A Morphometric Freeze-Fracture-Etch (FFE) Analysis on The Nature of Intramembrane Particles

We have recently completed (J. Cell Biol, in press) a morphometric analysis of FFE preparations of a membrane fraction from mammalian urinary bladder epithelial cells that gives a new insight into the nature of intramembrane particles (IMPs). The preparation contains vesicles of a membrane with paracrystalline arrays of external particles in placques 0.2-0.5μm in diameter. In transverse sections of placques (Fig. l) particles ~65Å thick by 100Å wide are seen spaced ~140Å center-to-center making the lattice membrane ~130Å thick. The interplacque membrane is an ordinary unit membrane ~75Å thick. We have prepared specimens for FFE study by centrifuging vesicles onto a glass cover slip cationized with alcian blue. The unattached membrane fragments were washed away with water and a small copper disc was placed on the cover slip before freezing by immersion in liquid propane by a special technique giving unusually high freezing rates. he specimens were fractured under liquig nitrogen and freeze-dried at -100° in a Denton FFE unit operated at 5 x 10-8 Torr. Platinum-carbon replicas were prepared by arc sublimation at -193°C. Unusual fracture planes sometimes occurred revealing EF, PF and PS surfaces as in Fig. 7. Two distinctly different patterns were observed in the EF faces: 1) placques consisting of globular particles each ~100-120Å in diameter spaced in a regular lattice corresponding to the external particle lattice (Fig. 4 - GP and Fig. 7). 2) placques entirely free of such particles in which a regular domain pattern was found with a lattice constant of 160Å (Fig. 1 - SP). The repeating unit in the latter was a domain consisting of a partial ring of metal ~20-25Å thick surrounding a shadow in the center of which there was a particle of metal about 20-25Å in diameter (filtered image inset to lower left in Fig. 4). Diffraction patterns from each type of lattice (insets Fig. 1) show the same lattice constants with evidence of shadowing directionality in the smooth lattice (absent diffraction spots) and prominent decoration in the globular lattice to the right (equal 1,0 spots). At the edges of placques the smooth lattice domains cast smooth shadows onto the glass (Fig. 5) whereas the globular particles cast spiked shadows (Fig. 6). Using the glass surface as a reference and employing stereoscopic techniques, measurements were made of the heights of the globular particle and the smooth domain lattices above the glass. The particle heights were analysed statistically and it was found that the mean globular particle height above the glass was 163(±24)Å. The total mean thickness of the unfractured lattice membrane was 146(±14)Å. The mean height of the smooth domain lattice above the glass was found to be 108(±17)Å. A model derived from thin sections corrected for 150Å shrinkage predicts the total lattice membrane thickness to be 1508 and the interplacque membrane to be 86Å. The model predicts the height of the EF face above the glass to be 107Å. The measurements indicate that the smooth domain lattice could represent a real structure and that the globular particles are artifacts. The PF faces in these membranes was studied in whole cells as well as membranes fractured on glass and found to be devoid of pits of sufficient size to accomodate the globular EF particles. The pattern in the PF faces (Fig. 2) consisted of domains in a hexagonal lattice with a lattice constant of 160Å. In this case each domain consisted of a complete ring of metal about 40Å thick surrounding a shadow around a central spot of metal about 608 in diameter. Fig. 3 is a filtered image of Fig. 2 showing this. The EF and PF faces are complementary in the sense that they contain similar structures; however, the ring shadows in each are mirror images. The globular EF lattice pattern was completely uncomplementary to the PF lattice in keeping with the conclusion that the globular particle is an artifact. It is probably produced by a combination of plastic deformation and decoration. Supported by NIH Grant #5 P01 GM 23911.