Hierarchy of interactions dictating the thermodynamics of real cell membranes: Following the insulin secretory granules paradigm up to fifteen-components vesicles
Graphical abstract
Introduction
The Insulin Secretory Granule (ISG) is a complex intracellular organelle of the pancreatic β-cell dedicated to insulin storage and secretion [1]. These subcellular vesicles have been characterized as phospholipid bilayer spheres of about 300 nm of diameter [2,3] containing a crystalline core of insulin and zinc surrounded by a halo region of amylin (also known as Islet Amyloid Polypeptide, IAPP) together with other additional molecules [4,5]. However, such a granule is far more than just a repository of the hormones in the cell since it is the site of insulin proteolytic activation and it is involved in intercellular communication. Moreover, its membrane contains a variety of proteins which are key components for secretion control, different catalytic activities and messenger functions [3].
However, cells and vesicles functionalities are not only dependent on protein-based mechanisms. Indeed, lipid composition of phospholipid membranes severely affect their thermodynamic stability, which in turn is able to influence membrane proteins’ functionality [[6], [7], [8]]. Moreover, among other external factors, the presence of Free Fatty Acids (FFAs) may severely affect the membrane thermotropic behaviour [9]. Low levels of FFAs are naturally present in biological membranes (around 0.3–10 % of total lipids) [10] as well as in plasma. However, altered FFAs levels are recurrent in diabetic and/or obese subjects and plasma FFAs concentration is generally high in both with levels that tend to be increasingly higher with weight gain [11].
Despite the numerous studies on model membranes carried out also including calorimetric techniques, such model systems were characterized by a simple composition which does not consider the complexity and the asymmetry of the real bilayers [12] and the lipid thermodynamic reorganization [13,14] that might be a key point for the correct interpretation of membrane-based mechanisms in cells since each lipid modification might produce a reorganization of the lipid molecules within such systems, which in turn might lead to variations of their physicochemical properties such as thermodynamic stability, permeability, fluidity, etc. [9,15].
In this frame, we started a stepwise study on the influence of the several parameters that may influence the IGS membrane thermodynamics. Indeed, in our previous work [9], we started from simple binary systems up to the preparation of a final model membranes whose phospholipids proportionally follow their abundancy and represent the 75 % of the phospholipids’ tails and the 50 % of the headgroups in real ISGs [16]. In particular, we dissect the effects of morphology (membrane curvature), lipid composition focusing on the acyl chains keeping the same headgroup (phosphocholines with different acyl chains length and also in presence of unsaturations) and the free fatty acids (FFAs) action. In synthesis, we evidenced that curvature effects are mainly relevant among one-component unilamellar vesicles, whereas become of minor importance increasing the vesicle complexity. Moreover, the saturated phospholipids’ contribution to the overall membrane stability is somehow “additive”, whereas the presence of unsaturated phospholipids, even in minor amounts, has a relevant overall destabilizing effect and enhance the homogeneousness on the phases distribution. The entropic effects were hierarchically summarized as phospholipid unsaturations > phospholipid tail length > membrane curvature. As for the influence of FFAs, saturated acids produce a mainly strong entropic and enthalpic stabilizing effects, whereas the unsaturated ones produce an overall destabilization of the membranes.
In this work, we continued this step-by-step dissection following the ISGs composition and gradually including the influence of the different phospholipid headgroups, 16:0 LysoPC, sphingomyelins (egg sphingomyelin was used in this case) and cholesterol. Namely, we performed a thermodynamic study of different Small Unilamellar Vesicles (SUVs) at physiological pH by using the micro-DSC technique. At the first, vesicles were prepared as mixed system of DPPC, DPPS and DPPE in order to study how phospholipid headgroups affect the membrane thermodynamics. Then, 16:0 LysoPC and egg sphingomyelin were added reproducing the 80 % of the headgroups and an average of 35 % of the tails in real ISGs [16]. In order to integrate the acyl chain distribution, vesicles were therefore obtained by mixing fourteen different lipids, achieving a composition which represents the 80 % of the headgroups and an average of 80 % of the tails in real ISGs. The inclusion of cholesterol was finally considered for the achievement of final ISG-like membrane. The influence of three FFAs (stearic, oleic and elaidic acids) was eventually investigated, in comparison with the previous study, highlighting the magnitude of the effects on such a detailed membrane.
Section snippets
Materials
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phospho-l-serine (DSPS, sodium salt), 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS, sodium
Phospholipid headgroups effects
In order to preliminary assess the mixing behaviour of systems with different phospholipid headgroup, we chose to use systems with same tail length (16:0). The micro-DSC thermograms of SUVs with binary composition prepared at 1:1 M ratio of DPPC:DPPE, DPPC:DPPS and DPPS:DPPE are reported in Fig. 1a.
We observed broad profiles typical of unilamellar vesicles with mixed composition, i.e. less cooperative respect to the single-component vesicles [9], corresponding to the main gel-to-liquid
Conclusions
In this paper we completed a stepwise thermodynamic study on the contributions of the phospholipid constituents in the cell membrane stability keeping the phospholipid bilayer of ISGs as reference system.
The results permitted to integrate the hierarchy of influence on membrane thermodynamic stability that may be resumed as: membrane curvature < phospholipid headgroup < phospholipid tail length < phospholipid tail unsaturation. Saturated phospholipids are thermodynamically compatible and their
CRediT authorship contribution statement
Francesca Saitta: Conceptualization, Methodology, Investigation, Validation, Writing - review & editing. Marco Signorelli: Investigation, Data curation. Dimitrios Fessas: Conceptualization, Methodology, Investigation, Writing - review & editing, Supervision, Resources.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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