Phospholipid mediated plasticity in exocytosis observed in PC12 cells

Abstract

Membrane composition serves to identify intracellular compartments, signal cell death, as well as to alter a cell's electrical and physical properties. Here we use amperometry to show that supplementation with the phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and phosphatidylserine (PS) can alter several aspects of exocytosis. Changes in the amperometric peak shape derived from individual exocytosing vesicles reveal that PC slows expulsion of neurotransmitter while PE accelerates expulsion of neurotransmitter. Amperometry data reveal a reduced amount of catecholamine released per event from PC-treated cells while electron micrographs indicate the vesicles in these cells are 50% larger than controls, thus providing evidence of pharmacological changes in vesicle concentration. Addition of SM appears to affect the rate of fusion pore expansion, indicated by slower peak rise times, but does not affect decay times or quantal size. Addition of PS results in a 1.7-fold increase in the number of events elicited by high-K + depolarization. Electron micrographs of PS-treated cells suggest that increased vesicle recruitment underlies enhanced secretion. We did not observe any effect of phosphatidylinositol (PI) treatment. Together these data suggest that differences in membrane composition affect exocytosis and might be involved in mechanisms of cell function controlling the dynamics of communication via exocytosis.

Phosphatidylserine Regulation of Ca2+-triggered Exocytosis and Fusion Pores in PC12 Cells

“…An important goal for future research is to design experiments that can distinguish between various alternative mechanisms by which PS can influence exocytosis. Many proteins regulate fusion pores ), but we know less about the effects of lipids (Uchiyama et al, 2007;Lam et al, 2008). In comparison with proteins, lipids are difficult to manipulate in living cells, due to the complex regulation of lipid-metabolizing enzymes (Vance and Vance, 2004).…”

“…As an initial attempt to test the role of PS in exocytosis, we added 100 M PS directly to the culture medium (Uchiyama et al, 2007). Two days later, lipid analysis with HPLC showed that this raised the total PS content of PC12 cells from 10.3 to 12.1% (Table 1).…”

The evidence for open and closed exocytosis as the primary release mechanism

“…The most direct evidence that the fraction released can be altered was presented by both the Amatore and Ewing groups who showed that the amount of neurotransmitter released per event can be quickly altered by influencing the lipid composition of the plasma membrane (Amatore et al 2006;Mellander et al 2012;Uchiyama et al 2007). In one case, they found that a shortterm incubation of PC12 cells with the inverted cone shaped lipid lysophosphatidylcholine (LPC) increased the average amount of dopamine released per exocytosis event while incubation with the cone shaped phosphatidylethanolamine (PE) decreased it.…”

Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation

“…Amatore et al recently showed that these lipids could each produce the opposite effect when added to the extracellular media (the noncontact side of the fusing membranes) of chromaffin cells undergoing exocytosis (33). In a similar experiment PC12 cells were incubated with dipalmitoylphosphatidylcholine, a lamellar lipid (close to no intrinsic curvature), resulting in a decrease in the rate of exocytosis (34). In summary, experiments with model systems show that physical manipulation can be used to redistribute membrane composition, and cell experiments show that altering composition can regulate the dynamics of the fusion event.…”

“…Our results suggest that function and structure dictate membrane composition rather than a mechanism where composition is altered in anticipation of function. This membrane composition is clearly important in determining cellular regulating factors, and recent observations have shown that incubation with lipids can alter the rate of chemical release via exocytosis (34,35). If, indeed, structure dictates the chemical composition of membranes, then it is convenient to speculate that membrane fusion events during exocytosis result in a subsequent change in local membrane composition.…”