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Title: A dynamin 1-, dynamin 3- and clathrin-independent pathway of synaptic vesicle recycling mediated by bulk endocytosis}
Article:
Abstract:
The exocytosis of synaptic vesicles( SVs) elicited by potent stimulation is rapidly compensated by bulk endocytosis of SV membranes leading to large endocytic vacuoles( ‘bulk’ endosomes). Subsequently, these vacuoles disappear in parallel with the reappearance of new SVs. We have used synapses of dynamin 1 and 3 double knock-out neurons, where clathrin-mediated endocytosis( CME) is dramatically impaired, to gain insight into the poorly understood mechanisms underlying this process. Massive formation of bulk endosomes was not defective, but rather enhanced, in the absence of dynamin 1 and 3. The subsequent conversion of bulk endosomes into SVs was not accompanied by the accumulation of clathrin coated buds on their surface and this process proceeded even after further clathrin knock-down, suggesting its independence of clathrin. These findings support the existence of a pathway for SV reformation that bypasses the requirement for clathrin and dynamin 1/3 and that operates during intense synaptic activity.
Introduction:
Synaptic vesicles( SVs) are the specialized organelles that store and secrete non-peptide neurotransmitters at synapses. Following exocytosis, their membranes are rapidly recaptured by endocytosis and recycled for the generation of new fusion-competent SVs( Ceccarelli et al., 1973; Heuser and Reese, 1973; Betz and Bewick, 1992). Strong evidence indicates that clathrin-mediated endocytosis( CME) plays a key role in this endocytic traffic( Heuser and Reese, 1973; Maycox et al., 1992; Granseth et al., 2006; Heerssen et al., 2008; Kasprowicz et al., 2008; Saheki and De Camilli, 2012). Endocytic vesicles generated by CME have a small homogenous size, in the same range of SVs( Shupliakov et al., 1997; Raimondi et al., 2011). While it had been originally hypothesized that such vesicles transit through an endosomal station( Heuser and Reese, 1973), subsequent studies have suggested that they may mature directly into SVs, a possibility consistent with their size( Takei et al., 1996). However, CME does not account for all SV recycling and the mechanisms of alternative pathways have been the object of intense debate. One proposed alternative recycling mechanism is so-called ‘kiss-and-run’, a process whereby SVs fuse only very transiently with the plasma membrane without collapsing into it, so that the SVs reform directly by the immediate closure of the fusion pore( Fesce et al., 1994; Alabi and Tsien, 2013). Yet another mechanism is bulk endocytosis, a high capacity pathway of plasma membrane internalization involving patches of membrane larger than the membrane of individual SVs. This mechanism predominates under intense neuronal activity( Miller and Heuser, 1984; Holt et al., 2003; Wu and Wu, 2007; Hayashi et al., 2008; Cousin, 2009; Wenzel et al., 2012; Kittelmann et al., 2013). Endocytic intermediates generated by bulk endocytosis, henceforth defined as ‘bulk’ endosomes, eventually disappear as new SVs appear, suggesting a precursor-product relationship and possibly a direct formation of SVs from bulk endosomes( Heuser and Reese, 1973). Studies in Caenorhabditis elegans have opened new questions on SV recycling mechanisms. Mutations expected to result in loss or strong defect of the function of clathrin or of its endocytic adaptor AP-2 were shown not to abolish synaptic function( Gu et al., 2008, 2013; Sato et al., 2009). Additionally, studies of worm synapses expressing channelrhodopsin and subjected to a very brief photostimulus revealed an ultrafast endocytic reaction mediated by uncoated invaginations larger than SVs( Watanabe et al., 2013a). More recently, similar results – an ultrafast endocytic reaction mediated by large uncoated invaginations in response to a single optogenetic stimulus – were observed at synapses of mouse hippocampal neurons in primary culture( Watanabe et al., 2013b). Both the molecular mechanisms underlying bulk endocytosis and those through which SVs are generated from bulk endosomes remain poorly understood. Like any other form of endocytosis, bulk endocytosis involves membrane remodeling and membrane fission. Thus, several studies of bulk endocytosis have addressed the potential involvement of dynamin, a GTPase known to mediate endocytic membrane fission in multiple contexts including, most prominently, endocytic fission at neuronal synapses( Koenig and Ikeda, 1989; Ramaswami et al., 1994; Ferguson and De Camilli, 2012). However, conflicting results have been reported. A role for dynamin, and more specifically for dynamin 1 has been supported by some studies( Clayton et al., 2009, 2010; Xue et al., 2011; Nguyen et al., 2012). Interestingly, dynamin 1 is constitutively phosphorylated in resting nerve terminals and its Ca2+ triggered dephosphorylation upon synaptic stimulation results in its binding to the F-BAR domain containing protein syndapin/pacsin( Anggono et al., 2006; Clayton et al., 2009; Koch et al., 2011), which has also been implicated in bulk endocytosis( Andersson et al., 2008). However, studies of dynamin 1 knock-out( KO) neurons have indicated the occurrence of robust bulk endocytosis even in the absence of dynamin 1( Hayashi et al., 2008), in spite of a strong impairment of CME( Ferguson et al., 2007; Lou et al., 2008). It remains possible that other dynamins, dynamin 3 in particular, which is the other predominantly neuronal dynamin, may substitute for dynamin 1 in these neurons( Ferguson et al., 2007; Raimondi et al., 2011). Drugs( dyngo-4a and dynasore) that impair dynamin activity were reported to block bulk endocytosis( Nguyen et al., 2012). However, the interpretation of such results is questioned by the more recent demonstration that these drugs can robustly affect plasma membrane dynamics by dynamin-independent mechanisms( Park et al., 2013). Concerning the conversion of ‘bulk’ endosomes into SVs, precise mechanisms have yet to emerge. Based on a study of ‘broken’ synaptosomes it was proposed that such a conversion occurs via the same clathrin-mediated budding reaction that drives clathrin-mediated budding from the plasma membrane( Takei et al., 1996). However, in that study bulk endosomes were exposed to conditions that can induce ectopic production of PI( 4, 5) P2( incubation with ATP and GTP or GTPγS)( Seaman et al., 1993; Takei et al., 1996; Krauss et al., 2003). Subsequent evidence that assembly of endocytic coats critically requires PI( 4, 5) P2 in the membrane from which they originate( Cremona et al., 1999; Höning et al., 2005; Zoncu et al., 2007; Idevall-Hagren et al., 2012) challenged these results as PI( 4, 5) P2, which is selectively concentrated at the plasma membrane, is generally rapidly depleted from endocytic membranes and is thus not expected to be concentrated on bulk endosomes( Cremona et al., 1999; Chang-Ileto et al., 2011; Milosevic et al., 2011). The goal of this study was to gain new information into the recycling of SV membranes via bulk endocytosis. To this aim we examined the impact of manipulations that affect dynamin-dependent endocytosis on this process using electron microscopy( EM) in combination with endocytic tracers. We demonstrate that the rapid, bulk endocytic recapture of SV membranes induced by a strong stimulus, either KCl or high frequency stimulation, is not impaired at synapses lacking both of the two predominant neuronal dynamins, dynamin 1 and dynamin 3, that is synapses where CME is severely perturbed( Ferguson et al., 2007; Raimondi et al., 2011; Ferguson and De Camilli, 2012). Conversion of bulk endosomes into new SVs does occur in these synapses, although less efficiently than in wild-type( WT) synapses. The partial impairment of this conversion is not further aggravated by the additional knock-down( KD) of clathrin, indicating the clathrin-independence of this process. These observations provide evidence for a pathway of SV reformation that predominates after strong stimulation and does not require clathrin mediated budding or dynamin 1 and 3.
Discussion:
While the revision of this manuscript was being finalized, a paper by Kononenko et al.( 2014) reported that bulk endocytosis in response to a strong stimulus is dynamin 1/3 dependent( based on RNA interference) and proposed an important role for clathrin in SV budding from endosomes. Our results, which are based on studies of dynamin 1/3 double KO synapses and on the ultrastructrual analysis of the time course of SV recovery from bulk endosomes in the presence or near depletion of clathrin lead to different conclusions.
Neurons propagate electrical signals from one cell to the next using small molecules called neurotransmitters . These molecules are held inside small compartments called synaptic vesicles . Once a neuron receives an electrical stimulus , the membranes that enclose the synaptic vesicles fuse with the plasma membrane that encloses the neuron . This releases the neurotransmitters , which then trigger an electrical signal in the neighboring cell . Once the neurotransmitters are released , the vesicle membrane is rapidly reinternalized from the plasma membrane in a process called endocytosis and then recycled , ready for the next round of signal transmission . The process of synaptic vesicle membrane endocytosis and recycling has been studied extensively , and several different mechanisms by which it occurs have been identified . The best understood relies on a protein called clathrin , and is thought to be essential for nervous system function . Recently , however , a mechanism of vesicle membrane endocytosis that does not involve clathrin was identified . This mechanism , called bulk endocytosis , involves reinternalizing large regions of the cell plasma membrane to generate large compartments called vacuoles , from which new synaptic vesicles eventually form . This mechanism has been observed when neurons fire at high frequency . The cellular processes underlying bulk endocytosis are not well understood , although several studies suggest proteins called dynamins are important . Wu et al . simulated the conditions a cell experiences during high levels of activity in neurons that lacked the two major dynamins present at the synapses between neurons—dynamin 1 and dynamin 3 . In these neurons , robust bulk endocytosis occurred , suggesting that these two major neuronal dynamins do not play a role in this process . Furthermore , formation of vesicles from the vacuoles generated by bulk endocytosis appeared to be clathrin-independent . These findings point to the occurrence of a pathway of synaptic vesicle recycling that bypasses the need for dynamin 1 and 3 as well as for clathrin . To reconcile these results with previously published work , Wu et al . propose that dynamins may only be required for processes that also require clathrin . But how are vesicles recycled during bulk endocytosis if dynamins are not involved ? There are currently few leads to base alternative mechanisms on . Further work is required to unravel this mystery , and to provide insights into how clathrin-dependent and independent recycling processes are linked during high neuronal activity .