Variations in Fusion Pore Formation in Cholesterol-Treated Platelets

Abstract

Exocytosis is a highly regulated intercellular communication process involving various membrane proteins, lipids, and cytoskeleton restructuring. These components help control granule fusion with the cell membrane, creating a pore through which granular contents are released into the extracellular environment. Platelets are an ideal model system for studying exocytosis due to their lack of a nucleus, resulting in decreased membrane regulation in response to cellular changes. In addition, platelets contain fewer granules than most other exocytosing cells, allowing straightforward measurement of individual granule release with carbon-fiber microelectrode amperometry. This technique monitors the concentration of serotonin, an electroactive molecule found in the dense-body granules of platelets, released as a function of time, with 50 μs time resolution, revealing biophysical characteristics of the fundamental exocytotic process. Variations in fusion pore formation and closure cause deviations from the classic current versus time spike profile and may influence diffusion of serotonin molecules from the site of granule fusion. Physiologically, the delivery of smaller packets of chemical messengers or the prolonged delivery of chemical messengers may represent how cells/organisms tune biological response. The goals of this work are twofold: 1) to categorize secretion features that deviate from the traditional mode of secretion and 2) to examine how changing the cholesterol composition of the platelet membrane results in changes in the pore formation process. Results herein indicate that the expected traditional mode of release is actually in the minority of granule content release events. In addition, results indicate that as the cholesterol content of the plasma membrane is increased, pore opening is less continuous.

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

“…Hence, enrichment of cholesterol seems to enhance membrane rigidity in the cell model membrane. Studies on cells are in agreement with our findings 24 26 27 and specifically one study on platelets has shown that cholesterol increases the percentage of total post spike feet in partial release 43 .…”

“…The foot current amplitude is larger with higher amount of membrane cholesterol, suggesting that a more rigid membrane is more difficult to bend, forcing the pore to stay open at bigger size. Similar results have been observed in live cells, where cholesterol creates larger fusion pores with similar dynamics as seen here 43 44 47 . The duration of the foot can in turn be related to the time it takes for the pore to shrink down in to a nanotube and it does in contrast not change with higher amounts of membrane cholesterol.…”

“…In this work, amperometric recordings of dopamine release from two different cell models exhibiting high and low membrane tension was employed to evaluate the influence of cholesterol on two distinct modes of exocytosis. The kinetic information from the current spike characteristics was used to deduce that cholesterol influences the fusion pore dynamics by stiffening the membrane of the fusion pore and widening the pore size, which is in agreement with recordings performed at live cells 43 44 .…”

The role of cholesterol in membrane fusion

“…Cholesterol’s direct effects on the dynamics of forming fusion pores has been measured in cells using amperometery (Ge et al, 2010; Wang et al, 2010; Koseoglu et al, 2011; Gruba et al, 2015; Finkenstaedt-Quinn et al, 2016). Recapituating these effects observed in cells, reconstitution of SNARE-mediated fusion in different biochemical settings also shows more efficient fusion when the cholesterol content is increased in the membrane (Tong et al, 2009; Kreutzberger et al, 2015; Stratton et al, 2016).…”

Artificial stabilization of the fusion pore by intra-organelle styrene-maleic acid copolymers

“…Inspired by the natural protein-mediated processes known for fusion pore stabilization 7,8,10,24,25 , we describe here an artificial alternative. We show that SMACs have the ability to stabilize the fusion pore in terms of its life-time.…”