Immunological purification of sea urchin egg tropomyosin.

The antiserum against lantern muscle tropomyosin of the sea urchin was prepared, and the presence of tropomyosin in the sea urchin egg was shown by immunodiffusion test between the antiserum and the egg tropomyosin fraction which was prepared according to the purification method for muscle tropomyosin. The sea urchin egg tropomyosin was isolated from the immuno-precipitate formed between the antiserum and the egg tropomyosin fraction. The subunit molecular weight of the egg tropomyosin was calculated to be 29,000.     

Wave of cortical actin polymerization in the sea urchin egg

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.     

Filamentous actin organization in the unfertilized sea urchin egg cortex

We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2-3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.     

pH regulates the polymerization of actin in the sea urchin egg cortex

The state of actin in the isolated cortex of the unfertilized sea urchin egg can be controlled by experimentally manipulating the pH of the isolation medium. Cortices isolated at the pH of the unfertilized egg (6.5--6.7) do not contain filamentous actin, while those isolated at the pH of the fertilized egg (7.3--7.5) develop large numbers of microvilli which contain bundles of actin filaments. Cortices that are isolated at pH 6.5 and then transferred to isolation medium buffered at pH 7.5 also develop actin filaments. However, the filaments are not arranged in bundles and microvilli do not form. Although the cortical granules in cortices isolated at pH 6.5 discharge at a free Ca++ concentration of approximately 10 micrometer, actin polymerization is not induced by increasing the Ca++ concentration of the isolation medium. These results suggest that the increase in cytoplasmic pH which occurs following fertilization induces the polymerization of actin in the egg cortex.     

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.     

Purification and properties of soluble actin from sea urchin eggs

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were homogenized in a buffer containing 0.1 M KCl and 2 mM MgCl2 at pH 6.85. About 50% of the actin was recovered in the high-speed supernate of the homogenate. More than 80% of the actin in this supernate was found to be monomeric upon gel filtration chromatography through a Sephadex G-150 column or by a DNase I inhibition assay. The critical concentration for polymerization of this actin prior to further purification was 0.3-0.9 mg/ml under various conditions. Actin was purified to near homogeneity from the Sephadex G-150 pool with high yield. The purified actin had a critical concentration for polymerization of 0.02-0.03 mg/ml. The isoelectric point of the crude actin and the purified actin was the same. Indeed, we found that there is only one isoelectric focusing species of actin in the sea urchin egg, and it has an isoelectric point more basic than rabbit skeletal muscle actin. The discrepancy between the polymerizability of the crude and purified actin may be due to the presence of factors in the crude fraction which inhibit the polymerization of actin.     

Polycation inhibition of exocytosis from sea urchin egg cortex

Summary The Ca²⁺-stimulated release of vesicle contents from cortical fragments prepared from sea urchin eggs is an in vitro model for exocytosis. Cortical fragments have been isolated either in suspension (cell surface complex, CSC preparation), or attached to polycation-coated surfaces (cortical lawn, CL preparation). CL, but not CSC, have been reported to undergo a rapid aging process whereby they fail to respond to micromolar free Ca²⁺. Since, in principle, the only difference between the two preparations is the use of polycations in the CL preparation, polycations were suspected of being inhibitory. This hypothesis was tested by evaluating the effects of polycation-containing buffers on the Ca²⁺ threshold, rate, and extent of exocytosis in CL prepared from the eggs ofStrongylocentrotus purpuratus. A sensitive microphotometric assay, based on light scattering by the individual cortical vesicles in the CL, was used to quantitate the exocytotic response. Buffers containing polylysine were found to be potent inhibitors of cortical exocytosis. The Ca²⁺ threshold of CL that had been treated for 15 min at room temperature with 50 g/ml of polylysine was more than three orders of magnitude greater than that of freshly prepared CL. The other polycations tested (protamine, spermine and neomycin) were also found to be inhibitory, but to a lesser degree than polylysine. Two lines of evidence suggested that the polycations used in the preparation of CL are responsible for the rapid aging phenomenon: (i) CSC fragments that had been affixed to polylysine-coated coverslips were shown to aquire aging characteristics similar to the CL preparations; control CSC that had been maintained in suspension did not. (ii) Radiolabeled poly-l-lysine was shown to dissociate from coated coverslips and redistribute onto CL.     

Inhibitory factor of microtubule assembly from sea urchin egg cortex. I. Preparation and characterization

A new inhibitory factor of the microtubule (MT) assembly system was isolated from unfertilized sea urchin egg cortex. This factor not only suppressed spontaneous brain MT assembly, but also induced depolymerization of the reconstituted MTs. The factor did not suppress initial MT growth initiated by ciliary outer fiber fragments but the assembled MTs were soon depolymerized with time. The inhibitory activity was heat-stable but sensitive to trypsin or urea. The mode of the inhibition was distinct from the inhibitory effects of RNA on the MT assembly. The inhibitory factor partially purified on DEAE-Sephadex A-50 completely inhibited tubulin polymerization in a factor: tubulin ratio of 0.013.     

Substructure of sea urchin egg cytoplasmic dynein

The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm X 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm X 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.     

Cytoskeleton of the unfertilized sea urchin egg

Unfertilized Paracentrotus lividus egg cytoskeleton is prepared by mild, nonionic detergent extraction at 4 degrees C in buffer systems containing either 2-methyl-2,4-pentanediol (hexylene glycol) or glycerol. These extractions allow the isolation of cytomatrices that maintain the egg form and are 70-80 micron in diameter. DNase inhibition assays show that actin is in polymerized form in these cytomatrices. Ultrastructural observations reveal that the cytoskeletons are made up essentially of 2 categories of filaments, 7-8-nm and 2-4-nm in diameter, respectively. After heavy meromyosin labelling, short, radiating actin filaments are seen in the cortical region, while longer actin filaments are found in the internal region of these cytomatrices. The 2-4-nm filaments of still unknown biochemical nature are organized in a meshwork. In contrast to results found with fertilized eggs, bundles of actin filaments and microtubules are absent; 8-13-nm filaments are not detected.     

Structure of F-actin needles from extracts of sea urchin oocytes

The mouse L-cell line LD maintains its mitochondrial DNA genome in the form of a head-to-tail unicircular dimer of the monomeric 16,000 base-pair species. This situation permits a comparison of the mechanism of replication of this dimeric molecule with our previous studies of replication of monomeric mouse L-cell mitochondrial DNA. Whereas monomeric mitochondrial DNA requires about one hour for a round of replication, the dimeric molecule requires almost three hours. Denaturing agarose gel electrophoretic analyses of replicative intermediates reveals several discrete size classes of partially replicated daughter strands of dimeric mitochondrial DNA. This suggests that replication occurs with specific discontinuities in the rate of daughter strand synthesis. The strand specificity of these daughter strands was determined by hybridization with ³²P-labeled DNA representing either the heavy or light strand mitochondrial DNA sequence. The sizes and strand specificities of these discrete daughter strands indicate that the same set of control sequences is functional in both dimer and monomer mitochondrial DNA replication.Immediately following a round of replication, the majority of dimeric mitochondrial DNA molecules contain displacement loops, as assessed by their sensitivity to nicking within the displaced DNA strand by single-strand DNA specific S₁ nuclease under conditions which leave supercoiled DNA intact. This result is in contrast with the conformation of newly replicated monomeric mitochondrial DNA molecules, which lack both superhelical turns and displacement loops. This indicates that dimeric mitochondrial DNA proceeds through a different series of post-replicative processing steps than does monomeric mitochondrial DNA. We postulate that intermediates at late stages of dimeric mitochondrial DNA replication contain displacement loops which remain intact following closure of the full-length daughter strands.     

Structure of actin filament bundles from microvilli of sea urchin eggs

The detailed substructure of actin filament bundles in microvilli of fertilized sea urchin eggs has been studied by analysing electron microscope images of negatively stained specimens. Transverse stripes which repeat about every 130 Å along the axis of a bundle, as previously observed by Burgess & Schroeder (1977), reflect the positions of cross-bridges that connect the filaments into a bundle. Analysis of optical transforms of the micrographs reveals that there are approximately 14 actin monomers between cross-overs of the two long-pitch helical strands of the actin filaments, with three cross-bridges in this interval. The structure is basically similar to that of the hexagonally packed bundles prepared in vitro from high speed supernatants of sea urchin eggs by Kane (1975) and analyzed by DeRosier et al. (1977). One clear difference, however, is that the in vivo microvillar filament bundles are supercoiled, giving rise to long axial repeats of 1500 to 2000 Å.Computationally filtered images of regions that were only slightly supercoiled reveal the relative alignment of filaments within the bundles and show that crossbridges appear to interact with four actin monomers, apparently linking two actin monomers on one strand of one filament to the nearest two monomers on a neighbouring filament. However, the cross-bridges are not spaced at equal intervals corresponding to four actin subunits, presumably because of the lack of hexagonal symmetry in the individual filaments, which have about 14 actin monomers between cross-overs. Instead, the cross-bridges are arranged quasiequivalently along the longitudinal axis of the bundles, in steps of four or five actin subunit spacings (28 Å each).     

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.