Molecular mechanisms of COMPLEXIN fusion clamp function in synaptic exocytosis revealed in a new Drosophila mutant
Introduction
It is widely accepted that SNARE proteins function at the core of the neurotransmitter release apparatus, where they promote exocytotic fusion of neurotransmitter-filled synaptic vesicles with the presynaptic plasma membrane (Jahn and Scheller, 2006). However, defining the mechanisms which provide precise and rapid regulation of synaptic vesicle fusion remains among the foremost problems in cellular and molecular neuroscience. The identification of CPX as a protein which binds and regulates SNARE complexes (Ishizuka et al., 1995, McMahon et al., 1995) has advanced our understanding of these mechanisms (Brose, 2008, Neher, 2010, Rizo and Rosenmund, 2008, Stein and Jahn, 2009, Südhof and Rothman, 2009). Notably, CPX can both promote SV fusion evoked by a presynaptic action potential and suppress or “clamp” spontaneous vesicle fusion. Recent models suggest that specific domains of CPX (Fig. 1A) contribute to different aspects of synaptic vesicle fusion (Hobson et al., 2011, Martin et al., 2011, Maximov et al., 2009, Reim et al., 2001, Strenzke et al., 2009, Tang et al., 2006, Xue et al., 2007, Xue et al., 2009, Xue et al., 2010, Yang et al., 2010). Whereas a “central helix” which binds SNARE complexes (Bracher et al., 2002, Chen et al., 2002) is absolutely required for CPX function, other domains appear to mediate specific aspects of CPX activity (Rizo and Rosenmund, 2008, Stein and Jahn, 2009). For example, recent studies have shown that the CPX C-terminus is specifically required for the clamping function (Buhl et al., 2013, Cho et al., 2010, Kaeser-Woo et al., 2012, Martin et al., 2011, Xue et al., 2009). Of particular relevance to the present study is a specific CaaX motif found at the extreme C-terminus of several mammalian and Drosophila CPX isoforms. This motif has been shown to mediate CPX prenylation [a form of lipid modification; (Omer and Gibbs, 1994, Resh, 2006)] and has been implicated in both targeting CPX to membranes (Reim et al., 2005) and the CPX clamping function (Cho et al., 2010, Xue et al., 2009). The form of prenylation demonstrated for mammalian CPX isoforms (CPX3 and 4) is farnesylation (Reim et al., 2005), consistent with previous studies indicating that one of several specific residues in the X position of the CaaX motif (A,C,M,Q,S) selectively mediates farnesylation (Omer and Gibbs, 1994).
This progress is extended by new insights gained from the present study, in which the isolation and characterization of a new cpx mutant further define the in vivo molecular basis of CPX functions and interactions within the neurotransmitter release apparatus. This study reveals a specific subcellular distribution for CPX within the presynaptic terminal and a role for C-terminal farnesylation in mediating both association of CPX with presynaptic membranes and CPX clamping of spontaneous synaptic vesicle fusion.
Section snippets
Genetic and molecular characterization of a new cpx mutant
Further genetic analysis to examine the in vivo molecular mechanisms of CPX function was pursued through a forward genetic screen for new mutant alleles of the single Drosophila cpx gene. To complement a previously reported cpx null mutant (Huntwork and Littleton, 2007), this screen was intended to recover hypomorphic and conditional alleles that may further define the in vivo molecular determinants of CPX function. A screen was performed using chemical mutagenesis and subsequent screening for
Discussion
Through characterization of a new Drosophila cpx mutant, the present study advances our understanding of the molecular mechanisms mediating CPX function in neurotransmitter release. Our findings provide new information about the subcellular distribution of CPX with respect to presynaptic membranes, as well as its molecular basis, and implicate CPX membrane association as a critical element in its clamping function and in vivo interactions with SNARE proteins.
As shown in Fig. 8, our working
Drosophila strains
Appl-GAL4 and w;;Ly/TM6c were from our laboratory stock collection. The cpxSH1 null mutant and the UAS-cpx transgenic line were generously provided by Troy Littleton (MIT, Cambridge, MA). Deficiency lines, Df(3L)GN34 and Df(3R)Exel6140, were obtained from the Bloomington Stock Center. UAS-EGFP-cpx and UAS-EGFP-cpx1257 transgenic lines were generated in the current study (see “Generation of transgenic lines”). Stocks and crosses were cultured on a conventional cornmeal–molasses–yeast medium at 20
Conflict of interest
The authors have no conflict of interest in submitting this manuscript.
Acknowledgments
A cpx null mutant stock, a UAS-cpx transgenic line and an anti-CPX antibody were generously provided by Troy Littleton (MIT, Cambridge, MA). We are also grateful to Noreen Reist (Colorado State University, Fort Collins, CO) and David Deitcher (Cornell University, Ithaca, NY) for providing anti-SYT and anti-SNAP25 antibodies, respectively. We thank Richard Ordway (Penn State University) for his continuous encouragement and invaluable discussion throughout this work. This study was supported by
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2023, Journal of Molecular BiologyCitation Excerpt :Mutations that disrupted prenylation of DmCpx7A eliminated its clamping function at the fly NMJ and switched biochemically isolated adult fly DmCpx from a detergent-soluble phase to an aqueous phase (Figure 3(B,C,E)).58,64,68 Notably, the specific subsynaptic location of CAAX variants such as DmCpx7A and mCpx3/4 is not yet entirely clear (e.g. SVs, AZ plasma membrane, ribbon, endosomes), although synaptic enrichment is sensitive to CT perturbations (Figure 3(D&F)).47,64,68–69 Loss of mCpx3/4 in retinal photoreceptor and bipolar cell ribbons or disruption of prenylation in fly Cpx led to subsynaptic alterations in SV tethering to ribbons/T-bars, supporting the notion that these CAAX variants target ribbons or T-bars via their lipid-modified C-termini.69–70
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2023, Journal of Molecular BiologyCitation Excerpt :The dissimilarities in the structural features of the mCpx1 AH-motif compared to that of wCpx1 may preclude such interactions when the mCpx1 AH-motif is substituted in living worms. There is a single dominant worm complexin, while there are two dominant splice isoforms in flies and four isoforms in mammals,6,18,25,46–48 all of which show wide sequence variation in their CTDs (Figure 9(B)). Complexin CTDs also feature known isoform-specific post-translational modifications (PTMs) with the potential to alter lipid or protein interactions, including farnesylation and phosphorylation.39,46–51
Complexin Suppresses Spontaneous Exocytosis by Capturing the Membrane-Proximal Regions of VAMP2 and SNAP25
2020, Cell ReportsCitation Excerpt :In addition, in vertebrates, such as Mus musculus, the stimulatory role seems to dominate, whereas in invertebrates, such as Drosophila melanogaster and Caenorhabditis elegans, the inhibitory function dominates (Hobson et al., 2011; Huntwork and Littleton, 2007; Lin et al., 2013; Lopez-Murcia et al., 2019; Maximov et al., 2009; Pang et al., 2006; Reim et al., 2001; Strenzke et al., 2009; Wragg et al., 2017; Xue et al., 2007). Complexin consists of an N-terminal stimulatory region (amino acids [aa] 1–25) that activates Ca2+-triggered release, an inhibitory accessory helix (aa 26–47) that suppresses spontaneous release, a central SNARE-binding helix (aa 48–74), and a largely unstructured C-terminal region (aa 75–134) (Buhl et al., 2013; Cho et al., 2010, 2014; Gong et al., 2016; Iyer et al., 2013; Kaeser-Woo et al., 2012; Kümmel et al., 2011; Lai et al., 2014, 2016; Martin et al., 2011; McMahon et al., 1995; Seiler et al., 2009; Snead et al., 2014; Wragg et al., 2013, 2017; Xue et al., 2007, 2010; Yang et al., 2015). The C-terminal region contains a short amphipathic helix that binds high-curvature membranes and modulates species-specific inhibition (Gong et al., 2016; Seiler et al., 2009; Snead et al., 2014; Wragg et al., 2013, 2017; Zdanowicz et al., 2017).
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2019, Journal of Biological ChemistryCitation Excerpt :Ultimately, the details of how the NTD and its membrane binding contribute to complexin function remain largely unclear (Fig. 4B). The intrinsically disordered CTD of complexin appears necessary for the inhibition of spontaneous SV fusion in worms (45, 64, 74), flies (85, 93–95), mice (96, 97), and in vitro fusion assays (87): elimination or perturbation of the CTD impairs complexin inhibitory function (96). The CTD may also, however, have some facilitatory function, based on observations in vitro (98, 99), and in C. elegans (45).
Complexins
2016, The Curated Reference Collection in Neuroscience and Biobehavioral Psychology