Cryo-electron microscopy of low density lipoprotein and reconstituted discoidal high density lipoprotein: imaging of the apolipoprotein moiety.

Cryo-electron microscopy was used to analyze the structure of low density lipoprotein from normolipidemic subjects (N-LDL), phospholipid-depleted N-LDL (PD-LDL), small dense LDL from hypertriglyceridemic subjects (SD-LDL), and reconstituted discoidal high density lipoproteins (rHDL). In different projections of N-LDL, a high density component of the particle was visible as two parallel bands or as a single ring. Projections of PD-LDL were very similar to those of N-LDL, indicating that the contribution of phospholipid headgroups to the observed high density structure is minor. In preparations of SD-LDL, projections with two high density bands or a single high density ring were rare. Instead, triangular and diamond-shaped projections were recognized. In different projections of discoidal rHDL, a high density component was visible as a single band or as a single ring. The present results indicate that cryo-electron microscopy reveals the distribution of apolipoproteins within lipoprotein particles. Thus, apolipoprotein B-100 (apoB) in N-LDL appears to be organized as a double ring around the particle, while apoB in SD-LDL is indicated to have a different conformation. Cryo-electron micrographs of rHDL are consistent with the presence of apolipoprotein A-I on the periphery of the lipoprotein disc.

A new type of highly polymerized yolk protein from the cochineal insect Dactylopius confusus.

A female specific protein was isolated from eggs and female hemolymph of cochineal insects, using density gradient ultracentrifugation, ammonium sulfate precipitation, and size exclusion column chromatography. The protein was found to consist of four different subunits with apparent molecular weights (Mr) 45,000, 49,000, 53,000, and 56,000, respectively. All four subunits were found to be glycosylated; no association of lipids was detected. Size exclusion column chromatography and non-denaturing polyacrylamide gel electrophoresis demonstrated that the native yolk protein exists as large polymers. Electron microscopy showed that these molecules are long, helical ribbons of variable size which are found in both hemolymph and eggs. Using cryo-electron microscopy, it was shown that the ribbons were 14.6 +/- 1.5 nm wide; the helix they form has a repeat distance of 104.9 +/- 11.3 nm and a diameter of 42.1 +/- 5 nm. A clear substructure of the ribbons was recognized. The newly identified protein is the major yolk protein of Dactylopius confusus and no other proteins resembling the more familiar vitellins of other insect species were detected. Moreover, the D. confusus yolk protein appears to be unique both in its subunit structure and in its polymerizing qualities. Thus, the cochineal yolk protein (CYP) is suggested to represent a new type of insect yolk protein.

Cryo-electron microscopy reveals human low density lipoprotein substructure.

In the present study, we have examined the structure of human low density lipoprotein (LDL) using cryo-electron microscopy. Human LDL particles were analyzed in a vitrified frozen-hydrated condition, without chemical fixation or any form of staining. Hence, the lipoproteins were visualized close to their native state. Contrary to current spherical models, the overall shape of human LDL is indicated to be discoidal. The observed LDL disks have a diameter of 21.4 +/- 1.3 nm and a height of 12.1 +/- 1.1 nm (mean +/- standard deviation). The average volume of LDL particles in cryo-electron microscopic preparations is estimated to be 4352 nm3. This value corresponds well with the LDL volume that has been determined by sedimentation equilibrium studies [4130-4803 nm3; Kahlon et al., 1982. Lipids. 17: 323-330]. Details of LDL ultrastructure, visible as a result of local differences in mass density, are indicated to reflect the distribution of protein within the lipoprotein particle. Thus, apolipoprotein B-100 (apoB) appears to form two ring-shaped structures that are organized around the perimeter of the LDL disk.

A new organelle related to osmoregulation in ultrarapidly frozenPelvetia embryos.

Freeze-fracture electron microscopy of the cortical cytoplasm of unfixed, uncryoprotected, ultrarapidly frozen embryos of the marine brown algaPelvetia fastigiata has demonstrated the presence of numerous 0.5-µm diameter, disc-shaped vesicles lying adjacent and nearly parallel to the plasma membrane. Some vesicles are fused with the plasma membrane through a narrow connection; this however appears to be a reversible attachment rather than an intermediate stage in the incorporation of the vesicle into the plasma membrane. The distribution of these connections in the plane of the membrane is not uniform; they tend to occur in patches. The fraction of vesicles that is fused with the plasma membrane at any one time appears to be related to a cell's perception of a stressful hypotonic imbalance between the internal and external concentrations of osmotically active compounds. Thus, a sudden 5% decrease in osmolarity of the artificial seawater medium just before freezing leads to a 38% increase in connections per unit membrane area, while a 20% decrease in osmolarity leads to a 75% increase in connections per unit area. Based on these findings and the corresponding ion-transport studies of R. Nuccitelli and L.F. Jaffe (1976, Planta131, 315-320), we postulate that the disc-shaped vesicles mediate short-term osmoregulation inPelvetia embryos by reversibly inserting chloride channels into the plasma membrane.

Improved specimen support cups and auxiliary devices for the Balzers high pressure freezing apparatus.

A new design for interlocking specimen support cups that permits reliable and reproducible freezing under high pressures of a range of different biological specimens is described. A stronger holder in which the support cups are tightly held during pressurization and freezing, and a sliding wedge for separating the cups after freezing are also described. Used in conjunction with the Balzers HPM 010 high pressure freezing apparatus, these modifications permit routine freezing of tissue for freeze-fracture and freeze-substitution for electron microscopy.

Roles of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes.

Unfertilized eggs of the medaka fish (Oryzias latipes) were injected with pH-buffered calcium buffers. Medaka egg activation is accompanied by a transient increase in cytoplasmic free calcium (Gilkey, J. C., L. F. Jaffe, E. B. Ridgway, and G. T. Reynolds, 1978, J. Cell Biol., 76:448-466). The calcium buffer injections demonstrated that (a) the threshold free calcium required to elicit the calcium transient and activate the egg is between 1.7 and 5.1 microM at pH 7.0, well below the 30 microM reached during the transient, and (b) buffers which hold free calcium below threshold prevent activation of the buffered region in subsequently fertilized eggs. Therefore an increase in free calcium is necessary and sufficient to elicit the calcium transient, and the calcium transient is necessary to activate the egg. Further, these results are additional proof that the calcium transient is initiated and propagated through the cytoplasm by a mechanism of calcium-stimulated calcium release. Finally, a normal calcium transient must propagate through the entire cytoplasm to ensure normal development. Unfertilized eggs were injected with pH buffers to produce short-term, localized changes in cytoplasmic pH. The eggs were then fertilized at various times after injection. In other experiments, unfertilized and fertilized eggs were exposed to media containing either NH4Cl or CO2 to produce longer term, global changes in cytoplasmic pH. These treatments neither activated the eggs nor interfered with the normal development of fertilized eggs, suggesting that even if a natural change in cytoplasmic pH is induced by activation, it has no role in medaka egg development. The injected pH buffers altered the rate of propagation of the calcium transient through the cytoplasm, suggesting that the threshold free calcium required to trigger calcium-stimulated calcium release might be pH dependent. The results of injection of pH-buffered calcium buffers support this conjecture: for a tenfold increase in hydrogen ion concentration, free calcium must also be raised tenfold to elicit the calcium transient.

A free calcium wave traverses the activating egg of the medaka, Oryzias latipes.

Aequorin-injected eggs of the medaka (a fresh water fish) show an explosive rise in free calcium during fertilization, which is followed by a slow return to the resting level. Image intensification techniques now show a spreading wave of high free calcium during fertilization. The wave starts at the animal pole (where the sperm enters) and then traverses the egg as a shallow, roughly 20 degrees-wide band which vanishes at the antipode some minutes later. The peak free calcium concentration within this moving band is estimated to be about 30 microM (perhaps 100-1,000 times the resting level). Eggs activated by ionophore A23187 may show multiple initiation sites. The resulting multiple waves never spread through each other; rather, they fuse upon meeting so as to form spreading waves of compound origin. The fertilization wave is nearly independent of extracellular calcium because it is only slightly slowed (by perhaps 15%) in a medium containing 5 mM ethylene glycol-bis[beta-aminoethyl ether]N,N'-tetraacetic acid (EGTA) and no deliberately added calcium. It is also independent of the large cortical vesicles, which may be centrifugally displaced. Normally, however, it distinctly precedes the well-known wave of cortical vesicle exocytosis. We conclude that the fertilization wave in the medaka egg is propagated by calcium-stimulated calcium release, primarily from some internal sources other than the large cortical vesicles. A comparison of the characteristics of the exocytotic wave in the medaka with that in other eggs, particularly in echinoderm eggs, suggests that such a propagated calcium wave is a general feature of egg activation.

Free calcium increases explosively in activating medaka eggs.

We have used the calcium-specific light-emitting protein aequorin to follow changes in free calcium concentration during fertilization and cleavage of eggs from medaka, a fresh-water fish. Aequorin-injected medaka eggs show a very low resting glow before they are fertilized, indicating a low calcium concentration in the resting state. Upon activation by sperm, the calcium-mediated light emission increases to a level some 10,000 times the resting level with a 1 to 2 sec time constant for an e-fold increase, and then slowly retruns to the resting level. Upon activation by the ionophore A23187, the early rise in luminescence is much slower, but once a threshold has been reached the subsequent rise becomes as rapid as the normal sperm-induced response. We infer that the explosive rise in calcium involves calcium-stimulated calcium release, and that a sperm normally triggers this rise by somehow inducing a more modest and localized rise in calcium.