Dr. John Bell, Department of Physiology and Developmental Biology
This proposal was designed to use fluorescence spectroscopy to identify in as much detail as possible physical properties of model membranes in effort to understand roles of cholesterol in biological membranes. The following questions were addressed: how does the effect of cholesterol differ between the inner and outer faces of the cell membrane; can fluorescence spectroscopy distinguish between homogenous and heterogeneous mixtures of membrane components; and what is the biophysical mechanism associated with one of the fluorescent probes used (merocyanine 540)? These questions were addressed with three specific aims. All three aims have been accomplished. One paper was published during 2008 based on aims 1 and 2 (with 5 undergraduate students as first and co-authors), and a second paper based on aim #3 is currently being prepared for submission in early 2009 (4 undergraduate students as first and co-authors). In addition, rapid completion of these aims allowed us to expand the scope of the original proposal to include two new directions in the laboratory studying the effects of cancer chemotherapeutic agents and endoplasmic reticulum stress on membrane properties. The information attained from the original aims of this proposal is important for enabling us to interpret the complex results attained with the chemotherapeutic agents and during endoplasmic reticulum stress. In addition to the two papers published or in preparation, three abstracts have been submitted for presentation at the annual international meeting of the Biophysical Society next February in Boston based directly on the results of this MEG (including 18 undergraduate students as co-authors), and three other papers are in review or in preparation based on the expanded scope of this MEG (also including 18 undergraduate students as co-authors).
- Generate two-dimensional phase maps for artificial membranes simulating the inner and outer leaflets of human erythrocytes. The phase maps will be based on three physical properties (inter-lipid spacing, lipid order, and lipid fluidity). The dimensions will be temperature and cholesterol concentration. The fluorescent probes to be used to construct the phase maps are merocyanine 540, laurdan, and diphenylhexatriene. Compare these phase maps and the average of the two to the whole erythrocyte maps. Make these comparisons on a probe-by-probe basis.
- Generate three-dimensional phase maps using the same fluorescent probes and assessing the same properties as in Aim 2. The dimensions will be cholesterol, saturated phosphatidylcholine, and unsaturated phosphatidylcholine concentrations. The physical measurements will be repeated at various temperatures to add a fourth dimension and thereby create a more complete thermodynamic description of the system.
- Use fluorescence anisotropy to determine whether the proposed mechanism for the fluorescence spectrum of MC540 is valid.
Aim #1: Figure 1 displays phase maps generated for human erythrocytes at various concentrations of membrane cholesterol. Cholesterol content was manipulated using methyl-β-cyclodextrin. Laurdan was used as a probe of lipid disorder in the membrane. Diphenylhexatriene (DPH) senses both lipid disorder and membrane fluidity. Merocyanine 540 detects the relative degree of spacing among adjacent lipids in the membrane. By overlaying the results with the three probes, five regions of distinct membrane properties were identified. Region I was characterized by relatively low spacing, low fluidity and high order. Region II displayed greater fluidity and spacing with a comparatively smaller reduction in order. Region III was observed at high temperature and when significant amounts of cholesterol are depleted from the membrane. Relative to Region II, it possessed lower order, higher fluidity, with the same level of apparent lipid spacing. Region IV, which seemed to dominate the phase map, described membrane behavior with diminished order compared to regions I and II but also with lower fluidity and tighter spacing than regions I and III. A fifth region was identified at moderate cholesterol concentrations (~30%). It appeared to have the same properties as Region III, but with greater spacing among phospholipids. Other color variations in the composite phase map presumably reflected smooth transitions among these regions.
An important issue with fluorescence measurements with erythrocytes is discerning whether the results observed represent the behavior of the outer membrane leaflet, the inner leaflet, or an average of the two. The primary purpose of aim #1 was to generate data with artificial membranes to help make that distinction. Since merocyanine 540 would have limited ability to cross the membrane and enter intact cells (verified for other cell types by confocal microscopy, not shown), we decided not to include it in this part of the study because its fluorescence reports only the outer leaflet. In contrast, the more hydrophobic laurdan and DPH do cross the cell membrane and presumably report behavior from both leaflets. In effort to provide some additional basis for interpretation of the data in Fig. 1, we created liposomes containing lipids representative of the outer or inner leaflets of the erythrocyte membrane and repeated the measurements with DPH and laurdan at both high and low cholesterol concentrations. As shown in Fig. 2, the results for the “inner leaflet” liposomes were qualitatively similar to those for the “outer leaflet” with respect to laurdan, although the overall level of disorder was greater than observed in the cells. Diphenylhexatriene responded more to cholesterol reduction for the “inner leaflet” liposomes than for the “outer.” Consequently, the hue for overlayed data switched from green-dominated for the “outer leaflet” as cholesterol concentration was increased, whereas it remained blue-dominated at both low and high cholesterol for the liposomes simulating the inner leaflet. These results suggest that the differences between the two leaflets with respect to the lipids are likely to be small, although the observations for the “outer” membrane resemble the data of Fig. 1 more than do those for the “inner” membrane. Moreover, evidence and analysis from studies comparing the leaflets of intact bilayers suggest that the two may be coupled thus reducing transbilayer differences in properties. These data were published during 2008 in the Journal of Lipid Research.
Aim #2: Phase maps were generated with liposomes containing equimolar mixtures of dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) combined with various concentrations of cholesterol. Rather than creating three-dimensional maps as originally proposed, we focused our attention on this combination of saturated (DPPC) and monounsaturated (DOPC) phospholipids which allows one to explore the behavior of the fluorescent probes under conditions of phase coexistence. At low cholesterol concentrations (below 10 mol %) the system consists of solid-ordered and liquid-disordered phases. Between 10 and 45 mol % cholesterol there is coexistence of liquid-disordered and liquid-ordered phases. At higher cholesterol contents, the liposomes would display solely liquid ordered properties. The results of these experiments are summarized as color phase maps in Fig. 3. Compared to phase maps generated with pure DPPC, there was much less diversity of fluorescence behavior across the cholesterol and temperature ranges tested. Most of the variations in fluorescence properties were attributed to the values of laurdan GP. Diphenylhexatriene anisotropy values followed similar trends to those of laurdan GP, but with a smaller range of variation. Merocyanine fluorescence was essentially constant throughout the range of experimental conditions.
These data, together with those from a previous MEG using sphingomyelin liposomes facilitate interpretation of the data for cells in Fig. 1. For example, regions I and II compare well with the two types of liquid-ordered phase observed in the sphingomyelin liposomes. Likewise, region III in the erythrocytes displays relative behavior reminiscent of the liquid-disordered phase. In fact, it is likely that all three regions with decreased cholesterol reflect lipid properties influenced heavily by disordered unsaturated lipids (III, IV, and V). Comparison to the DPPC/DOPC vesicles (Fig. 3) supports this idea, especially in terms of the smooth transition from green- dominated to blue-dominated hues in the combined laurdan and DPH data. Nevertheless, the absolute level of membrane order displayed by the erythrocytes was less than the average level observed for the liposomes, regardless of the presence of unsaturated phospholipids (Fig. 1). Furthermore, it is certain that the observed properties, regardless of cholesterol concentration are impacted by the heterogeneity of membrane lipids, the presence of integral membrane proteins, the transbilayer asymmetry of phospholipid species, and interactions with the cytoskeleton. These data were published during 2008 in the Journal of Lipid Research.
Aim #3: This aim was based on the hypothesis that merocyanine 540 binds to phospholipid membranes in two different configurations. In one configuration, the probe resides as a monomer superficially in the membrane aligned parallel with the phospholipid side chains. In the second configuration, it resides in the membrane interior as a dimer aligned perpendicular to the lipid chains. These two configurations are thought to emit light at different energy levels and to exhibit different levels of rotational movement. This hypothesis was tested using steady-state fluorescence spectroscopy, wavelength-dependent steady-state anisotropy measurements, and wavelength-dependent fluorescence lifetime measurements in DPPC liposomes at different temperatures. In the solid-ordered phase, where both configurations are thought to be detectable, anisotropy and lifetime measurements showed strong wavelength dependence. In contrast, at high temperatures in the liquid-disordered phase, virtually no wavelength dependence was observed. These results substantiated the hypothesis. Subsequent experiments with cultured lymphocytes revealed the relevance of these observations to biological membranes. These data are currently being prepared for submission published during 2008 in the Journal of Lipid Research.
Additional scope: The goals of the original proposal were completed much more quickly than anticipated. As a result, we had sufficient resources to pursue additional projects that continue the long-term research objective of the lab to understand the relationship between membrane physical behavior and the physiological and pathological status of cells. Two related avenues have been initiated. The first involves changes in the cell membrane during apoptosis (programmed cell death) induced by endoplasmic reticulum stress. This stress is created experimentally using the drug thapsigargin. The results of this study have shown that the cell membrane undergoes several significant changes during stress-induced apoptosis that include changes in composition, hydrolysis by secretory phospholipase A2, biphasic changes in membrane order and fluidity, and eventual disruption of membrane integrity. These changes are relevant to potential inflammatory responses during stress-induced apoptosis.
Experiments with chemotherapeutic agents have revealed similar alterations to the cell membrane. Thus, it appears that cell death involves progression of several interrelated changes in the cell membrane regardless of the means by which death is initiated. So far, the drugs used include daunorubicin and methotrexate. These experiments will be expanded greatly in the next MEG proposal.
Names of undergraduate students are bolded.
Stott, B.M., Vu, M.P., McLemore, C.O., Lund, M.S., Gibbons, E., Brueseke, T.J., Wilson-Ashworth, H.A., and Bell, J.D. (2008) Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes. J. Lipid Res. 49, 1202-1215.
Bailey, R.W., Nguyen, T.T., Robertson, L., Gibbons, E., Keller, J.N., Christensen, R.E., Bell, J.P., Judd, A.M., and Bell, J.D. Sequence of Physical Changes to the Cell Membrane during Glucocorticoid-Induced Apoptosis in S49 Lymphoma Cells. Biophys. J. (in review).
Franchino, H.A, Tajhya, R.B., Neeley, S., Johnson, B., and Bell, J.D. Resolution of subpopulations of membrane-bound merocyanine by anisotropy. (in preparation).
Gibbons, E., Griffiths, K., Warcup, A.O., Streeter, M., Askew, C., Yeung, C.H.-Y., Judd, A.M., and Bell, J.D. Alterations in membrane physical properties during apoptosis induced by endoplasmic reticulum stress. (in preparation).
Nelson, J, Berbert, A., Beck, O., Eshenroder, N., Pruitt, M., Neeley, K., Thompson, K., Thurber, B., Barlow, K., Damm, K., Judd, A.M., and Bell, J.D. Secondary effects of chemotherapeutic agents on membrane physical properties. (in preparation).
Abstracts presented at the Biophysical Society in 2008:
Stott, B.M., Franchino, H.A., Tajhya, R.B., Vu, M.P., Yeung, C.H.-Y., Wilson- Ashworth, H.A., and Bell, J.D. (2008) A complete phase diagram for palmitoylsphingomyelin-cholesterol liposomes. Biophys. J. 94 (suppl): (#393).
Nelson, J., Ellis, C.S., Robertson, L., and Bell, J.D. (2008) Investigating and modeling possible mechanisms by which healthy cell membranes become resistant to hydrolysis by secretory phospholipase A2. Biophys. J. 94 (suppl): (#2062).
Olson, E.D., Nguyen, T.T., Judd, A.M., and Bell, J.D. (2008) Analysis of hydrolysis kinetics among sPLA2 isoforms during apoptosis in S49 lymphoma cells. Biophys. J. 94 (suppl): (#2838).
Bailey, R.W., Gibbons, E., Robertson, L., Nguyen, T.T., Nelson, J., Judd, A.M., and Bell, J.D. (2008) Biophysical changes in the plasma membrane during glucocorticoid-stimulated apoptosis promote hydrolysis by secretory phospholipase A2. Biophys. J. 94 (suppl): 2841.
Abstracts for presentation at the Biophysical Society in 2009:
Franchino, H.B., Johnson, B.C., Neeley, S.K., Tajhya, R.B., Bell, J.D. (2009) Assessment of Merocyanine Subpopulations in DPPC Vesicles using Anisotropy and Lifetimes Measurements. Biophys. J. (submitted).
Gibbons, E., Askew, C.E., Griffith, K. R., Streeter, M. C., Warcup, A. O., Yeung C. H.-Y., Judd, A. M., Bell, J.D. Membrane Changes during Apoptosis: Part of the Process or Characteristics of the Corpse? Biophys. J. (submitted).
Nelson, J., Barlow, K., Beck, D.O., Berbert, A.M., Damm, K., Eshenroder, N., Neeley, K., Pruitt, M., Thompson, K., Thurber, B.W., Yeung C.H.-Y. Chemotherapeutic Apoptosis: Who assailed the membrane, the inducer, or the induced? Biophys. J. (submitted).
Additional Mentoring Activities
Six undergraduate students mentored by this MEG traveled with me to the annual meeting of the Biophysical Society in Long Beach during February of 2008 where they presented their research findings in four posters as part of the regular scientific sessions. Four undergraduate students will be traveling to Boston to present data at the same meeting in March, 2009. Two undergraduate students had the opportunity to attend the Laboratory for Fluorescence Dynamics (LFD) at the University of California, Irvine as part of their experience during August 2009. The experience at the LFD was particularly rewarding because it allowed the students to meet some of the world’s experts in the field of fluorescence and to see how research is conducted at another university. The details of student participation are listed below.
The following students were mentored at some level through this award.
Olin Beck (ug) 1 1 Nate Eshenroder (ug) 1 1 Mark Pruitt (ug) 1 1 Rajeev Tajhya (ug) 1 1 Steve Neeley (ug) 1 1 Brett Johnson (ug) 1 1 Kristina Neeley (ug) 1 1 Kyle Thompson (ug) 1 1 Kristen Barlow (ug) 1 1 Brian Thurber (ug) 1 1 Kelly Damm (ug) 1 1
|Name||Co-author on published paper||Co-author on paper in review||Co-author on papers in preparation||Co-author on presentations at Biophysical Society Meeting in 2008||Co-author on presentations at Biophysical Society Meeting in 2009||Attended Biophysical Society Meeting in 2008||Will attend Biophysical Society Meeting in 2009||Attended LFD|
|Liz Gibbons (ug and grad)||1||1||1||1||1||yes||yes||yes|
|Thao Nguyen (ug)||1||2||yes||yes|
|Brian Stott (ug)||1||1||yes||yes|
|Hannabeth Franchino (ug)||1||1||1||yes||yes||yes|
|Celestine Yeung (ug)||1||1||1||yes||yes||yes|
|Katalyn Griffith (ug)||1||1||yes||yes|
|Ashley Obray (ug)||1||1||yes||yes|
|Mike Streeter (ug)||1||1||yes|
|Caitlin Askew (ug)||1||1|
|Amanda Berber (ug)||1||1|