Heterogeneity of milk fat globule membrane structure and composition as observed using fluorescence microscopy techniques
Introduction
Fat globules in milk are surrounded by a thin layer of surface-active material, which is called the milk fat globule membrane (MFGM). Upon secretion of the fat droplet from the mammary secretory cell, the structure of the MFGM is trilaminar, consisting of a surface-active inner layer that surrounds the intracellular fat droplet, then a dense proteinaceous coat and finally a true bilayer membrane originating from the secretory cell apical plasma membrane (Keenan & Mather, 2002). Following secretion of the fat globule by the secretory cell, the MFGM is believed to change to some degree, e.g., by partial loss of the bilayer membrane, although the extent of the loss is unclear (Evers, 2004a).
Much of the current knowledge of the structure and molecular organisation of the MFGM has been obtained using morphological techniques such as (immuno)electron microscopy applied to fat globules that had been isolated from the original milk sample (Buchheim, 1982, Franke et al., 1981, Henson et al., 1971, Horisberger et al., 1977, Mather et al., 2001, Monis et al., 1975, Pinto da Silva et al., 1980, Robenek et al., 2006, Sasaki and Keenan, 1979, Schmidt and Buchheim, 1992, Welsch et al., 1988, Wooding, 1971). We were particularly intrigued by recently published freeze-fracture electron microscopy results (Robenek et al., 2006), which revealed variability in the physical structure of the surface of the MFGM of individual globules. However, electron microscopy results apply to fat globules that may, or may not, have undergone substantial physical and/or chemical modification, and the environment of which has been drastically changed. Hence, the degree to which the results represent the structure of the membranes of fat globules in the original sample remains uncertain.
Fluorescence microscopy can be used to study fat globules without isolating them from the milk sample matrix. The commercially available fluorescent dyes Nile Blue and Nile Red have been used to study fat globules in milk and milk products, using either conventional fluorescence microscopy or confocal laser scanning microscopy (CLSM; note that this abbreviation is also used for the confocal laser scanning microscope, as defined by the context) (Everett and Olson, 2003, Evers, 2004b, Herbert et al., 1999, Lopez and Briard-Bion, 2007, Lopez et al., 2008, Lopez et al., 2007, Michalski et al., 2004, Sutheerawattananonda et al., 1997, Trachoo, 2003, Ye et al., 2005). However, these neutral fat dyes stain the fat core of the globules and, therefore, provide no information about the MFGM. The fluorescent dye Acridine Orange has been used to visualise fat globules (King, 1958, Scolozzi et al., 2003) or cytoplasmic crescents associated with fat globules (Janssen and Walstra, 1982, Patton and Huston, 1988), but this dye is presumed to stain cytoplasmic matter rather than the MFGM itself (Scolozzi et al., 2003). Immunofluorescence techniques have revealed generally patchy distributions of butyrophilin and other membrane proteins (McManaman, Palmer, Wright, & Neville, 2002), but these techniques are known to introduce artefacts (Robenek et al., 2006).
It is in this context that we sought to develop new techniques that would specifically probe, in real time, the structural and compositional details of the MFGM in its native environment (i.e., milk) in a non-destructive manner. Thus, a range of commercially available fluorescent probes, individually and in combination, were screened by means of conventional fluorescence microscopy and CLSM. In this paper, we present staining patterns of the MFGM by two lipophilic probes, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid (DiIC18(3)-DS) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64), and by the lectin wheat germ agglutinin (WGA).
Section snippets
Chemicals
Reverse osmosis water was purified using a Milli-Q system (Millipore Corporation, Bedford, MA, USA). Fluorescent probes (solids for reconstitution; molecular structures given in Fig. 1) were obtained from Molecular Probes (Eugene, OR, USA): DiIC18(3)-DS, FM4-64 and wheat germ agglutinin (WGAxyz, where xyz indicates the particular Alexa Fluor® conjugate, e.g., WGA488). Single-probe stock concentrations were 1 mg mL−1 Milli-Q water, unless indicated otherwise. A dual-probe stock solution contained
Lipophilic probes
An ethanolic DiIC18(3)-DS stock solution immediately resulted in a small proportion (estimated <5%) of the fat globules being brightly stained. Subsequent incubation at room temperature for several hours resulted in the staining of further fat globules, but not as brightly as those stained initially. DiIC18(3)-DS was located exclusively, but often heterogeneously, in the MFGM (Fig. 2). Because of the risk of ethanol perturbing the membrane structure by denaturing membrane proteins, aqueous
WGA controls
Conflicting results for WGA control studies using GlcNAc have been reported in the literature. In washed bovine milk fat globules and human milk fat globules, GlcNAc appeared to completely inhibit WGA binding (Farrar, Harrison, & Mohanna, 1980). Similarly, non-specific binding of WGA in the presence of N-acetylchitopentaose was reported to be very low (Horisberger et al., 1977). However, in equine milk, GlcNAc had no inhibiting effect (Welsch et al., 1988).
To our knowledge, no studies of WGA
Conclusions
Fluorescence microscopy using MFGM-specific probes has revealed chemical and structural heterogeneity at the micron and submicron scale in the MFGM, not only of individual globules but also among globules. In providing chemical as well as physical information on MFGMs in their native environment, our results confirm, complement and substantially extend previously reported morphological results for the MFGM of fat globules isolated from milk. The techniques developed show significant potential
Acknowledgements
We thank Elizabeth Nickless for support with CLSM. We thank Massey University No. 1 Dairy Farm staff, Kingsmeade sheep farm staff and the human volunteer for providing milk samples. This research was funded in part by the New Zealand Tertiary Education Commission through a Bright Future scholarship (J. M. Evers). Dr T. Coolbear is acknowledged for critical evaluation of this manuscript.
References (49)
Ultrastructural observations on the milk fat globule envelope of cow's milk
Journal of Dairy Science
(1972)- et al.
Free oil and rheology of Cheddar cheese containing fat globules stabilized with different proteins
Journal of Dairy Science
(2003) The milkfat globule membrane – compositional and structural changes post secretion by the mammary secretory cell
International Dairy Journal
(2004)The milkfat globule membrane – methodologies for measuring milkfat globule (membrane) damage
International Dairy Journal
(2004)- et al.
Comparison of lectin receptors on the surface of human and bovine milk fat globule membranes
Comparative Biochemistry and Physiology
(1980) - et al.
Intracellular origin and secretion of milk fat globules
European Journal of Cell Biology
(2005) - et al.
Physicochemical analyses of bovine milk fat globule membrane. II. Electron microscopy
Journal of Dairy Science
(1971) - et al.
Location of glycoproteins on milk fat globule membrane by scanning and transmission electron microscopy, using lectin-labelled gold granules
Experimental Cell Research
(1977) - et al.
Factors related to the formation of cytoplasmic crescents on milk fat globules
Journal of Dairy Science
(1990) - et al.
Milk fat globule membrane