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Plasma membrane rafts of rainbow trout are subject to thermal acclimation

John K. Zehmer^* and Jeffrey R. Hazel

Biology Department, Arizona State University, Tempe, AZ 85287, USA

View larger version (35K): [in a new window]   Fig. 1. Detergent analyses. (A) Diagrammatic representation of detergent–membrane interaction. Loosely packed raft-depleted plasma membrane (RDPM) accommodates more detergent prior to saturation (higher saturation point; plate 1) than tightly packed raft (plate 2). After saturation occurs, mixed micelles form, dissolving the membrane. Lipids in RDPM are more readily incorporated into micelles (steeper solubilization slope; plate 3) than are raft lipids (plate 4). (B) Detergent assay. Membrane dissolution is followed as changes in optical density (OD) over time. End points are recorded as optical density just prior to detergent addition (arrow). Inset: normalized end points as a function of detergent/lipid (D/L) ratios with sigmoidal regression. (C) Analysis. Sigmoidal regression with D/L values of 2nd-derivative peaks marked (points a and b) with the linear regression between points a and b superimposed. Inset: 2nd derivative with peaks marked (points a and b). Saturation point (i.e. where solubilization becomes dominant over detergent incorporation) is defined by point a. Solubilization slope is defined as the slope of the linear regression between points a and b.

View larger version (47K): [in a new window]   Fig. 2. Western blots. (A) Distribution of caveolin in trout kidney (Kid), spleen (Spl), adipose (Adi) and liver (Liv). All lanes, 15 µg total protein. (B–E) Samples loaded: 30 µg crude homogenate (Hom) and 15 µg plasma membrane (PM), raft-depleted PM (RDPM) and raft-enriched PM (raft). Probed with (B) antibodies against ß2 adrenergic receptor (ß2AR), (C) adenylyl cyclase (AC), (D) insulin receptor ß subunit (IRß) and (E) clathrin (Clath).

View larger version (19K): [in a new window]   Fig. 3. Compositional analyses of membrane fractions. (A) Compositional comparisons of membrane fractions from cold-acclimated (open bars) and warm-acclimated (filled bars) fish expressed as ratios. (A) Cholesterol/protein (Ch/Pr). (B) Phospholipid/protein (PL/Pr). (C) Cholesterol/phospholipid (Chl/PL). Values are means ± S.E.M., N=6. Bars with a common lower-case letter do not differ significantly. PM, plasma membrane; RDPM, raft-depleted plasma membrane.

View larger version (21K): [in a new window]   Fig. 4. Effects of temperature and cholesterol content on saturation point. (A) Symmetric methylene vibrational rates of myristoylpalmitoyl phosphatidylcholine (MPPC) vesicles as a function of temperature (circles). Saturation points of MPPC vesicles as a function of temperature (bars). Values are means ± range, N=2. (B) Saturation points of palmitoyloleoyl phosphatidylcholine (POPC) and POPC + 30 mol% cholesterol vesicles at 25°C. Values are means ± range, N=2.

View larger version (16K): [in a new window]   Fig. 5. Effects of temperature and cholesterol content on solubilization slope. Values are means ± range, N=2. Absolute values of slopes of near-linear regions between 2nd-derivative peaks of fitted sigmoidal curves for (A) myristoylpalmitoyl phosphatidylcholine (MPPC) vesicles as a function of temperature and (B) palmitoyloleoyl phosphatidylcholine (POPC) and POPC + 30 mol% cholesterol vesicles at 25°C.

View larger version (13K): [in a new window]   Fig. 6. Detergent solubility curves. Decline in normalized optical density (OD) as a function of detergent/lipid (D/L) ratios. Values are means ± S.E.M., N=4. Cold-acclimated samples assayed at 5°C (other data not shown). PM, plasma membrane; RDPM, raft-depleted plasma membrane.

View larger version (15K): [in a new window]   Fig. 7. Saturation points. Bars with a common lower-case letter do not differ significantly. (A) Effects of acclimation group and fraction (data collapsed across assay temperature). Cold-acclimated, open bars; warm-acclimated, filled bars. Values are means ± S.E.M., N=8. (B) Effects of assay temperature (data collapsed across fraction and acclimation group). Values are means ± S.E.M., N=24. PM, plasma membrane; RDPM, raft-depleted plasma membrane.

View larger version (21K): [in a new window]   Fig. 8. Saturation points, all comparisons. Values are means ± S.E.M., N=4. Cold-acclimated, open bars; warm-acclimated, filled bars. Dots denote measurements made at physiological temperatures. PM, plasma membrane; RDPM, raft-depleted plasma membrane.

View larger version (22K): [in a new window]   Fig. 9. Saturation points, elevated assay temperature. Values are means ± S.E.M., N=4. Asterisks denote significant difference from values at 20°C (P<0.05). RDPM, raft-depleted plasma membrane.

View larger version (17K): [in a new window]   Fig. 10. Slopes. Absolute values of slopes of near-linear regions between 2nd-derivative peaks of fitted sigmoidal curves (data collapsed across assay temperature). Cold-acclimated, open bars; warm-acclimated, filled bars. Values are means ± S.E.M., N=8. Bars with a common lower-case letter do not differ significantly. PM, plasma membrane; RDPM, raft-depleted plasma membrane.