Hannabeth Franchino and Dr. John Bell, Physiology and Developmental Biology

This study investigated the effects of lipid phase on monomer and dimer fluorescence of merocyanine 540. Emission and anisotropy spectra were assessed at multiple temperatures covering all four lamellar phases of pure dipalmityolphosphadtidylcholine. The probe segregates in the bilayer into two populations: monomers (emission maximum~585 nm) and dimers (emission maximum~621 nm). Induction of the crystalline (LC) phase by extended pre-incubation at 4 °C produced a strong wavelength dependence of anisotropy values (0.33 at 580 nm, 0.14 at 625 nm). Wavelength dependence was strong at 15 and 25 °C, weak at 38 °C, and absent above the main phase transition (>41.4 °C) or after returning the temperature to from 46 to 25 °C. Average anisotropy values across the complete temperature range revealed both the sub- and main phase transitions. The temperature dependence of total fluorescence intensity likewise displayed both transitions, along with the pre-transition. In contrast, changes in the shape of the emission spectrum were sensitive to the sub- and pre-transitions but not the main phase transition. These changes were quantified by calculating the ratio of intensity at the two peaks in the emission spectrum (585 and 621 nm). Additionally, these three characteristics were assayed in vesicles containing cholesterol, allowing for the creation of a phase diagram. Variance in the measurements in membranes with high cholesterol concentrations indicates lack of uniformity in the liquid-ordered phase (Lo). These results indicate that dimer fluorescence mostly vanishes at the pre-transition because the spectrum shape was unchanged above 35 °C. Thus, the absence of wavelength dependence of anisotropy at higher temperatures was largely due to the loss of fluorescence from probe dimers. These observations are consistent with a model in which merocyanine dimers are localized to the region between membrane leaflets where their motion is greater than that of the monomers, which reside among the packed head groups. Moreover, dimer fluorescence intensity is enhanced by constraints on its movement by highly-ordered lipids.

I am using my research from my ORCA project for both an Honors Thesis (for my upcoming April 2010 graduation) and for a paper for publication in the Biophysical Journal. The above paragraph will be submitted as the abstract for both projects. Below, I have included several figures which I intend to use for each publication.

Figure 1 (A- left; B- right). MC540 anisotropy values for vesicles in the Lc phase (15 °C) depended strongly on emission wavelength. The decreasing trend in anisotropy between 580 nm and 625 nm was statistically significant based on linear regression. The anisotropy measurements were repeated at 25 °C, a temperature above the subtransition from the Lc to the LB (gel) phase. When this was done, the wavelength dependence persisted, although the overall anisotropy values were lower compared to those at 15 °C. However, once the sample passed through the pre-transition from the LB to the PB′ phase, changes across the wavelength spectrum were significantly reduced (38° C) and were nearly absent above the main phase transition (46 °C; tm = 41.4 °C).
One of the characteristics of the Lc phase is that the kinetics of the transition to and from it are slow such that significant hysteresis is observed. We used this phenomenon to determine whether the wavelength dependence at low temperature was due to the Lc phase by returning the temperature to 25 °C after the vesicles had passed through the main transition. The loss of wavelength dependence at 25° C following a temperature reversal indicates that the vesicles were affected by their recent thermal history suggesting that the original data at 25 °C reflected vestigial effects of the Lc phase caused by hysteresis through the subtransition. This interpretation was confirmed by experiments with vesicles that were stored at room temperature continuously since their manufacture (Figure 1B). Similar to the data for vesicles returned to 25 °C after passing through high temperature (violet symbols in Figure 1A), the anisotropy trend at 25 °C matched the trend at 38 °C.

Figure 2 displays a detailed temperature profile of MC540 anisotropy for vesicles stored previously at 4 °C (“refrigerated”). The hysteretic recovery of anisotropy from the sub transition was evident for the data from 20 to 32 °C. The triangle and dotted line indicate the anisotropy level upon return of the vesicles from high temperature. The main phase transition was also apparent in the data at 40–42 °C.

Figure 3. Evidence for all three phase transitions was observed in the temperature profile of emission intensity (585 nm, Figure 3). The data shown in black were obtained from refrigerated vesicles. The red symbols represent vesicles equilibrated at 50 °C prior to measurements. The rising intensity from 29 to 35 °C in the black curve appeared due to both the sub- and the pre-transition, as indicated by comparison to the data from equilibrated vesicles. The difference in starting intensity between the refrigerated and high-temperature equilibrated vesicles presumably reflected the subtransition as argued above for the anisotropy data. Since the equilibrated vesicles would have been in the Lβ′ at 25 °C, the intensity elevation at 29–35 °C corresponded to the pre-transition. The main transition was evident by the change in intensity between 40 and 42 °C.

Figure 4 (A- right, B- left). As shown in Figure 4A the emission spectrum shape differed between high and low temperatures. The low temperature spectrum consisted of two peaks, reflecting separate populations of MC540 in fluorescent monomer (585 nm) and dimer (621 nm) conformations. At temperature above the main phase transition, the dimer subpopulation no longer fluoresces, and the spectrum observed here accordingly consisted of a single peak, reflecting only monomer fluorescence. Previously, the spectrum showing both monomers and dimers was attributed to the gel phase (Lβ) of the lipids. However, the emission spectra at 25 °C from vesicles equilibrated at high temperature mostly lacked the dimer peak, indicating that the membrane configuration in the Lc phase may contribute more to dimer fluorescence than that of the Lβ phase (Figure 4B). Figure 5. Differences in the shape of the spectra were quantified by calculating the ratio of fluorescent intensity at the two spectral peaks (Figure 5). Comparison of the detailed temperature profile between refrigerated and equilibrated ratio calculations revealed similarities to the pair of curves for the normalized intensity. The equilibrated ratio returned to a point that sits midway along the 29 to 35 °C increase of the refrigerated vesicles. Therefore, both the sub- and the pre-transition appeared to contribute to the temperature dependence between 29 and 35 °C. However, unlike the emission intensity (Figure 3), the ratio of intensities remained constant as the lipids passed through the main transition, indicating no change in the shape of the emission spectra above the pre-transition (Figure 5).