Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.     

Ultrastructure of the zonula adherens revealed by rapid-freeze deep-etching

The zonula adherens (ZA) in adult chicken retinal pigment epithelium was examined with cryo-electron microscopic methods. Deep-etching of the cross-fractured ZA showed globules in the intercellular space. These globules apparently correspond to the electron-dense structure seen in thin sections. Deep-etching of obliquely fractured ZA further revealed rod-like structures extending from the extracellular surface into the intercellular space. These rods (mean approximately 9 nm thick, approximately 20 nm long) were straight and sometimes divided into two or three segments. The rods typically canted at approximately 60 degrees with respect to the plasma membrane, and they were often connected to the intercellular globules at their distal ends. When the rods are compared with the isolated cadherins reported previously, it is suggested that a combination of a rod and a globule may represent an extracellular part of cadherin. Membrane particles were observed on the P-face of the ZA plasma membrane, and their distribution density was approximately seven times that of the rods. The freeze-etching also revealed a characteristic particle complex on the ZA cytoplasmic surface, which may represent the cytosolic proteins linking cadherins to actin bundles.     

Filamentous structure of the external surface of abalone eggs revealed by quick-freeze deep-etch technique

The external surface of abalone eggs was examined by thin section and quick-freeze, deep-etch electron microscopy. In thin sections, networks of fine filaments were found interconnecting the adjacent microvilli on the surface of unfertilized eggs. Quick-freeze, deep-etch electron microscopy revealed the three-dimensional structure of these networks of filaments on the external surface of the egg. Mainly two networks of filaments were identified; one was composed of thicker (14–19 nm) filaments interconnecting with the neighboring microvilli nearly horizontally, and the other was composed of thinner (8–14 nm) branched filaments closely surrounding the microvilli surface as well as highly interconnecting neighboring microvilli in a polygonal pattern. The overall structure of the filamentous network on the egg surface showed no distinct alteration after fertilization. These networks of filaments observed on the egg surface may play a key role in sperm–egg interaction.     

Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.     

Calorimetric studies of bovine rod outer segment disk membranes support a monomeric unit for both rhodopsin and opsin

The photoreceptor rhodopsin is a G-protein coupled receptor that has recently been proposed to exist as a dimer or higher order oligomer, in contrast to the previously described monomer, in retinal rod outer segment disk membranes. Rhodopsin exhibits considerably greater thermal stability than opsin (the bleached form of the receptor), which is reflected in an ∼15°C difference in the thermal denaturation temperatures (T_m) of rhodopsin and opsin as measured by differential scanning calorimetry. Here we use differential scanning calorimetry to investigate the effect of partial bleaching of disk membranes on the T_m of rhodopsin and of opsin in native disk membranes, as well as in cross-linked disk membranes in which rhodopsin dimers are known to be present. The T_ms of rhodopsin and opsin are expected to be perturbed if mixed oligomers are present. The T_m remained constant for rhodopsin and opsin in native disks regardless of the level of bleaching. In contrast, the T_m of cross-linked rhodopsin in disk membranes was dependent on the extent of bleaching. The energy of activation for denaturation of rhodopsin and cross-linked rhodopsin was calculated. Cross-linking rhodopsin significantly decreased the energy of activation. We conclude that in native disk membranes, rhodopsin behaves predominantly as a monomer.     

An investigation of mitochondrial inner membranes by rapid-freeze deep- etch techniques

Physical fixation by rapid freezing followed by freeze-fracture and deep-etching has provided the means for potentially seeing the three-dimensional arrangement in the native state of particles on mitochondrial inner membranes. We have used these techniques to study the tubular cristae of Paramecium in the hope of determining the arrangement of F1 complexes, their abundance, and location in the membranes. We also sought information regarding other respiratory complexes in these membranes. Our results, supported by stereo pairs, show that F1 complexes are arranged as a double row of particles spaced at 12 nm along each row as a zipper following the full length of the outer curve of the helically shaped tubular cristae. There are an average of 1,500 highly ordered F1 complexes per micrometer squared of 50-nm tubular cristae surface. The F1 complexes definitely lie outside the membranes in their native state. Other particle subsets, also nonrandomly arrayed, were seen. One such population located along the inner helical curve consisted of large 13-nm-wide particles that were spaced at 30 nm center-to-center. Such particles, because of their large size and relative abundance when compared to F1 units, resemble complex I of the respiratory complexes. Any models attempting to understand the coupling of respiratory complexes with F0F1 ATPase in Paramecium must take into account a relatively high degree of order and potential immobility of at least some of these integral membrane complexes.     

Ultrastructural organization of the glomerular basement membrane as revealed by a deep-etch replica method

Summary The fine structure of the glomerular basement membrane was re-evaluated by using a deep-etch replica method.The structure of the laminae rarae interna and externa of the rat glomerular basement membrane was basically identical in that 6 to 8 nm fibrils were interconnected to form a three-dimensional, polygonal network. The size of the mesh was quite variable but most often ranged from 20 to 25 nm in width. In addition, a zipper-like substructure of the epithelial slit diaphragm was observed. By contrast, the lamina densa was composed of closely packed particles.After exposure of the bovine glomerular basement membrane to ultrasonic waves or trypsin, the particles of the lamina densa were effectively removed. The underlying structure showed the fibrillar network closely resembled that seen in the laminae rarae of the rat glomerular basement membrane.The glomerular basement membrane thus revealed was as principally composed of a fibrillar network, which might be regularly arranged units of type-IV collagen. Numerous fine particles, most likely proper components of the glomerular basement membrane, were attached onto this basic fibrillar structure, giving rise to a morphologic appearance different from that of the laminae rarae.     

Ultrastructure of detergent-resistant cytoskeletons in the noncortical domain of sea urchin eggs as revealed by the quick-freeze deep-etch technique.

The ultrastructure of detergent-resistant cytoskeletons in the noncortical cytoplasm of sea urchin eggs was studied by quick-freeze, deep-etch electron microscopy. Two different cytoskeletal organizations were identified in the detergent-treated sea urchin eggs. They were distinguished by the presence or the absence of long actin filaments and probably correspond to the cortex and the noncortical cytoplasm, respectively. The non-cortical cytoplasm was composed of a complex network (designated here as the ground network) of filaments 6 to 13 nm in diameter, that interconnected aggregates of small globular materials, yolk granules and a meshwork of uniform filaments (8-9 nm in diameter). The 6 to 13 nm filaments comprising the ground network were branched and associated with filaments of the same or other sizes, resulting in the formation of an extremely complex network. The meshwork of 8-9 nm filaments was homogeneous in composition and constitutes a novel structure which has not been previously described. The 8-9 nm filaments were connected to one another at their ends, forming a meshwork of polygons. Meshworks, ranging up to 3 microns in diameter, were distributed throughout the non-cortical cytoplasm of the egg. Similar cytoplasmic structures were also observed in fertilized eggs.     

The structure and thermotropism of thymocyte plasma membranes as revealed by laser-raman spectroscopy.

1. Plasma membranes from rabbit thymocytes have been analyzed by laser-Raman spectroscopy over the 800-3000 cm-1 region and the spectra compared with those of endoplasmic reticulum, as well as relevant liposome systems. 2. Evaluation of the Amide I and Amide III regions indicates that thymocyte plasma membranes, but not endoplasmic reticulum, contain appreciable beta-structure peptide. This conclusion is supported by infrared spectroscopy. 3. Evaluation of the 2890 cm-1: 2850 cm-1 intensity ratio of plasma membranes as a function of temperature, using an integration technique, demonstrates a thermotropic lipid transition centered near 23 degrees C. This transition is less sharp than one observed with egg lecithin in this temperature range. 4. The significance of the thermotropic transition is evaluated in view of the lack of thermotropic lipid-protein segregation detectable by freeze-fracture electron microscopy (Wunderlich, F., Wallach, D.F.H., Speth, V. and Fischer, H. (1973) Biochim. Biophys. Acta 373, 34-43).     

New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy.

The structure of the flagellar base in Salmonella typhimurium has been studied by rapid-freeze techniques. Freeze-substituted thin sections and freeze-etched replicas of cell envelope preparations have provided complementary information about the flagellar base. The flagellar base has a bell-shaped extension reaching as far as 50 nm into the bacterial cytoplasm. This structure can be recognized in intact bacteria but was studied in detail in cell envelopes, where some flagella lacking parts of the bell were helpful in understanding its substructure. Structural relationships may be inferred between this cytoplasmic component of the flagellum and the recently described flagellar intramembrane particle rings as well as the structures associated with the basal body in isolated, chemically fixed flagella.     

Substructure of cisternal organelles of neuronal perikarya in immature rat brains revealed by quick-freeze and deep-etch techniques

Summary Membrane-bounded organelles possessing cisternae, i.e., rough endoplasmic reticulum and Golgi apparatus, in immature rat central neurons were examined by quick-freeze and deep-etch techniques to see how their intracisternal structures are organized and how ribosomes are associated with the membrane of the endoplasmic reticulum. Cisternae of endoplasmic reticulum, 60–100 nm wide, were bridged with randomly-distributed strands (trabecular strands, 12.5 nm in mean diameter). Luminal surfaces of cisternae of the endoplasmic reticulum were decorated with various-sized globular particles, some as small as intramembrane particles, and others as large as granules formed by soluble proteins seen in the cytoplasm. A closer examination revealed much thinner strands (3.3. nm in mean diameter). Such thin strands were short, usually winding toward the luminal surface, and sometimes touching the luminal surface with one end. Ribosomes appeared to be embedded into the entire thickness of cross-fractured membranes of endoplasmic reticulum, that is, their internal portions appeared to be situated at almost the same level as the cisternal luminal surface. From the internal portion of ribosomes, single thin strands occasionally protruded into the lumen, suggesting that these thin strands were newly synthesized polypeptides. A horizontal separation within ribosomes appeared to occur at the same level as the hydrophobic middle of the membrane of the endoplasmic reticulum. Interiors of the Golgi apparatus cisternae, which were much narrower than cisternae of endoplasmic reticulum, were similarly bridged with trabecular strands, but the Golgi trabecular strands were thinner and more frequent. Their cisternal lumina were also dotted with globular particles. No identifiable profiles corresponding to the thin strands in the endoplasmic reticulum were observed. Golgi cisternae showed a heterogeneous distribution of membrane granularity; the membrane in narrow cisternal space was granule-rich, while that in expanded space was granule-poor, suggesting a functional compartmentalization of the Golgi cisternae.     

Monomeric CFTR in plasma membranes in live cells revealed by single molecule fluorescence imaging

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel. There is indirect and conflicting evidence about whether CFTR exists in cell membranes as monomers, dimers, or higher order oligomers. We measured fluorescence intensities and photobleaching dynamics of distinct fluorescent spots in cells expressing functional CFTR-green fluorescent protein (GFP) chimeras. Intensity analysis of GFP-labeled CFTR in live cells showed single-component distributions with mean intensity equal to that of purified monomeric GFP, indicating monomeric CFTR in cell membranes. Fluorescent spots showed single-step photobleaching, independently verifying that CFTR is monomeric. Results did not depend on whether GFP was added to the CFTR N terminus or fourth extracellular loop or on whether CFTR chloride conductance was stimulated by cAMP agonists. Control measurements with a CFTR chimera containing two GFPs showed two-step photobleaching and a single-component intensity distribution with mean intensity twice that of monomeric GFP. These results provide direct evidence for monomeric CFTR in live cells.     

Molecular motions and hydration of purple membranes and disk membranes studied by neutron scattering

Fast stochastic equilibrium fluctuations (time scale: 10^–10–10^–13 seconds) in purple membranes (PM) and in disk membranes (DM) have been measured with quasielastic incoherent neutron scattering. The comparison of predominantly stochastic motions occurring in purple membranes and in disk membranes revealed qualitatively similar dynamical behaviour. Models of internal motions within restricted volumes have been shown to be useful to fit the spectra from both samples. From fits using these models we found “amplitudes” 15 to 20% larger for motions in DM samples compared to PM samples. This indicates a higher internal flexibility of the DM. Because the dynamical behaviour is very sensitive to the hydration of the protein-lipid complex, we also performed neutron diffraction experiments to determine lamellar spacings as a measure of level of hydration and as a function of temperature. From these studies the interaction of solvent molecules with the surface of the protein-lipid complex appears to be qualitatively similar for both types of membranes. Received: 12 February 1998 / Revised version: 18 March 1998 / Accepted: 27 March 1998     

Accumulation of immunoreactive opsin on plasma membranes in degenerating rod cells of rd/rd mutant mice

Immunoreactive opsin was detectable in the apical portion of normally developing photoreceptor cells on postnatal day 3 by the indirect enzyme-labeled antibody method. Immunoreactivity increased and had extended from the central retina to the periphery by the advanced stages of development. In the rd mutant retinas, accumulated opsin was present in the apical portion and in the outer nuclear layer on postnatal day 8. Immunoreactive opsin mainly was present in the outer nuclear layer by day 14, even being detectable on day 28. No immunoreactivity was present in the remaining cones. Electron microscopic immunocytochemistry confirmed the association of immunoreactive opsin with the persistent rod cell plasma membrane. Molecular weight of immunoreactive opsin in 14-day-old rd mutant mouse retina, as estimated by gel filtration chromatography, was large and did not seem to be degraded. These findings indicate that accumulated rhodopsin continues to function in the plasma membrane because an electroretinogram could be made after day 14 for the rd mutant mouse retina.     

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.     

The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique

Using the quick-freeze, deep-etch technique, we have analyzed the structure of the intact cell wall of Chlamydomonas reinhardi, and have visualized its component glycoproteins after mechanical shearing and after depolymerization induced by perchlorate or by the wall-disrupting agent, autolysin. The intact wall has previously been shown in a thin-section study (Roberts, K., M. Gurney-Smith, and G. J. Hills, 1972, J. Ultrastruct. Res. 40:599-613) to consist of a discrete central triplet bisecting a meshwork of fibrils. The deep-etch technique provides additional information about the architecture of each of these layers under several different experimental conditions, and demonstrates that each layer is constructed from a distinct set of components. The innermost layer of the central triplet proves to be a fibrous network which is stable to perchlorate but destabilized by autolysin, disassembling into fibrillar units we designate as "fishbones." The medial layer of the triplet is a loose assemblage of large granules. The outer layer is a thin, crystalline assembly that is relatively unaffected by autolysin. It depolymerizes into two glycoprotein species, one fibrous and one globular. The wall glycoproteins prove to be structurally similar to two fibrous proteins that associate with the flagellar membrane, namely, the sexual agglutinins and the protomers of a structure we designate a "hammock." They are also homologous to some of the fibrous components found in the extracellular matrices of multicellular plants and animals. The quick-freeze, deep-etch technique is demonstrated to be a highly informative way to dissect the structure of a fibrous matrix and visualize its component macromolecules.     

Fusion between disk membranes and plasma membrane of bovine photoreceptor cells is calcium dependent.

Disk membranes and plasma membrane vesicles were prepared from bovine retinal rod outer segments (ROS). The plasma membrane vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C and in the presence of micromolar calcium, an increase in R18 fluorescence with time was observed when R18-labeled plasma membrane vesicles were introduced to a suspension of disks. This result was interpreted as fusion between the disk membranes and the plasma membranes, the fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes as a result of lipid mixing during membrane fusion. While the disk membranes exposed exclusively their cytoplasmic surface, plasma membrane vesicles were found with both possible orientations. These vesicles were fractionated into subpopulations with homogeneous orientation. Plasma membrane vesicles that were oriented with the cytoplasmic surface exposed were able to fuse with the disk membranes in a Ca(2+)-dependent manner. Fusion was not detected between disk membranes and plasma membrane vesicles oriented such that the cytoplasmic surface was on the interior of the vesicles. ROS plasma membrane-disk membrane fusion was stimulated by calcium, inhibited by EGTA, and unaffected by magnesium. Rod photoreceptor cells of vertebrate retinas undergo diurnal shedding of disk membranes containing the photopigment rhodopsin. Membrane fusion is required for the shedding process.