Localization of photosynthetic reaction centers by antibody binding to chromatophore membranes from Rhodopseudomonas spheroides strain R26

Rabbit antiserum against highly purified reaction center preparations was shown to react specifically with a single component of chromatophore membranes from Rhodopseudomonas spheroides strain R-26. The conjugate of purified gamma globulin and ferritin prepared with toluene diisocyanate was used to determine the localization of reaction centers in the chromatophore membranes. Virtually no antibody was bound by intact membranes. After removing the 9 nm ATPase from these membranes by dilute EDTA treatment, a considerable amount of antibody was bound to the exposed outer membrane surface. The reaction center binding sites were estimated to be uniformly distributed with approx. 1 reaction center per 200 nm² of membrane surface. These results indicate that the reaction centers are located near the outer membrane surface but below the ATPase particles. Since the distribution of reaction centers and particles on rough faces seen by freeze-fracture electron microscopy are similar, it is suggested that the freeze-fracture particle may be a complex of a reaction center and other electron transfer components localized within the hydrophobic region of the membrane.     

The sarcolemma of Aplysia smooth muscle in freeze-fracture preparations

Smooth muscle cells in the sheath covering the visceral ganglion of Aplysia californica were examined with the techniques of freeze-fracture and conventional electron microscopy. The sarcolemma of these muscle cell invaginates to form myriad caveolae that have an intrinsic marker within their membrane. This intrinsic structure of the caveolar membrane is revealed by freeze-fracture and consists of rows of large particles in the outer half and matching grooves on the complementary inner half of the membrane. In thin plastic sections, parallel striations or shelves within the caveolar membrane appear to be the equivalent of the particles and grooves of the fractured membrane. Physical fixation of some specimens by rapid freezing in supercooled liquid nitrogen or in liquid helium suggests that in their natural state, the caveolar ostia are not uniform in size and that at any given moment a number of caveolae are flattened. When segments of the connective nerves which link the visceral ganglion to the cephalic ganglia are stretched in vitro two to three times their in situ length, the caveolae lose their invaginated shape and are fully exposed to the extracellular space. The caveolar membrane, so stretched, is pulled into the line of fracture with the result that the large particles rather than the ostia appear on the cleaved surface. This flattening of the caveolae is reversible and suggests that they might serve as miniature stretch-receptors within the membrane of the smooth muscle cells. The caveolae are accessible to extracellular horseradish peroxidase but do not appear to pinocytose the protein.     

Freeze-fracturing and deep-etching with the volatile cryoprotectant ethanol reveals true membrane surfaces of kidney structures

Summary With the conventional freeze-fracture technique applied to biological specimens, cell membranes split along an interior plane and two membrane faces are produced. True membrane surfaces remain hidden and can only be uncovered by deep-etching. To date, deep-etching could not be satisfactorily performed in the presence of cryoprotective agents since conventional cryoprotectants do not sublime due to their low vapour pressure. This lack of suitable volatile cryoprotectants has limited deep-etching so far to very small objects which can be cryofixed without cryoprotectants. As a consequence, our freeze-fracture knowledge of cell surfaces is still poor.The present study shows that ethanol is a suitable volatile cryoprotectant for the freeze-fracture technique, and provides a novel approach to the routine deep-etching of freeze-fracture specimens without the need for special equipment. With ethanol deep-etching, true outer cell-surfaces are demonstrated within the kidneys of rat and Psammomys.     

Detection of surface-bound ligands by freeze-fracture autoradiography

This article describes a new freeze-fracture autoradiographic technique for the detection of radioactive ligands associated with the surface of cells in monolayer or suspension culture. Since freeze-fracture replicas are produced in the conventional way, all membrane features normally seen in freeze-fracture are retained, and autoradiographic grains produced by the labeled ligands are seen superimposed on unaltered exoplasmic membrane fracture faces. To assess the feasibility and resolution of this technique, we compared the surface distribution of alpha 2-macroglobulin and cholera toxin, labeled either with 125I or with colloidal gold, on 3T3-L1 fibroblasts. Both by autoradiography and cytochemical gold labeling, alpha 2-macroglobulin was associated specifically with coated pits, whereas cholera toxin was preferentially found over smaller, apparently non-coated membrane invaginations. Together with data on the surface localization of 125I-transferrin on HL-60 myelomonocytic cells, these results demonstrate the application of this technique for the accurate determination of ligand distribution over large areas of plasma membrane. The simplicity and reproducibility of the method should now allow freeze-fracture autoradiography to become a standard technique for investigating the distribution of both endogenous and exogenous cell surface-associated molecules, as well as the redistribution of such molecules under different experimental conditions.     

Label-fracture of cell surfaces by replica staining

We introduce replica-staining label-fracture, a method for the cytochemical mapping of membrane surfaces. This method is a corollary of the rationale of label-fracture (Pinto da Silva and Kan, 1984: J Cell Biol 99:1156). After freeze-fracture the exoplasmic halves of the membrane remain attached to the replica. We show that cytochemical labeling of cell surfaces can be performed by direct post-fracture staining of freeze-fracture replicas. This new variant of label-fracture leads to miniaturization of labeling procedures and allows standardization of labeling conditions and simultaneous processing of different specimens.     

Morphological features of the surface of the sea urchin (Arbacia punctulata) egg: Oolemma-cortical granule association

Scanning microscopy and transmission electron microscopy of sectioned specimens and freeze-fracture replicas revealed the presence of slightly elevated regions, approximately one-fourth to one-half the diameter of microvilli, which were situated along the surface of unfertilized Arbacia eggs. These modifications of the surface of the egg were observed in areas occupied by cortical granules and were greatly reduced in number following the cortical granule reaction. Few such modifications were present in immature and urethane-treated ova, in which cortical granules were located in regions of the egg other than the cortex. Freeze-fracture replicas of unfertilized eggs revealed a significantly higher density of intramembranous particles within the plasmalemma when compared to replicas of the membrane surrounding cortical granules. Areas characteristic of the cortical granule membrane, i.e., sparsely laden with particles, were not observed within the plasmalemma of the fertilized egg. Hence, following its fusion with the egg plasma membrane there is a dramatic reorganization in particle distribution of the membrane derived from cortical granules.     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

Freeze-fracture autoradiography. Progress towards a routine technique

Freeze-fracture autoradiography was introduced in 1976 as a new technique for the autoradiography of diffusible compounds at the electron microscope level. With the original approach coating of the frozen replicated specimens was performed in a cryostat at atmospheric pressure. Ice contamination of the specimen surface acting as an outstanding source of artifacts was thereby not excluded. With the use of a specially designed coating device and volatile spreading substances it was made possible to coat the frozen replicated specimens in the maintained vacuum of the freeze-fracture plant. In this complicated technique we have recently extended the freeze-fracture autoradiography to labeled frozen-dried "half" membranes of red blood cells.     

Comparative electron microscopic studies of partially negatively stained, freeze-dried and freeze-fractured cytochrome reductase membrane crystals

Membrane crystals of ubiquinol: cytochrome c reductase have been studied by electron microscopy of one-sided negatively stained, freeze-dried and freeze-fractured specimens. The results confirm the unequal distribution of protein on both membrane surfaces which was found by three-dimensional image reconstruction. During freeze-drying, however, a considerable change in the lattice constants occurred. Furthermore, the apparent position of protein relative to the bilayer was changed by interaction with the support. The freeze-fracture plane was found to lie within the hydrophobic middle of the bilayer, splitting the membrane crystal into two equal leaflets.     

Surface-induced lamellar orientation of multilayer membrane arrays. Theoretical analysis and a new method with application to purple membrane fragments.

The orientation of membrane fragments into a lamellar array by a flat surface is analyzed. This analysis includes processes such as centrifugation and drying and physical effects due to membrane fragment steric interactions, finite size, elasticity, and thermal fluctuations. Several model calculations of optimal orientational order in multilayer membrane arrays are presented. The predictions of a smectic A model agree quantitatively with the measured spatial dependence of the fluctuations in layer orientation in a multilamellar arrays. A new technique, based in part on this analysis, for the preparation of well-oriented multilamellar arrays of natural and artificial membranes, isopotential spin-dry centrifugation, is described. The method involves the use of specially designed inserts for the buckets of a standard vacuum ultracentrifuge. The membrane fragments to be oriented are sedimented from solution or suspension onto a substrate of a convenient material which forms a gravitational isopotential surface at high g. Sedimentation is accompanied by removal of the suspending medium at high g to produce oriented films with a selected degree of solvation. In addition, a method is described whereby small solute molecules can be maintained in constant concentration with the membrane fragments during this process. Initial application of the method to the orientation of purple membrane fragments is described. The degree of orientation obtained in this system is evaluated using freeze-fracture and scanning electron microscopy, optical birefringence, linear dichroism, and microscopy.     

Morphological detection of filipin-sterol complexes in the cytoplasmic membrane of staphylococcal L-form

Filipin, a sterol-specific antibiotic, and freeze-fracture electron microscopy were used to study the presence and distribution of sterol in the cytoplasmic membrane of stable staphylococcal L-form cells. Fixed cells were treated with filipin, and then observed by freeze-fracture electron microscopy. Freeze-fractured profiles of the L-form cells treated with filipin demonstrated irregular distribution of protuberances or pits of 25-30 nm, representing filipin-sterol complexes, on the proto-plasmic fracture face (PF) and exoplasmic fracture face (EF) of the cytoplasmic membrane. In contrast, no such structure was detected in the filipin-treated parent cells or protoplasts. The results suggest that some sterol molecules, which are usually not found in staphylococcal or other bacterial cells, emerged on the cytoplasmic membrane after the cells were converted to the stable L-form.     

Structure, isolation, and protein composition of the pellicle of Sarcocystis muris cystozoites (Protozoa, Coccidia)

The molecular organization of the Sarcocystis muris cystozoite pellicle has been investigated by freeze-fracture electron microscopy and by electrophoresis of the proteins of isolated pellicles. Freeze-fracture revealed a highly ordered organization of the inner membrane complex similar to the one described in other coccidian zoites. Purification of pellicles was achieved by French Press homogenization followed by sucrose gradient floatation. Electron microscopy of the pellicle fraction demonstrated the partial preservation of the triple-membrane structure whereas freeze-fracture showed the disorganization of the particle arrangements of the inner membrane complex. The SDS-PAGE of the fraction revealed a complex protein composition with one major protein of 31,000 daltons, not labeled by lactoperoxidase-catalyzed surface iodination of living cystozoites.     

Simulcast: contiguous views of fracture faces and membrane surfaces in a single cell

We introduce "simulcast", a new method that combines the advantages of freeze-fracture with those of "fracture-flip" (Anderson-Forsman, C., P. Pinto da Silva, J. Cell Sci. 90, 531-541 (1988)) to provide images of the fracture faces and membrane surfaces of the same membrane in a single cell. The method involves low-angle unidirectional shadowing, careful azimuthal reorientation of the replicas, flipping, and re-shadowing. Simulcast relates, in a single image, the freeze-fracture morphology of fracture faces, the nanoanatomy of membrane surfaces and the topochemistry of surface receptors/antigens.     

SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application

 Recently, we have developed a quick-freezing/freeze-fracture replica labeling technique, sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL), to study the two-dimensional distribution of cytochemical labeling on the membrane surface and the relationship of this distribution to images of freeze-fracture replicas created by platinum shadowing. In SDS-FRL, unfixed, quick-frozen cells, after freeze-fracture and platinum/carbon shadowing, are treated with SDS. The detergent dissolves unfractured areas of the cell membranes, with the release of the cytoplasmic contents. The cytoplasmic and exoplasmic membrane surfaces can be then labeled cytochemically. Integral membrane proteins, revealed as intramembrane particles by freeze-fracture replication, which are indistinguishable on a purely morphological basis, can be selectively labeled by SDS-FRL with specific antibody. In addition, this approach can be applied to examine the transmembrane phospholipid distribution in various cell and intracellular membranes. In this review, we describe the practical procedure for SDS-FRL in detail, present its application to labeling of various membrane components, and briefly discuss the possibility of a combination of SDS-FRL with atomic force microscopy. Accepted: 1 November 1996     

The ultrastructural organization of gap junctions between follicle cells and the oocyte in Xenopus laevis

The cellular contact sites between the full-grown oocyte of Xenopus laevis and the surface extensions of surrounding follicles cells were analysed by electron microscopy of ultrathin sections, freeze-fracture replicas and critical point-dried specimens. Evidence is given for the presence of clusters of intramembranous particles (IMPs) at the P-face which represent gap junctions in diverse forms. Most common are maculae (phi 0.2-0.5 micron) of densely packed IMPs (phi 12 +/- 2 nm) which represent focal gap junctions generally found at the tips of follicle cell surface extensions. Inside many maculae an IMP-free area occurs which appears as a smooth disk (phi 70-80 nm) at both fracture faces. Occasionally a few IMPs are trapped within the smooth disks. Beside the maculae, networks of arrayed IMPs occur that enclose several smooth disks. These latter gap junctions probably are more frequent in side-to-side contacts between surface extensions of the oocyte and the follicle cells. The possible function of these IMP networks is discussed as being related to similar membrane specializations in excitable cells. In addition, indirect evidence was found that the extensions of the follicle cells transport yolky material.     

Fusion of mitochondrial inner membranes by electric fields produces inside-out vesicles. Visualization by freeze-fracture electron microscopy

The fusion of vesicular-shaped mitochondrial inner membranes was observed by a new approach which combines freeze-fracture electron microscopy and electric field-induced fusion. Results show that membrane events caused by the exposure to the electric field can be time-coordinated with sample freezing for subsequent analysis by freeze-fracture electron microscopy.     

Fusion of mitochondrial inner membranes by electric fields produces inside-out vesicles: Visualization by freeze-fracture electron microscopy

The fusion of vesicular-shaped mitochondrial inner membranes was observed by a new approach which combines freeze-fracture electron microscopy and electric field-induced fusion. Results show that membrane events caused by the exposure to the electric field can be time-coordinated with sample freezing for subsequent analysis by freeze-fracture electron microscopy.     

Organization of calcium pump protein dimers in the isolated sarcoplasmic reticulum membrane.

The arrangement of the calcium pump protein in the isolated sarcoplasmic reticulum (SR) membrane was examined by optical diffraction of freeze-fracture electron micrographs. Several states of protein particle organization were observed: random, hexagonal and tetragonal packing, and a mixture of hexagonal and tetragonal packing. This suggests that the time-averaged positions of protein particles in the plane of the SR membrane are weakly defined. In addition, there appears to be a greater degree of local or short-range order compared to long-range order within the field of freeze-fracture particles. We utilized measurements from tetragonally or hexagonally packed arrays to determine a unit cell area occupied by each freeze-fracture particle and its associated lipid matrix. When these unit cell areas and the stereologically determined area per freeze-fracture particle were compared to the cross-sectional area occupied by a single calcium pump protein and its associated lipid, obtained by x-ray and neutron diffraction methods, we concluded that each freeze-fracture particle probably represents a dimer of pump protein molecules in the plane of the SR membrane.     

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum “cast” intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.     

Electron microscopic and biophysical studies of liposome membrane structures to characterize similar features of the membranes of Streptomyces hygroscopicus

To characterize the novel non-planar plasma membrane structure of bacteria (wafer structure), liposome membranes from the bacterial lipid mixture and individual lipid fractions were prepared and investigated by freeze-fracture electron microscopy, microcalorimetry and 31P-NMR spectroscopy. The phospholipid content of the membranes is essential for the formation of the non-planar membrane structure and there is no indication that the formation of the structure is connected with temperature-induced lipid phase transition processes. An exaggerated form of the wafer structure (raspberry structure) is also visible and additionally, in both cases, many small spherical vesicles are observed. We suggest that both membrane features of the liposomal and bacterial membranes are induced by these vesicles, forming a hexagonal or cubic organization of vesicles on the cytoplasmic surface of the biological membrane, and in between the multilamellae in the artificial membranes.