Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis

Abstract

Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release.

Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry

“…Neither intracellular injection of the D4 peptide nor transfection of cells with the mCherry-D4-encoding plasmid produced significant labeling of the cytoplasmic leaflet of the PM (Figure S1). In MβCD-treated (10 mM, 30 min) cells (Najafinobar et al, 2016; Rituper et al, 2013a), the PM D4 staining was strongly reduced (Figure 2B, right), indicating, together with the results in Figure S1, that D4 associates with the cholesterol-rich domains in the outer PM leaflet. The extent of D4 labeling was determined by delineating a band overlaying the PM (Figure S2) and calculating the percentage of D4-positive pixels, relative to all the pixels in the region of interest.…”

“…Although the fusion pore was also modeled to be exclusively lined by lipids (Nanavati et al, 1992), it is more likely to be a mixture of proteolipids (Gasman and Vitale, 2017; Sharma and Lindau, 2018). There has been interest in how cholesterol initiates and regulates the stages of regulated exocytosis (Churchward et al, 2005a; Cookson et al, 2013; Mahadeo et al, 2015; Najafinobar et al, 2016; Rituper et al, 2012; Rogasevskaia and Coorssen, 2006; Stratton et al, 2016; Wang et al, 2010; Xu et al, 2017); however, direct evidence that membrane lipids define the dynamics of single fusion pore stages is missing. Our results establish the role of a cholesterol-dependent radial force that constricts the fusion pore (Figures 5 and 6).…”

Interplay between Membrane Curvature and Cholesterol: Role of Palmitoylated Caveolin-1

“…Although counterintuitive, the increased cholesterol on the opposing leaflet appears to be critical in inducing membrane curvature. In fact, spontaneous negative curvature of cholesterol is believed to favor negative curvature regions of the membrane (60)(61)(62)(63). We suggest that the increased cholesterol density in the opposing leaflet plays an important role in cav-1 modulated membrane topology.…”

Cellular cholesterol regulates monocyte deformation

“…Our data show that the cellular cholesterol content governs cellular deformability as a consequence of PKC phosphorylation, which directly orchestrates several important downstream signaling pathways. The changes in cellular deformability can have significant implications in monocyte immune response including interaction with the vascular endothelium, transmigration into tissues, and phagocytosis [31–33] . Monocytes are vital components of the immune system and assessment of biomechanical response at the cellular level can help better understand the mechanistic aspects of infection and inflammation.…”