BD, Kessels MM & Qualmann B (2005). EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling. Mol Biol Cell 16, 3642–3658.
Conner SD & Schmid SL (2003). Regulated portals of entry into the cell. Nature 422, 37–44.
Cousin MA & Robinson PJ (2001). The dephosphins: dephosphorylation by calcineurin triggers synaptic vesicle endocytosis. Trends Neurosci 24, 659–665.[CrossRef][Medline]
Evans GJ & Cousin MA (2007). Activity-dependent control of slow synaptic vesicle endocytosis by cyclin-dependent kinase 5. J Neurosci 27, 401–411.
Gad H, Low P, Zotova E, Brodin L & Shupliakov O (1998). Dissociation between Ca2+-triggered synaptic vesicle exocytosis and clathrin-mediated endocytosis at a central synapse. Neuron 21, 607–616.[CrossRef][Medline]
Grabs D, Slepnev VI, Songyang Z, David C, Lynch M, Cantley LC & De Camilli P (1997). The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that defines a new SH3 binding consensus sequence. J Biol Chem 272, 13419–13425.
Harata NC, Aravanis AM & Tsien RW (2006). Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion. J Neurochem 97, 1546–1570.[CrossRef][Medline]
Holt M, Cooke A, Wu MM & Lagnado L (2003). Bulk membrane retrieval in the synaptic terminal of retinal bipolar cells. J Neurosci 23, 1329–1339.
Itoh T, Erdmann KS, Roux A, Habermann B, Werner H & De Camilli P (2005). Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev Cell 9, 791–804.[CrossRef][Medline]
Kessels MM & Qualmann B (2002). Syndapins integrate N-WASP in receptor-mediated endocytosis. EMBO J 21, 6083–6094.[CrossRef][Medline]
de Lange RP, de Roos AD & Borst JG (2003). Two modes of vesicle recycling in the rat calyx of Held. J Neurosci 23, 10164–10173.
Lee SY, Voronov S, Letinic K, Nairn AC, Di Paolo G & De Camilli P (2005). Regulation of the interaction between PIPKI and talin by proline-directed protein kinases. J Cell Biol 168, 789–799.
Leenders AG, Scholten G, de Lange RP, Lopes Da Silva FH & Ghijsen WE (2002). Sequential changes in synaptic vesicle pools and endosome-like organelles during depolarization near the active zone of central nerve terminals. Neuroscience 109, 195–206.[CrossRef][Medline]
Marxen M, Volknandt W & Zimmermann H (1999). Endocytic vacuoles formed following a short pulse of K+-stimulation contain a plethora of presynaptic membrane proteins. Neuroscience 94, 985–996.[CrossRef][Medline]
Murthy VN & De Camilli P (2003). Cell biology of the presynaptic terminal. Annu Rev Neurosci 26, 701–728.[CrossRef][Medline]
Paillart C, Li J, Matthews G & Sterling P (2003). Endocytosis and vesicle recycling at a ribbon synapse. J Neurosci 23, 4092–4099.
Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR & McMahon HT (2004). BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499.
Richards DA, Guatimosim C & Betz WJ (2000). Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals. Neuron 27, 551–559.[CrossRef][Medline]
Richards DA, Rizzoli SO & Betz WJ (2004). Effects of wortmannin and latrunculin A on slow endocytosis at the frog neuromuscular junction. J Physiol 557, 77–91.
Robinson PJ, Liu JP, Powell KA, Fykse EM & Südhof TC (1994). Phosphorylation of dynamin I and synaptic vesicle recycling. Trends Neurosci 17, 348–353.[CrossRef][Medline]
Royle SJ & Lagnado L (2003). Endocytosis at the synaptic terminal. J Physiol 553, 345–355.
Sihra TS, Bogonez E & Nicholls DG (1992). Localized Ca2+ entry preferentially effects protein dephosphorylation, phosphorylation, and glutamate release. J Biol Chem 267, 1983–1989.
Takei K, Mundigl O, Daniell L & De Camilli P (1996). The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin. J Cell Biol 133, 1237–1250.
Tan TC, Valova VA, Malladi CS, Graham ME, Berven LA, Jupp OJ, Hansra G, McClure SJ, Sarcevic B, Boadle RA, Larsen MR, Cousin MA & Robinson PJ (2003). Cdk5 is essential for synaptic vesicle endocytosis. Nature Cell Biol 5, 701–710.[CrossRef][Medline]
Teng H, Lin MY & Wilkinson RS (2007). Macroendocytosis and endosome processing in snake motor boutons. J Physiol (in press 10.1113/jphysiol.2007.130989).
Teng H & Wilkinson RS (2000). Clathrin-mediated endocytosis near active zones in snake motor boutons. J Neurosci 20, 7986–7993.
Tsujita K, Suetsugu S, Sasaki N, Furutani M, Oikawa T & Takenawa T (2006). Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J Cell Biol 172, 269–279.
Wigge P, Vallis Y & McMahon HT (1997). Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain. Curr Biol 7, 554–560.[CrossRef][Medline]
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Acknowledgements |
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