Anionic lipids in Ca²⁺-triggered fusion

Abstract

Anionic lipids are native membrane components that have a profound impact on many cellular processes, including regulated exocytosis. Nonetheless, the full nature of their contribution to the fast, Ca²⁺-triggered fusion pathway remains poorly defined. Here we utilize the tightly coupled quantitative molecular and functional analyses enabled by the cortical vesicle model system to elucidate the roles of specific anionic lipids in the docking, priming and fusion steps of regulated release. Studies with cholesterol sulfate established that effectively localized anionic lipids could contribute to Ca²⁺-sensing and even bind Ca²⁺ directly as effectors of necessary membrane rearrangements. The data thus support a role for phosphatidylserine in Ca²⁺ sensing. In contrast, phosphatidylinositol would appear to serve regulatory functions in the physiological fusion machine, contributing to priming and thus the modulation and tuning of the fusion process. We note the complexities associated with establishing the specific roles of (anionic) lipids in the native fusion mechanism, including their localization and interactions with other critical components that also remain to be more clearly and quantitatively defined.

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.