TI - Effects of Cav1 expression on membrane structure in adherent cells . AB - Biochemical assay may not reflect preexisting rafts in intact , adherent cells ( 6 , 9 , 14 ) . Hence , we assessed the effect of Cav1 expression on the physical state of cell membranes in intact cells labeled with the fluorescent probe Laurdan ( 36 ) . The probe is sensitive to membrane structure , with its peak emission shifting from ~500 nm in fluid to ~430 nm in ordered membrane . A normalized ratio of the two emission regions , defined as generalized polarization ( GP ) , reflects membrane order with +1 indicating the most ordered and -1 the most fluid membranes . Recently , we showed that Cav1 is associated with ordered domains in WT MEFs , yielding a mean GP value of 0.382 + - 0.070 ( from n = 27 images ) , while the nonraft marker , transferrin receptor , colocalizes with fluid domains ( GP 0165 + - 0066 , n = 13 ) ( 35 ) . The subunit B of cholera toxin , which specifically binds to the ganglioside GM1 and is frequently used as a raft marker , also colocalized to ordered domains in both WT and Cav1-/- cells ( GP 0434 + - 0069 , n = 11 WT MEFs ; 0442 + - 0099 , n = 10 Cav1-/- MEFs ) . The similarity in subunit B of cholera toxin binding and association with domains of high order ( GP ) in both cell types suggests that noncaveolar raft domains may be independent of Cav1 expression . For a more general analysis of the effects of Cav1 on membrane structure , we compared the global GP distribution in MEFs , shown in Fig. 6A for WT MEFs . Two populations , fluid ( Pf , dark gray ) and ordered ( Po , light gray ) , were identified . Each population has a characteristic mean GP value and abundance ( or coverage ) that equate to the area under the curve ( 34 ) . The mean GP values of fluid membranes , Pf , were similar in WT and Cav1-/- MEFs (Fig 6B) , but in Cav1-/- cells covered a greater proportion of the membrane surface ( 919 + - 37% in Cav1-/- MEFs versus 714 + - 57% in WT MEFs ; y axis in Fig 6B ) . In contrast , the ordered membranes were relatively more fluid and less abundant in Cav1-/- MEFs than WT MEFs (Fig 6C) , suggesting that Cav1 does contribute to the lipid order of raft-like domains in adherent cells . To demonstrate that the differences in membrane structure were directly related to Cav1 expression and function , Cav1-/- MEFs were transfected with either WT Cav1 or with Y14F Cav1 . Expressing WT Cav1 in Cav1-/- MEFs restored both order and relative surface coverage of ordered domains to WT levels . Mutating the only tyrosine PHOSphorylation site in Cav1 (Y14F Cav1) has no effect on caveolae formation but inhibits raft internalization ( 46 ) and decreases membrane order at focal adhesions ( 34 ) . Expression of Y14F Cav1 only partially rescued ordered domains when expressed in Cav1-/- MEF (Fig 6C) . WT Cav1 and Y14F Cav1 correspondingly decreased the proportion of fluid domains in Cav1-/- MEF (Fig 6B) . Overall , the data support a contribution of Cav1 to the generation of ordered domains in adherent , intact cells . The differential effect of Cav1 expression on biochemically identified domains and those observed by microscopy is an indication that cell architecture plays a greater role in lipid raft abundance than subtle changes in lipid composition .