Cell membranes freeze-fractured

The dimer chains of Ca -ATPase can also be observed by freeze-fracture electron microscopy [119,165,166,172-174], forming regular arrays of oblique parallel ridges on the concave P fracture faces of the membrane, with complementary grooves or furrows on the convex E fracture faces. Resolution of the surface projections of individual Ca -ATPase molecules within the crystalline arrays has also been achieved on freeze-dried rotary shadowed preparations of vanadate treated rabbit sarcoplasmic reticulum [163,166,173,175]. The unit cell dimensions derived from these preparations are a = 6.5 nm b = 10.7 nm and 7 = 85.5° [175], in reasonable agreement with earlier estimates on negatively stained preparations [88]. 

The process of infection of lupine nodule cells by Rhizobia was examined by the thin-section electron microscopic technique, as well as the freeze-fracture technique. Different membranes such as infection thread membranes, peribacterioid membranes, plasma membranes, membranes of cytoplasmic vesicles, and membranes of the Golgi bodies and ER were stained with uranium-lead, silver, phosphotungstic acid, and ZIO (31). ZIO stained the membranes of the proximal face of the Golgi bodies and endoplasmic reticulum. ZIO staining has given good contrast in thick sections such as a cotyledon cell, a root cell, and an aleurone layer for ER, dictyosomes cisternae, mitochondria, and nuclear envelopes (17,32-37). 

Diaz JAM, De Souza W (1997) Purification and biochemical characterization of the hydrogenosomes of the flagellate protist Tritrichomonas foetus. Eur J Cell Biol 74 85-91 Diniz JA, Benchimol M (1998) Monocercomonas sp. cytochemistry and fine structure of freeze-fractured membranes. J Eukaryot Microbiol 45 314-322 Embley TM, Horner DA, Hirt RP (1997) Anaerobic eukaryote evolution hydrogenosomes as biochemically modified mitochondria Trends Ecol Evol 12 437-441 Embley TM, van der Gienzen M, Horner DA, Hirt RP, Dyal PL, Bell S, Foster PG (2003) Hydrogenosomes, mitochondria and early eukaryotic evolution. IUBMB Life 55 387-395... 

Pinto da Silva, P., Peixoto de Menezes, A. and Mather, I. H. 1980. Structure and dynamics of the bovine milk fat globule membrane viewed by freeze fracture. Exp. Cell Res. 125, 127-139. 

The freeze-fracture immunolabeling technique can be used in two ways. In the first, the cells are immunolabeled and freeze-fractured. This shows immunolabeling of the outer surface of the cell membrane superimposed on a high-reso-lution replica of the inner surface of the membrane. In the second, the cells are fractured before immunolabeling. This permits immunolabeling to be correlaled w ith large areas of the fractured faces. 

Integral proteins are usually free to move in the plane of the bilayer by lateral and rotational movement, but are not able to flip from one side of the membrane to the other (transverse movement). Immunofluorescence microscopy may be used to follow the movement of two proteins from different cells following fusion of the cells to form a hybrid heterokaryon. Immediately after fusion the two integral proteins are found segregated at either end of the heterokaryon but with time diffuse to all areas of the cell surface. The distribution of integral proteins within the membrane can be studied by electron microscopy using the freeze-fracture technique in which membranes are fractured along the interface between the inner and outer leaflets. 

Simpson, D.J. 1983. Freeze-fracture studies on barley plastid membranes. VI. Location of the P700 chlorophyll a-protein 1. Eur. J. Cell Biol. 31, 305-314. 

Spicule formation takes place within the intracellular environment. The spicules, however, are much larger than individual cells. This is achieved by many cells fusing their membranes to enclose an extended space (called a syncytium) (Fig. 1.9). Spicule formation takes place inside a membrane delineated vacuole within this space [70, 71]. The size and the shape of the syncytium constantly increase and change during growth of the spicule [72, 73]. In fact, a freeze-fracture TEM study of the relation between the membrane and the growing spicule shows that the membrane is always juxtaposed to the spicule surface. There is thus no bulk solution within which the spicule forms [74], Thus spicule... 

Freeze-fracture electron microscopy studies of the membranes of E. coli and A. vinelandii by Reusch et al.24 provide evidence of structural changes that support the fluorescence data (Figure 10). Freeze-fracture micrographs of log-phase cells show a typical mosaic of particles and pits on both concave and convex surfaces of the plasma membranes. However, as complexed PHB was increasingly incorporated into the membranes, as determined by analysis of the purified membranes and evidenced by the intensity of the thermotropic transition at - 56 °C, the micrographs revealed the formation of small semi-regular plaques in the plasma membranes (arrows) that possess shallow particles. The plaques grew in size and frequency as the concentration of membrane PHB and intensity of the PHB/polyP transition increased. 

Figure 10. Representative freeze-fracture electron micrograph of competent E. coli DH1. The micrograph shows the typical appearance of small semi-regular plaques (arrows) in the plasma membranes of E. coli DH1 cells after treatment to make them genetically transformable by the method of Hanahan.146 These cells have sharp thermotropic transitions at -56 °C when examined as in Figure 9A.24...

Although information on the spatial distribution of membrane lipids can be important (3), most mass-spectrometric approaches used in lipidomics focus currently on the determination of total lipid composition. An advanced mass-spectrometric surface analysis technique, however, secondary ion mass spectrometry (SIMS) determines lipid compositions within different areas of freeze-fractured cells (12). This technique and related imaging techniques will become important tools in lipidomics. 

Indeed, these situations can be observed in living cell structures. During electron microscopic observation of freeze-thawed cell nuclei, rapidly frozen specimens had the original tissue structure. When these specimens were thawed rapidly, no difference could be seen in comparison with the control. Only a minor increase in the affinity to dyes and a slight condensation of chromosome have been observed. On the contrary, when specimens are thawed slowly many large cavities are observed, which indicates that the cellular materials are forced out by ice crystals. In these specimens, a very serious rupture of nuclear membrane was also observed. Similar results have been obtained with freeze-fractured electron microscopic observation of rapidly frozen red blood cells. In these experiments, membrane structure had been damaged in the regions where ice crystals and red blood cell membranes were in close contact. 

Figure 5.10 Electron micrographs of L-cells of Streptomyces hydroscopicas [73]. The freeze-fractured texture shows the periodically curved lipid bilayer. The curvature is weakly expressed in a and very distinct in fi. Two attached lamellar bodies and the imderlying membrane are shown in fe. The bar (in fi) is 500 nm.

One method of communication between cells is by passage of chemical substances through special junctions which, because of their appearance in electron micrographs of thin sections (Fig. 1-15, G) are known as gap junctions. T45,146 Q p junctions may cover substantial areas of the cell interface. In cross section, a thin 3-4 nm gap between the adjacent cell membranes is bridged by a lattice-like structure, which may appear in freeze-fractured surfaces as a hexagonal array of particles (Fig. 1-15, F, lower junction). These particles or connexons are each thought to be composed of six protein subunits. A central channel in the connexon is able to pass molecules of molecular mass up to about 500 Small molecules may be able to pass... 

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