Figure 1.
Differential scanning calorimetry of pure sphingomyelins and ceramide-1-phosphates.
Thermogram of A) C12SM (green) and C12Cer-1-P (red). B) Thermogram of egg SM (green) and and C16Cer-1-P (red).
Figure 2.
LAURDAN GP of C12SM∶C12Cer-1-P mixtures at 22°C.
The LAURDAN GP of suspensions of mixed C12SM and C12Cer-1-P at different ratios was measured. Pure C12Cer-1-P could not be extruded so the LAURDAN GP measurement was taken directly on the lipid suspension.
Figure 3.
Pre-mixed C12SM∶C12Cer-1-P domains in GUVs.
Representative images of domains in individual vesicles composed of 23 mol% C12Cer-1-P visualized with (A) DiIC18 which is excluded from the C12Cer-1-P-enriched domains, B) LAURDAN intensity image at the pole of a giant vesicle where photoselection prevents probe excitation in the more ordered domains and, C) LAURDAN GP analysis of domains in an equatorial section showing areas of low dipolar relaxation (high GP, top histogram) and higher dipolar relaxation (lower GP, bottom histogram). Histograms were determined opposite to each other to avoid any bias caused by photoselection. Bars are 5 µm.
Figure 4.
Kinetics of SMD activity on large unilamellar vesicles.
The main panel shows a representative trace of the kinetics of SMD activity on LUV preparations of C12SM (red), egg SM (green) and POPC (black) at 22°C. Inset: Specific activity of SMD on C12SM and egg SM LUVs. The difference in specific activity is statistically significant (P<0.0001).
Figure 5.
Dynamic Light Scattering of C12SM LUVs under the action of SMD.
Representative evolution of the size (as mean diameter in nm) of C12SM vesicles treated with 50 ng/ml (red) and 1 µg/ml (green) SMD. The open black circles show the scattering behavior of untreated vesicles, which was similar to control POPC vesicles treated with SMD and C12SM vesicles treated with inactive Lb3 (not shown).
Figure 6.
Förster resonance energy transfer (FRET) of LAURDAN- and DiIC18-labeled LUVs under the action of SMD.
Separately labeled vesicles were mixed at a 1∶1 ratio and treated with SMD (1 µg/ml). Emission at the DiIC18 maximum (with excitation at 374 nm) was followed in time relative to a reference of an identical liposome mixture without enzyme. The ordinate is the normalized ratio of fluorescent emission of the treated over the untreated control.
Figure 7.
Fluorescence correlation spectroscopy of C12SM LUVs under the action of SMD.
A) Representative normalized autocorrelation plots for the untreated vesicles (green circles), the same preparation treated with 1 µg/ml SMD (red circles) and a control with Triton X-100 (open black circles). B) Diffusion coefficient (D2) in time calculated from fitting the autocorrelation plot for the enzyme-treated sample. The bottom panels represent fluorescence images taken at 24 hours of C) sedimented material after enzyme treatment (1 µg/ml SMD) and D) untreated C12SM LUVs. The images are 30 µm×30 µm.
Figure 8.
LAURDAN GP image of sedimented material from C12SM LUVs treated with SMD.
A) Sedimented control 100 nm liposomes after 24 hours (without SMD) at the bottom of the slide. Average GP value is −0.029±0.129. B) Sedimented material of LUVs treated with 1 µg/ml SMD after 24 hours at the bottom of the slide. Average GP value is 0.228±0.088. C) Same as B) but 5 µm above the slide surface. Average GP of 0.269±0.176 with areas of GP of 0.452±0.124. D) Sedimented material from the 50 mol% pre-mixed lipids without enzyme. Average GP of −0.044±0.079. Fields are 19 µm×19 µm.
Figure 9.
Representative kinetics of LAURDAN GP of C12SM LUVs treated with SMD.
LAURDAN-labeled C12SM LUVs were treated with different concentrations of SMD: 100 ng/ml (green triangles), 1 µg/ml (blue squares) and 2.5 µg/ml (red circles). The black open circles are the same vesicles before addition of enzyme and the black crosses are non-substrate POPC LUVs with 2.5 µg/ml SMD. The arrow indicates the time of SMD addition.
Figure 10.
Representative fluorescence microscopy images of the action of SMD on C12SM giant unilamellar vesicles.
A) DiIC18-labeled GUVs. Top left shows a typical vesicle before SMD addition and bottom left a GUV after 17 hours of incubation with inactive Lb3. The center panel shows domain formation in several GUVs 1–3 hours after SMD addition. On the right a collapsed vesicle with extruded tubes. B) Changes in LAURDAN GP induced by SMD. The left panel shows an untreated GUV, homogeneously fluid. The center panel shows domains of different GP value (indicated by arrows). The right panel shows a GUV with tubular extrusions (left) and a collapsed one (right). Bars are 5 µm.
Figure 11.
Protein fluorescence spectra of SMD.
The main panel shows the normalized fluorescence emission spectrum of SMD (excitation at 280 nm). The black trace is SMD alone, mixed with POPC vesicles (green) and with C12SM LUVs (red). Inset: fluorescence polarization values for the same samples. The differences in polarization are statistically significant (P<0.003).
Figure 12.
Effect of product on relative enzyme specific activity and on vesicle size.
A) Specific activity of SMD on C12SM LUVs with increasing molar fractions of C12Cer-1-P. With the exception of pure C12SM and the 50 mol% mixture, all other differences are statistically significant (P<0.003). B) Mean diameter in nm versus time obtained by DLS of C12SM∶C12Cer-1-P LUVs: The black open circles represent pure C12SM untreated vesicles, the green triangles represent untreated C12SM∶C12Cer-1-P vesicles (23 mol%), the closed red squares are C12SM∶C12Cer-1-P vesicles (50 mol%) treated with 1 µg/ml SMD and the open red squares are the same but without enzyme.
Figure 13.
Effect of cholesterol on relative enzyme specific activity and on vesicle size.
A) Effect of cholesterol on the specific activity of SMD. With the exception of pure C12SM and the 9 mol% mixture, all other differences are statistically significant (P<0.003). B) Mean diameter in nm versus time obtained by DLS of C12SM∶Cholesterol LUVs: The black circles represent pure C12SM vesicles with SMD, in green C12SM∶cholesterol vesicles (23 mol%) with (closed squares) and without (open squares) SMD and in red C12SM∶cholesterol vesicles (50 mol%) with SMD (closed triangles) and without (open triangles). Enzyme concentration was 1 µg/ml.
Figure 14.
Representative fluorescence images of the effect of SMD on GUVs composed of DOPC∶eggSM∶cholesterol 2∶1∶1 mol.
A) Domains in a DiIC18-labeled GUV exposed to inactive Lb3 for 16 hours. B) Representative DiIC18-labeled GUVs before and after treatment with SMD. C) Two LAURDAN-labeled GUVs before (left) and after (right) exposure to SMD. Before treatment two domains are apparent (average GP values of 0.1 and 0.5 indicated by the green and red arrows, respectively), after SMD action the membrane becomes uniform (average GP value of 0.3). Bars are 5 µm.