Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. in and monolayers. Enriching the monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a flat structure). Rather, such effects 864070-44-0 support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut. solution; the top, solution was grounded. When the holding potential was 0 mV, at least two sine waves of different frequencies were simultaneously applied so that pore conductances could be calculated. The in-phase (Y0), out-of-phase (Y90), and DC (YDC) components of admittance were calculated on-line with a software-based phase detector (Ratinov et al., 1998) and used to calculate pore conductance (Melikyan et al., 1995solution (i.e., the bilayer bends in a path opposing the curvature from the cells at their stage of get in touch with). This path of bending happens if positive spontaneous monolayer curvature real estate agents (e.g., lysophosphatidylcholine) are contained in the leaflet or adverse spontaneous curvature substances are put into just the leaflet of the in any other case symmetrical bilayer. We define the bilayer curvature as adverse if it bulges in to the solution. That is achieved either with the addition of adverse spontaneous monolayer curvature real estate agents inside the leaflets or positive spontaneous monolayer curvature real estate agents in the leaflets. Data Evaluation Several features of fusion activity had been acquired to characterize the results of changing spontaneous monolayer curvature (discover Fig. ?Fig.2).2). (was plotted as and leaflet with 20 mV put on the perfect solution is (leaflet with ?20 mV (leaflet, flickering happened for prolonged intervals and a pore never fully enlarged often. Fusion pores linking a cell and planar bilayer are attracted as toroidal instead of like a pore within a hemifusion diaphragm. As can be elaborated and demonstrated in the dialogue, open up time distributions recommend a toroidal rather than hemifusion structure in the pore flickering stage. Demonstrated will be the ideal moments from triggering fusion by acidification to pH 4.9 before appearance from the first fusion pore, and 0.05 where may be the attained degree 864070-44-0 of significance as examined by the non-parametric two-sample Kolmogorov-Smirnov check. outcomes R18 and DiI Promote Negative Curvatures R18 and DiI are positively charged amphiphiles with one or two C18-saturated hydrocarbon chains, respectively, causing them to partition into membranes. The manner by which R18 and DiI affect monolayer curvature was assessed by 864070-44-0 measuring the direction and extent by which they shift the bilayer to hexagonal phase transition temperature (TH) of a model phosphatidylethanolamine membrane. A plot of TH of DEPE as a function of the mole fraction of dye added to the membrane is shown in Fig. ?Fig.1.1. Both probes exhibited a linear relationship SMOC1 between the lowering of TH and the mole fraction of probe added to the membrane. This indicates that both probes facilitated the formation of inverted phases with unfavorable curvature. At a mole fraction of 0.01, DiI lowered the TH of DEPE by 3.6 0.3C, while R18 lowered this transition temperature by 1.4 0.1C. That is, a mole fraction of 0.01 R18 and 0.005 DiI lowered TH by approximately the same amount. For the fusion studies, we used 5 mol% R18 and 3 mol% DiI in the planar membrane. Thus, at these concentrations, the two probes should have altered spontaneous curvatures to roughly the same extent. Open in a separate window Physique 1 The bilayer to hexagonal phase transition of DEPE was measured by differential scanning calorimetry 864070-44-0 as a function of the mole fraction of R18 and DiI added to the phospholipid. Both probes facilitated the formation of the unfavorable curvature inverted phase. A linear relationship was exhibited between the amount of probe added and the observed transition temperature. Linear.