Exocytosis is a complex series of overlapping, integrated molecular interactions by which vesicles in different intermediate states/pools progress to fusion. The challenge then is to study a given step of the pathway without having inter-connected steps contaminate the functional assays or the identification of components critical to mechanism. Unfortunately, few model systems can directly assess individual stages in exocytosis at both functional and
Anionic lipids in Ca2+-triggered fusion
Introduction
Lipids comprise a class of structurally and functionally diverse biomolecules, and as major components of biological membranes have profound impact on many cellular processes, including regulated exocytosis. This fundamental cellular process involves the mutual efforts of proteins and lipids, culminating in Ca2+-triggered fusion, in which the roles of membrane lipids dominate. Thus, lipid composition of the local membrane matrix is a critical factor that can modulate the fusion process by promoting or inhibiting formation of the fusion pore [1], [2]. In particular, it has been quantitatively established that lipids having a specific intrinsic negative curvature are favored to initiate formation of the transient intermediate structures that effect bilayer merger, and therefore play a crucial role in the late stages of triggered exocytosis [1], [3], [4], [5], [6], [7], [8]. Cholesterol (CHOL) is one such critical component that also functions in priming by contributing to the formation of microdomains that maintain other critical docking/fusion components in the close proximity and conformations necessary to ensure the efficiency of Ca2+-triggered fusion [3], [4], [5], [7], [9], [10], [11], [12], [13]. It has also been quantitatively established that lipids having an intrinsic negative curvature comparable to or greater than that of CHOL, including phosphatidylethanolamine (PE), diacylglycerol (DAG), and α-tocopherol, can facilitate the initiation of native membrane merger [3], [4], [5], [7].
Some lipidic membrane components predominantly exist as deprotonated negatively charged species under cellular conditions. It has been recognized since the 1960s that these anionic lipids can bind various di- and trivalent cations particularly Ca2+, the endogenous trigger for the fast membrane fusion steps of regulated exocytosis [14]. Since these original observations, there have been a vast number of studies characterizing these binding reactions and the subsequent effects on bilayer membranes [14], [15], [16], [17], [18]. Ca2+ can dehydrate anionic lipid head groups, thus bringing membranes into close proximity, as required to initiate membrane fusion [19]. Ca2+ binding to negatively charged lipidic species can also (i) increase the surface tension of lipid bilayers and alter the membrane curvature to be more conducive to membrane fusion; and (ii) alter the lipid density/packing in the local membrane region [17]. Detailed studies over the past 30+ years, mainly using phosphatidylserine (PS) as a model anionic lipid, have identified Ca2+ as the most effective divalent cation for binding to these charged lipids (both in cis and trans configurations). In particular, binding in trans (i.e. between PS headgroups on apposed bilayer membranes) results in lateral phase separation of membrane components and the formation of a collapsed, dehydrated Ca(PS)2 phase; neighboring neutral lipids are also dehydrated by the energy released during the formation of this phase [20], [21]. It was established that this Ca2+–PS binding could take place at the low micromolar concentrations of free Ca2+ associated physiologically with triggering of the regulated fusion pathway [22]. This originally led to speculation that localized anionic lipids at the fusion site, tempered by excess neighboring neutral and zwitterionic lipids (in particular DAG, CHOL, and PE), could contribute to the native fusion reaction by binding Ca2+, and thereby contribute to overcoming local hydration repulsion as well as promoting potentially necessary local phase separations. In addition, using protein kinase C as the original example, it was also speculated that neutral/zwitterionic lipids such as DAG and PE could modify the Ca2+ binding at a patch of anionic lipids to promote protein binding and active conformations [23]. A large range of proteins involved in a plethora of biological functions are now recognized to bind specifically to anionic lipids, ensuring localized and optimal activity [24], [25], [26], [27], [28], [29], [30], [31].
Using the well-characterized cortical vesicle (CV) model system, we have previously assessed the roles of phosphatidic acid (PA) and the polyphosphoinositides (PIP) in fast, Ca2+ triggered fusion [4], [5]. Due to its charge and spontaneous negative curvature, it had been speculated that PA could be a localized Ca2+ sensor for triggered fusion. In stark contrast, we found that exogenously supplied PA, or native PA generated in the CV membrane via treatment with exogenous phospholipase D, consistently inhibited all three parameters of triggered fusion – extent, Ca2+ sensitivity, and kinetics – and further compounded the inhibition seen after depletion of CV cholesterol [4]. This effect was comparable to that seen upon addition of the positive curvature (i.e. fusion-inhibiting) lipid, lysophosphatidylcholine [3]. The data were most consistent with an upstream role for PA in vesicle attachment and/or priming reactions, likely via protein binding [3], [4], [32], [33]; if PA were at the fusion site, it would seem more likely to function either as a negative regulator, inhibiting the formation of high curvature fusion intermediates, or as an annular lipid maintaining the localization and/or conformation of a specific protein [33]. Quantitative analysis of the PIP also suggests an upstream role, with phosphatidylinositol-3-kinase activity likely defining the last priming step(s) necessary to establish the full fusion readiness of a docked vesicle; this priming function may also promote the rate of the subsequent triggered fusion reaction [5], [34]. Thus, while a myriad of literature in this area, including our own work, suggests that anionic lipids form part of the physiological fusion machine (PFM) any potential roles in the fundamental fusion mechanism (FFM) [10] remain to be quantitatively established in a native membrane system.
Here we extend these studies to test whether phosphatidylinositol (PI) and PS have specific roles in the fast, Ca2+-triggered steps of regulated exocytosis as occurs in oocytes, neurons, and neuroendocrine cells. As touched on above, much of the earlier work in this area involved liposomes or multi-lamellar systems of defined lipid composition (i.e. both in terms of lipid species and proportions); these have yielded critical insights concerning potential effects on local membrane structure and the energies associated with membrane hydration, adhesion, and the transient focal lipid reorganizations that ultimately enable bilayer mixing and subsequent fusion pore opening. More recent work has largely involved proteoliposomes containing SNAREs and associated proteins (reviewed in [10]), providing critical physico-chemical information concerning potential protein interactions and the influence of some lipids on the structure and function of these, particularly with regard to the known priming role of the inter-membrane SNARE complex [35], [36], [37], [38], [39], [40], [41]. Initial amperometric studies in the neuroendocrine-like PC12 cell have also suggested roles for some anionic lipids during fusion pore opening and expansion [42], [43].
Here we capitalize on the tightly coupled quantitative molecular and functional analyses enabled by the CV model system; these high purity secretory vesicles are easily isolated from their fully docked, fusion-ready state, and require only an increase in the free Ca2+ concentration to trigger fast membrane fusion in the absence of any cytosolic factors. A previous review effectively diagrammed the process for isolating CV and the different functional preparations that can be isolated from the sea urchin egg (see Fig. 1 in [44]). Prior to reaching the fusion-ready state, vesicles must be targeted, tethered, and docked to the plasma membrane. The tethering and docking conditions are characterized by a certain minimal distance (<30 nm and <10 nm, respectively) between the approaching membranes; in and of itself, simple contact of two lipid bilayers is insufficient for the fusion process to occur [45], [46]. Docked vesicles are subject to a maturation process to become fusion competent [39]; although not well understood, priming represents the readiness or competence of membranes for fusion, and in part involves ATP-dependent processes like the biosynthesis of polyphosphoinositides and the consequent rearrangement and activation of critical proteins [46]. In essence, after all docking and priming reactions have occurred, this system is ‘locked’ at a step just prior to Ca2+ triggering and fusion. Thus, devoid of other cellular processes and modulatory influences, this is an ideal system with which to assess the specific roles of different membrane components in the PFM and native FFM. Furthermore, as the CV are isolated, there is little if any chance for metabolic processes to interfere with the molecular manipulations used, and this is particularly important in effectively assessing the local role of lipids. Thus, this highly fusogenic native membrane model enables us to determine whether molecular components are minimally essential to the focal fusion step per se (i.e. the FFM) and/or whether they more broadly influence the PFM, including roles in vesicle recruitment, tethering/attachment, docking, and priming. Our data indicate more complicated effects than perhaps previously realized but appear to support notions of local Ca2+-sensing for PS, as well as upstream, co-factor roles for proteins that implicate both PI and PS in the physiologically important modulation and tuning of the fusion process.
Section snippets
Materials
Sea urchins (Strongylocentrotus purpuratus) were purchased from Westwind Sea Laboratories (Victoria, BC). Phospholipid and neutral lipid standards (all the esters of oleic acid 18:1(Δ9)) for high-performance thin-layer chromatography (HPTLC), including CHOL, dioleoylphosphatidylserine (DOPS) and PI (bovine liver) were obtained from Avanti Polar Lipids (Alabaster, AL). Cholesterol sulfate, hexadecane, neomycin sulphate (Neo), methyl-β-cyclodextrin (mβcd) and 2-hydroxypropyl-β-cyclodextrin
Exogenous PI and PS
The roles of PI and PS in the fast Ca2+-triggered fusion pathway were assessed using acute treatments of isolated fusion-ready CV. To understand the extent to which exogenous PI and PS can affect the fusion process, CV were treated with 100–500 μM of either liver PI or DOPS. In both cases, these treatments resulted in similar concentration-dependent decreases in the extent of fusion (i.e. the ability to fuse), inhibition of initial fusion kinetics, and rightward shifts in Ca2+-sensitivity; these
Discussion
For over 100 years, the sea urchin egg has served as a critical model system, yielding original insights into fundamental cellular processes. For over 35 years, model systems derived from the unfertilized egg have enabled an unsurpassed focus on the highly conserved Ca2+-triggered fusion pathway, providing the most direct route to identifying minimally essential membrane components as well as those providing modulatory influences. Thus, studies in this system have yielded seminal contributions
Conflict of interest statement
The authors have no conflicts to declare.
Acknowledgements
TPR and JRC acknowledge the support of the Faculty of Medicine and the Hotchkiss Brain Institute at the University of Calgary. JRC acknowledges the support of the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Alberta Heritage Foundation for Medical Research, and the University of Western Sydney; MAC held an NSERC Doctoral Award at the time this research was carried out. Special thanks to Drs. R.G. Hanshaw and B.D. Smith for
References (103)
Non-bilayer lipids and biological fusion intermediates
Chem. Phys. Lipids
(1996)- et al.
Membrane hemifusion: crossing a chasm in two leaps
Cell
(2005) - et al.
Specific lipids supply critical negative spontaneous curvature – an essential component of native Ca2+-triggered membrane fusion
Biophys. J.
(2008) - et al.
The influence of cholesterol on phospholipid membrane curvature and bending elasticity
Biophys. J.
(1997) - et al.
Lipid raft association of SNARE proteins regulates exocytosis in PC12 cells
J. Biol. Chem.
(2005) - et al.
Fusion step-specific influence of cholesterol on SNARE-mediated membrane fusion
Biophys. J.
(2009) - et al.
Morphological responses to calcium-induced interaction of phosphatidylserine-containing vesicles
Biophys. J.
(1986) - et al.
Structural effects of neutral lipids on divalent cation-induced interactions of phosphatidylserine-containing bilayers
Biophys. J.
(1995) - et al.
Anionic phospholipids are involved in membrane targeting of PI 3-kinase gamma
Biochem. Biophys. Res. Commun.
(2001) - et al.
Specific binding of phosphatidylinositol 4,5-bisphosphate to calcium-dependent activator protein for secretion (CAPS), a potential phosphoinositide effector protein for regulated exocytosis
J. Biol. Chem.
(1998)
Phosphatidic acid- and phosphatidylserine-binding proteins
Biochim. Biophys. Acta
Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells 1
Neuron
Revisiting the role of SNAREs in exocytosis and membrane fusion
Biochim. Biophys. Acta
Phospholipid mediated plasticity in exocytosis observed in PC12 cells
Brain Res.
A stage-specific preparation to study the Ca(2+)-triggered fusion steps of exocytosis: rationale and perspectives
Biochimie
Priming in exocytosis: attaining fusion-competence after vesicle docking
Biochimie
New reagents for phosphatidylserine recognition and detection of apoptosis
Bioorg. Med. Chem.
Interaction of calcium and cholesterol sulphate induces membrane destabilization and fusion: implications for the acrosome reaction
Biochim. Biophys. Acta
Actin is not an essential component in the mechanism of calcium-triggered vesicle fusion
Int. J. Biochem. Cell Biol.
Calcium-triggered membrane fusion proceeds independently of specific presynaptic proteins
J. Biol. Chem.
The isolation of intact cortical granules from sea urchin eggs: calcium ions trigger granule discharge
Dev. Biol.
Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion
J. Biol. Chem.
The C2 domains of Rabphilin3A specifically bind phosphatidylinositol 4,5-bisphosphate containing vesicles in a Ca2+-dependent manner. In vitro characteristics and possible significance
J. Biol. Chem.
Phosphatidic acid domains in membranes: effect of divalent counterions
Biophys. J.
Mechanism of alpha-cyclodextrin-induced hemolysis. 1. The two-step extraction of phosphatidylinositol from the membrane
J. Pharm. Sci.
Identification of secretory granule phosphatidylinositol 4,5-bisphosphate-interacting proteins using an affinity pulldown strategy
Mol. Cell. Proteomics
Synapsin I-associated phosphatidylinositol 3-kinase mediates synaptic vesicle delivery to the readily releasable pool 16
J. Biol. Chem.
CAPS-1 and CAPS-2 are essential synaptic vesicle priming proteins
Cell
Calcium-triggered fusion of exocytotic granules requires proteins in only one membrane
J. Biol. Chem.
Ca2+-independent, protein-mediated fusion of chromaffin granule ghosts with liposomes
Biochim. Biophys. Acta
Point-like protrusion as a prestalk intermediate in membrane fusion pathway 6
Biophys. J.
Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes
Biochem. Biophys. Res. Commun.
Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes
J. Biol. Chem.
Synaptotagmin 1 modulates lipid acyl chain order in lipid bilayers by demixing phosphatidylserine
J. Biol. Chem.
Docking and fast fusion of synaptobrevin vesicles depends on the lipid compositions of the vesicle and the acceptor SNARE complex-containing target membrane
Biophys. J.
Cholesterol facilitates the native mechanism of Ca2+-triggered membrane fusion
J. Cell Sci.
A new approach to the molecular analysis of docking, priming, and regulated membrane fusion
J. Chem. Biol.
Sphingomyelin-enriched microdomains define the efficiency of native Ca(2+)-triggered membrane fusion
J. Cell Sci.
Effects of cholesterol on the structural transitions induced by diacylglycerol in phosphatidylcholine and phosphatidylethanolamine bilayer systems
Biochem. Cell Biol.
Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion
EMBO J.
Cholesterol, regulated exocytosis, and the physiological fusion
Biochem. J.
SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis
EMBO J.
Membrane fusion
Subcell. Biochem.
Fusion of Phospholipid Vesicles induced by Divalent Cations and Protons. Modulation by Phase Transition, Free Fatty Acids, Monovalent Cations, and Polyamines
Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers
Biochemistry
Effects of Na+, K+, and Ca2+ on the structures of anionic lipid bilayers and biological implication
J. Phys. Chem. B
Calcium binding and head group dipole angle in phosphatidylserine–phosphatidylcholine bilayers
Langmuir
Mechanisms of membrane fusion
Annu. Rev. Biophys. Biomol. Struct.
Calcium ion binding between lipid bilayers: the four-component system of phosphatidylserine, phosphatidylcholine, calcium chloride, and water
Biochemistry
Mechanism of activation of protein kinase C: roles of diolein and phosphatidylserine
Biochemistry
Cited by (18)
Arachidonic acid and lysophosphatidylcholine inhibit multiple late steps of regulated exocytosis
2019, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Additionally, cholesterol acts as a key local contributor of negative curvature for progression to the hemifusion stage [4,22,25], while optimal levels of sphingolipids contribute to both the efficiency and capacity for fusion [23,26]. Other Ca2+ binding sites and effectors that may be localized at the fusion site as ‘efficiency’ factors include PS, cholesterol sulfate, and synaptotagmin [4,23,26,27]. Apart from modifying membrane biophysical properties, exogenous lipids, including (arachidonic acid; ARA) and LPC, alter protein-protein interactions [28–31], membrane enzyme activities [32,33] and membrane lipid homeostasis [11,32,34,35].
Sphingolipids modulate docking, Ca<sup>2+</sup> sensitivity and membrane fusion of native cortical vesicles
2018, International Journal of Biochemistry and Cell BiologyPhospholipase A<inf>2:</inf> Potential roles in native membrane fusion
2017, International Journal of Biochemistry and Cell BiologyCitation Excerpt :PA, on the other hand seems to have a role in vesicle docking and/or priming (Rogasevskaia and Coorssen, 2015; Starr et al., 2016 Starr et al., 2016). In contrast, cholesterol has both an intrinsic negative curvature and defines membrane microdomains; it can directly facilitate membrane merger by lowering the energy barrier in the proximal monolayer mixing (Churchward and Coorssen, 2009; Churchward et al., 2008; Churchward et al., 2005), and could also promote the efficiency of the fusion mechanism by maintaining other key components in a localized, functional state – the physiological fusion machine (Churchward et al., 2005; Rogasevskaia et al., 2012). Studies have suggested a role for PLA2 in regulated exocytosis, and perhaps in membrane merger in neuroendocrine cells, as treatment with PLA2 inhibitors resulted in blockade of ARA production and a parallel inhibition of triggered exocytosis (Ray et al., 1993).
The role of phospholipase D in regulated exocytosis
2015, Journal of Biological ChemistryCitation Excerpt :In addition to comparable Ca2+ activity curves, a well established and rigorously tested mathematical model of CV-PM fusion also describes CV-CV fusion (42, 43, 46, 64). Considering that (i) full docking is critical to efficient fusion and (ii) the rigorous mathematical model was originally derived for fully docked, fusion-ready CV at the PM, the data here are consistent with previous conclusions that the CV membrane contains the minimal essential machinery for docking, Ca2+-sensing, and fusion (reviewed in detail in Refs. 19, 45, and 65–67). Thus, although the docking/settle assay used here involves a modification of the CV-CV fusion assay, this strongly reflects native docking.
Secretory vesicle cholesterol: Correlating lipid domain organization and Ca<sup>2 +</sup> triggered fusion
2015, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :The role of cholesterol in heterotypic (vesicle–plasma membrane) and homotypic (vesicle–vesicle) fusion has been rigorously analyzed using the CV model system. Well established sigmoidal Ca2 + activity curves for CV fusion (Fig. 1) reflect both the ability of vesicles to fuse (extent) and the efficiency of the reaction (Ca2 + sensitivity) [12–14,27,31,32,34,65,67]. Cholesterol depletion results in both a decrease in the extent and Ca2 + sensitivity of fusion without altering the underlying shape of the Ca2 + activity curve.
Cholesterol stabilizes fluid phosphoinositide domains
2014, Chemistry and Physics of Lipids
- 1
Current address: Centre for Neuroscience, University of Alberta, 116 St and 85th Ave, Edmonton, AB, T6G 2R3 Canada.