One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.

TOTAL REFLECTION FLUORESCENCE 249 for angles of incidence 0 > Oc and light wavelength in vacuum Ao. Depth d is independent of the polarization of the incident light and decreases with increasing e. Except for e � ec (where d-+ (0), d is on the order of Ao or smaller. The intensity at z = 0, 10, depends on both the incidence angle e and the incident beam polarization. lo is proportional to the square of the amplitude of the evanescent electric field E at z = 0. 1 (These expressions are given in the next subsection.) For incident electric field intensities f ll .1-with polarizations parallel and perpendicular, respectively, to the plane of incidence, the evanescent intensities Ig• .1 are Ig = f ll. 4 cos 2 e(2 sin 2 e-n 2) n4 cos 2 e+sin 2 (} _ n 2 and .1 _ J .1. 4 cos2 () 10

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Abstract. Using immunohistochemical methods, we have investigated the role of transforming growth factor-13 (TGF-13) in the development of the mouse embryo. For detection of TGF-13 in ll-18-d-old embryos, we have used a polyclonal antibody specific for TGF-13 type 1 and the peroxidase-antiperoxidase technique. Staining of TGF-fl is closely associated with mesenchyme per se or with tissues derived from mesenchyme, such as connective tissue, cartilage, and bone. TGF-13 is conspicuous in tissues derived from neural crest mesenchyme, such as the palate, larynx, facial mesenchyme, nasal sinuses, meninges, and teeth. Staining of all of these tissues is greatest during periods of morphogenesis. In many instances, intense staining is seen in mesenchyme when critical interactions with adjacent epithelium occur, as in the development of hair follicles, teeth, and the submandibular gland. Marked staining is also seen when remodeling of mesenchyme or mesoderm occurs, as during formation of digits from limb buds, formation of the palate, and formation of the heart valves. The presence of TGF-13 is often coupled with pronounced angiogenic activity. The histochemical results are discussed in terms of the known biochemical actions of TGF-13, especially its ability to control both synthesis and degradation of both structural and adhesion molecules of the extracellular matrix.

The theoretical basis of a new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labeled solute molecules at solid surfaces has been developed. The technique combines total internal reflection fluorescence (TIR) with either fluorescence photobleaching recovery (FPR) or fluorescence correlation spectroscopy (FCS). A laser beam totally internally reflects at a solid/liquid interface; the shallow evanescent field in the liquid excites the fluorescence of surface adsorbed molecules. In TIR/FPR, adsorbed molecules are bleaching by a flash of the focused laser beam; subsequent fluorescence recovery is monitored as bleached molecules exchange with unbleached ones from the solution or surrounding nonilluminated regions of the surface. In TIR/FCS, spontaneous fluorescence fluctuations due to individual molecules entering and leaving a well-defined portion of the evanescent field are autocorrelated. Under appropriate experimental conditions, the rate constants and surface diffusion coefficient can be readily obtained from the TIR/FPR and TIR/FCS curves. In general, the shape of the theoretical TIR/FPR and TIR/FCS curves depends in a complex manner upon the bulk and surface diffusion coefficients, the size of the iluminated or observed region, and the adsorption/desorption/kinetic rate constants. The theory can be applied both to specific binding between immobilized receptors and soluble ligands, and to nonspecific adsorption processes. A discussion of experimental considerations and the application of this technique to the adsorption of serum proteins on quartz may be found in the accompanying paper (Burghardt and Axelrod. 1981. Biophys. J. 33:455).