Thin-section and critical-point-dried fracture-labeled preparations are used to determine the distribution and partition of glycophorin-associated wheat germ agglutinin (WGA) binding sites over protoplasmic and exoplasmic faces of freeze-fractured human erythrocyte membranes. Most wheat germ agglutinin binding sites are found over exoplasmic faces . Label is sparse over the protoplasmic faces . These results contrast with previous observations of the partition of band 3 component where biochemical analysis and fracture-label of concanavalin A (Con A) binding sites show preferential partition of this transmembrane protein with the protoplasmic face . Presence of characteristic proportions of WGA and Con A binding sites over each fracture face is interpreted to indicate the operation of a stochastic process during freezefracture . This process appears modulated by the relative expression of each transmembrane protein at either surface as well as by their association to components of the erythrocyte membrane skeleton .In freeze-fracture, splitting of the bilayer membrane continuum with differential partition of integral membrane proteins can be considered a process of structural dissection . Recently, we developed "fracture-label" techniques for the direct cytochemical investigation of the distribution and partition of integral and peripheral membrane components as indicated by the presence of chemical groups and lectin binding sites on the faces of freeze-fractured plasma and intracellular membranes . The results can be observed both in thin sections of freezefractured material ("thin section fracture-label") (1, 2) and in platinum-carbon replicas ofcritical-point-dried specimens after freeze-fracture ("critical point drying fracture-label") (3) .Initial application of thin-section fracture-label to the cytochemical investigation of the fracture faces of human erythrocyte membranes showed that concanavalin A (Con A) binding sites are preferentially labeled on the protoplasmic faces (1) . Labeling of the P face by Con A-ferritin conjugates implies that band 3 component (the Con A binding transmembrane protein ; see references 4-8) generally partitions with the inner membrane half, a process that must involve dragging of its Con A binding heterosaccharides across the outer membrane half. These results are consistent with the biochemical analysis of half-membrane preparations (9) and, in addition, they indicate that Con A labeling of the E face, although minor, appears significant .Much less is known about the partition of glycophorin . Biochemical studies indicate the presence of glycophorin molecules and shorter sialoglycopeptides in preparations of exoplasmic membrane halves (9). These studies, however, were not designed to assess the degree of partition of glycophorin with the inner membrane half, nor could this partition be inferred from our initial fracture-label studies (1) showing the presence of cationized ferritin or colloidal iron on both P and E faces, as these markers cannot identify the molecul...

We used freeze fracture electron microscopy to study the fine structure of Mycobacterium avium inside phagosomes of murine macrophages. M. avium-susceptible C57BL/6 mice were infected with M. avium by intraperitoneal inoculation of 108 viable bacilli. We studied the microanatomy of the mycobacteria in 3-month infections of mice, a situation in which bacillary multiplication is extensive. In these samples, freeze fracture revealed that intraphagosomal bacilli were surrounded by a multilamellar coat that was apposed to the cell wall. In thin sections, in contrast, the area corresponding to the coat showed no substructure and was electron transparent (the so-called electron-transparent zone that'has been previously reported by others). The multiple lamellae resembled an onionlike assembly that was inserted in between the mycobacterial wall outer surface and the phagosomal membrane. Each lamella of the M. avium coat was made up of parallel straight fibrils with a width of 5 nm. A variable number of lamellae, sometimes up to 10 or more elements, coated individual bacilli. The multilamellar coat was absent around both extracellular M. avium and intramacrophagic M. avium after short-term (45-min) inoculation of mice. The supramolecular organization of the M. avium lamellar coat as viewed here by freeze fracture is similar to that of purified mycoside C (P. Draper,