Strongylocentrotus Purpuratus Eggs Research Articles

Speract, a sea urchin egg peptide that regulates sperm motility, also stimulates sperm mitochondrial metabolism

Sea urchin sperm have only one mitochondrion, that in addition to being the main source of energy, may modulate intracellular Ca2+ concentration ([Ca2+]i) to regulate their motility and possibly the acrosome reaction. Speract is a decapeptide from the outer jelly layer of the Strongylocentrotus purpuratus egg that upon binding to its receptor in the sperm, stimulates sperm motility, respiration and ion fluxes, among other physiological events. Altering the sea urchin sperm mitochondrial function with specific inhibitors of this organelle, increases [Ca2+]i in an external Ca2+ concentration ([Ca2+]ext)-dependent manner (Ardón, et al., 2009. BBActa 1787: 15), suggesting that the mitochondrion is involved in sperm [Ca2+]i homeostasis. To further understand the interrelationship between the mitochondrion and the speract responses, we measured mitochondrial membrane potential (ΔΨ) and NADH levels. We found that the stimulation of sperm with speract depolarizes the mitochondrion and increases the levels of NADH. Surprisingly, these responses are independent of external Ca2+ and are due to the increase in intracellular pH (pHi) induced by speract. Our findings indicate that speract, by regulating pHi, in addition to [Ca2+]i, may finely modulate mitochondrial metabolism to control motility and ensure that sperm reach the egg and fertilize it.

Effects of sea urchin hyalin on gastrulation

The sea urchin hyaline layer is an extracellular matrix that develops soon after fertilization of Strongylocentrotus purpuratus eggs. The major constituent of this layer is the fibrillar glycoprotein hyalin that is essential for maintenance, organization and adhesion of cells during morphogenesis. Here, hyalin was isolated from newly fertilized S. purpuratus embryos via disruption of the vitelline envelope with dithiothreitol, followed by removal of the hyaline layer from the embryo surface with ice-cold NaCl-KCl. Hyalin was collected as the supernatant from the settled embryos and its purity was assessed on 2 percent agarose gels. 25 hr S. purpuratus embryos were incubated for 25 hrs at 15 C with normal sea water, hyalin/low calcium sea water, NaCl-KCl/ low calcium sea water or low calcium sea water alone in 96 well microplates. The experiment was repeated 10 times. The hyalin-treated and control samples were observed live and formaldehyde fixed using a Zeiss Axiolab photomicroscope. 87 percent (average) of all the controls developed complete archenterons. In the hyalin treated samples, 48 percent displayed no invagination, 49 percent showed incomplete, unattached archenterons, and 3 percent developed compete archenterons. Many studies, including this one, suggest that hyalin may be involved in gastrulation events such as initiation of invagination. The assay used here, because it allows precise quantification of the effects of substances on specific morphogenetic events, should help us understand exactly how molecules, such as hyalin, affect one specific event versus another (supported by NIH NIGMS SCORE, RISE, MARC, ITQ program and the Joseph Drown Foundation).

Regulated Proteolysis by Cortical Granule Serine Protease 1 at Fertilization

Cortical granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, cortical granule serine protease 1 (CGSP1), has been identified in the cortical granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the granule, and is inactive at pH 6.5 or below. We determined the pH of the cortical granule by fluorescent indicators and micro-pH probe measurements and found the granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native cortical granule targets of the protease are beta-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the granule are not proteolyzed by CGSP1. Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the cortical granules, and it has a small population of targets for cleavage within the cortical granules.

26] Bioassay for determining endogenous levels of cyclic ADP-ribose

Publisher Summary This chapter describes a bioassay for cyclic ADP-ribose (ADPR) on the basis of its Ca 2+ -releasing activity in homogenates prepared from sea urchin eggs. Substances that could potentially interfere with the Ca 2+ release bioassay, such as Ca 2+ , inositol 1,4,5-triphosphate, and NAD + , must be removed from the sample. Cyclic ADP-ribose in the extracts is purified by a two-step system consisting of phenylboronate chromatography followed by anion-exchange chromatography on AG MP-1. Strongylocentrotus purpuratus eggs are obtained by stimulating ovulation of a female sea urchin with a 1-ml injection of 0.5 M KCI and washed once in artificial seawater, twice in Ca 2+ -free seawater containing 1 m M ethylene glycol tetraacetic acid (EGTA), twice in Ca 2+ -free seawater without EGTA, once with the homogenization buffer, and resuspended with the same medium to 25%. The sensitivity of the bioassay can be increased by pretreating the homogenate with caffeine, which is known to potentiate cADPR-induced Ca 2+ release.

Characterization of a large conductance, cation-selective channel from sea urchin eggs that is sensitive to sulfhydryl reducing agents.

Vesicles containing large conductance cation selective channels were isolated from sea urchin (Strongylocentrotus purpuratus) eggs. Addition of the vesicles to one side of lipid bilayer led to the rapid appearance of 200 or more identical channels. These channels would then inactivate within 2 to 10 min. The inactivation could be prevented by the addition of sulfhydryl reducing agents (e.g., dithiothreitol or glutathione) to the cis side of the membrane. Only one channel type is present. The channel is cation selective, with a conductance of 572 ps in symmetrical 0.5 M KCl. The relative cation selectivity is K (1.0) > Cs (0.53) approximately < Na (0.52) > Li (0.2). The permeability ratio (Px/Pk) is 1.37 (Li) > 1.27 (Na) > 0.57 (Cs). Most organic cations (choline, tetraethylamine, tetrabutylamine, gallamine, lysine, histidine, arginine, etc.) and multivalent cations (La+3, alkali earth family, Zn+2, Eu+3, etc.) produced a significant channel block. The highest observed affinity was for La+3 which produced a 50% decrease in conductance in 500 mM KCl at a concentration of 8 microM. The biophysical properties of this channel are similar to those of a non-selective channel found in ascidian egg plasma membrane (Dale & DeFelice, 1984). A soluble extract of the egg supernatant can also prevent the inactivation of the channels. Using deactivated channels reconstituted into a planar lipid bilayer as an assay, this factor was partially purified. It is heat and acetone stable with a molecular weight of between 10 and 20 K. One of the major bands remaining in the purest fraction cross reacted with antibodies raised against E. coli thioredoxin.

The Isolation of Acrosome-Reaction-Inducing Glycoproteins from Sea Urchin Egg Jelly

The solubilized jelly coat of Strongylocentrotus purpuratus eggs was boiled in SDS and mercaptoethanol, precipitated in ethanol, washed in 70% ethanol, lyophilized, and redissolved in seawater. The sample retained its activity to induce the sperm acrosome reaction (AR), showing that the AR inducer was resistant to harsh denaturing conditions. Egg jelly (EJ) was separated into its component macromolecules by denaturing polyacrylamide gel electrophoresis and the gel silver-stained. Several macromolecules ranging from approximately 30 to 380 kDa were consistently observed. These macromolecules were not seen by Coomassie blue staining. With the exception of the fucose sulfate polymer (FSP; 380 kDa) and the 300-kDa component, the other molecules were of minor abundance. Sephacryl-500 gel-filtration chromatography in detergents and disulfide reducing agents separated EJ into three fractions. The FSP and 300-kDa components were purified to homogeneity. A third fraction consisted of components of 30, 82, 116, and 138 kDa. The purified FSP and 300-kDa components had insignificant AR-inducing activity. Substantial AR activity was found only in the 30 to 138-kDa fraction. Ion-exchange chromatography and Centricon filtration separated the 82- and 138-kDa glycoproteins from the other components, but these two components could not be separated from each other. There may be other AR-inducing molecules in sea urchin EJ that were not visible after silver staining of gels. Also, some EJ components are so large that they did not enter the stacking gel. Of those macromolecules that were visible on gels after silver staining, only the fraction containing the 80- and 138-kDa proteins had significant AR-inducing activity.

The sea urchin egg receptor for sperm: isolation and characterization of the intact, biologically active receptor.

The species-specific binding of sea urchin sperm to the egg is mediated by an egg cell surface receptor. Although earlier studies have resulted in the cloning and sequencing of the receptor, structure/function studies require knowledge of the structure of the mature cell surface protein. In this study, we report the purification of this glycoprotein to homogeneity from a cell surface complex of Strongylocentrotus purpuratus eggs using lectin and ion exchange chromatography. Based on the yield of receptor it can be calculated that each egg contains approximately 1.25 x 10(6) receptor molecules on its surface. The receptor, which has an apparent M(r) of 350 kD, is a highly glycosylated transmembrane protein composed of approximately 70% carbohydrate. Because earlier studies on the partially purified receptor and on a pure, extracellular fragment of the receptor indicated that the carbohydrate chains were important in sperm binding, we undertook compositional analysis of the carbohydrate in the intact receptor. These analyses and lectin binding studies revealed that the oligosaccharide chains of the receptor are sulfated and that both N- and O-linked chains are present. Functional analyses revealed that the purified receptor retained biological activity; it inhibited fertilization in a species-specific and dose-dependent manner, and polystyrene beads coated with it bound to acrosome-reacted sperm in a species-specific manner. The availability of biochemical quantities of this novel cell recognition molecule opens new avenues to studying the interaction of complementary cell surface ligands in fertilization.

Cortical granule matrix disassembly during exocytosis in sea urchin eggs

Cortical granule exocytosis in sea urchins was studied using hyperosmotic and polymer-containing seawater to halt granule matrix dispersal. Addition of Na 2SO 4-containing seawater (2.5 osmole/kg) to Strongylocentrotus purpuratus eggs 10 to 40 sec after insemination resulted in arrest of the exocytic wave during propagation. EM examination of these eggs revealed that matrix disassembly occurred in distinct stages. In the earliest stage, granule-plasma membrane fusion had occurred, but the matrix remained completely intact. This early stage was observed in hyperosmotic media, either ionic or nonionic, suggesting that matrix hydration is required for disassembly and exocytic pore widening, but not for membrane fusion. Subsequent stages, in which partially disassembled matrices remained within ω-configured pockets, were captured by activating eggs in 30% dextran in seawater. Stability of these intermediates stages required the presence of Ca 2+ and Mg 2+; in the absence of divalent cations the matrices completely disassembled and the exocytic pockets flattened. Divalent cations appeared to prevent fragmentation of the matrix lamellae. Late stages of matrix disassembly, in which the lamellae fragmented and formed small particles, were inhibited by media of high ionic strength. Hyperosmolality alone, provided by sucrose, was unable to halt these late stages suggesting that water availability does not play an important role once a critical point in matrix dispersal has been reached.

Sea urchin egg villin: Identification of villin in a non-epithelial cell from an invertebrate species

Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subsequent formation of a brush border-like cortical cytoskeleton. A 110 x 10(3) Mr (110K) actin binding protein has been purified from extracts of unfertilized Strongylocentrotus purpuratus eggs. Analysis of polymerization kinetics using fluorescence and viscometry assays demonstrated that 110K accelerated the nucleation phase of actin assembly only in the presence of elevated Ca2+. The Ca(2+)-mediated effects were correlated with a decrease in sedimentable polymer and a decrease in average filament length. Addition of Ca2+ to solutions of 110K and F-actin, polymerized in the presence of EGTA, resulted in a precipitous drop in viscosity and the decreased viscosity was fully reversible upon chelation of Ca2+. The Ca2+ threshold for 110K activation was in the 10(-6) to 10(-7) M range. Nucleated assembly experiments using Limulus sperm acrosomal processes demonstrated that egg 110K capped the barbed ends of actin filaments. In the absence of Ca2+, 110K organized actin filaments into bundles at pH values less than 7.4. Anti-egg 110K antibody crossreacted with chicken intestinal epithelial cell villin and anti-porcine villin headpiece monoclonal antibody crossreacted with 110K. Further, 110K possesses an approximately 10 x 10(3) Mr terminal polypeptide segment that is immunologically related to villin headpiece. These studies demonstrate that sea urchin egg 110K is functionally, immunologically and structurally related to villin, an actin binding protein expressed in specific epithelial tissues in vertebrates. Consequently, this finding provides insight into the potential mechanisms that might determine the genesis of the cortical brush border cytoarchitecture in sea urchin eggs and further sheds light on the evolution of the villin protein family.

Negative spatial regulation of the lineage specific CyIIIa actin gene in the sea urchin embryo

The CyIIIa.CAT fusion gene was injected into Strongylocentrotus purpuratus eggs, together with excess ligated competitor sequences representing subregions of the CyIIIa regulatory domain. In this construct, the chloramphenicol acetyltransferase (CAT) reporter gene is placed under the control of the 2300 nucleotide upstream regulatory domain of the lineage-specific CyIIIa cytoskeletal actin gene. CAT mRNA was detected by in situ hybridization in serial sections of pluteus stage embryos derived from the injected eggs. When carrier DNA lacking competitor CyIIIa fragments was coinjected with CyIIIa.CAT, CAT mRNA was observed exclusively in aboral ectoderm cells, i.e. the territory in which the CyIIIa gene itself is normally expressed (as also reported by us previously). The same result was obtained when five of seven different competitor subfragments bearing sites of DNA-protein interaction were coinjected. However, coinjection of excess quantities of either of two widely separated, nonhomologous fragments of the CyIIIa regulatory domain produced a dramatic ectopic expression of CAT mRNA in the recipient embryos. CAT mRNA was observed in gut, mesenchyme cells and oral ectoderm in these embryos. We conclude that these fragments contain regulatory sites that negatively control spatial expression of the CyIIIa gene.

Hyperosmolality inhibits exocytosis in sea urchin eggs by formation of a granule-free zone and arrest of pore widening

Hyperosmolality is known to inhibit membrane fusion during exocytosis. In this study cortical granule exocytosis in sea urchin eggs is used as a model system to determine at what step this inhibition occurs. Strongylocentrotus purpuratus eggs were incubated in hyperosmotic seawater (Na2SO4, sucrose or sodium HEPES used as osmoticants), the eggs activated with 20 microM A23187 to trigger exocytosis, and then quick frozen or chemically fixed for electron microscopy. Thin sections and freeze-fracture replicas show that at high osmolality (2.31 osmol/kg), there is a decrease in cortical granule size, a 90% reduction in granule-plasma membrane fusion, and formation of a granule-free zone between the plasma membrane and cortical granules. This zone averages 0.64 micron in thickness and prevents the majority of granules from docking at the plasma membrane. The remaining granules (approximately 10%) exhibit early stages of fusion which appear to have been stabilized; the matrix of these granules remains intact. We conclude that exocytosis is blocked by two separate mechanisms. First, the granule-free zone prevents granule-plasma membrane contact required for fusion. Second, in cases where fusion does occur, opening of the pocket and dispersal of the granule contents are slowed in hyperosmotic media.

High molecular weight polymers block cortical granule exocytosis in sea urchin eggs at the level of granule matrix disassembly.

Recently, we have shown that high molecular weight polymers inhibit cortical granule exocytosis at total osmolalities only slightly higher than that of sea water (Whitaker, M., and J. Zimmerberg. 1987. J. Physiol. 389:527-539). In this study, we visualize the step at which this inhibition occurs. Lytechinus pictus and Strongylocentrotus purpuratus eggs were exposed to 0.8 M stachyose or 40% (wt/vol) dextran (average molecular mass of 10 kD) in artificial sea water, activated with 60 microM of the calcium ionophore A23187, and then either fixed with glutaraldehyde and embedded or quick-frozen and freeze-fractured. Stachyose (2.6 osmol/kg) appears to inhibit cortical granule exocytosis by eliciting formation of a granule-free zone (GFZ) in the egg cortex which pushes granules away from the plasma membrane thus preventing their fusion. In contrast, 40% dextran (1.58 osmol/kg) does not result in a GFZ and cortical granules undergo fusion. In some specimens, the pores joining granule and plasma membranes are relatively small; in other cases, the exocytotic pocket has been stabilized in an omega configuration and the granule matrix remains intact. These observations suggest that high molecular weight polymers block exocytosis because of their inability to enter the granule matrix: they retard the water entry that is needed for matrix dispersal.

Immunolocalization of the Sea Urchin Sperm Receptor in Eggs and Maturing Ovaries1

The sperm receptor from Strongylocentrotus purpuratus eggs is a high molecular weight proteoglycan-like molecule that inhibits fertilization species-specifically in a competition bioassay. A preparation of highly active sperm receptor that contained one major N-terminal sequence was used to generate polyclonal antibody in rabbits. This antibody species-specifically inhibited fertilization at low concentrations without interfering with fertilization envelope elevation. On immunofluorescence microscopy, the antibody recognized determinants on the mature egg cell surface and in the cortical granules just beneath the surface. Preabsorption of the antibody with the calcium-soluble fraction of the exudate from cortical granules rendered the antibody specific for the cell surface in mature eggs and still able to inhibit fertilization at the same concentrations as before treatment with cortical granule exudate. With antibody preabsorbed with cortical granule and by counting antibody-gold particles viewed by electron microscopy, sperm receptor was almost undetectable on the cell surface of immature oocytes in preseason ovaries, present on the cell surface and intracellularly in immature oocytes of ovaries collected at the beginning or at the height of the spawning season, and present only on the cell surface of mature oocytes in the lumen of the ovaries. Our results indicate that receptor is synthesized early in oogenesis and is rapidly moved to the egg cell surface.

Sea urchin sperm as a vector of foreign genetic information

Paracentrotus lividus Strongylocentrotus purpuratus and Arbacia lixula sperm is capable of incorporating homologous as well as heterologous macromolecules and vehiculating them into eggs of the same species. If Strongylocentrotus purpuratus eggs are fertilized with sperm previously incubated with pSV2-cat or pRSV-cat plasmids, chloramphenicol acetyl transferase (CAT) activity is found in the embryos at the swimming blastula stage, showing that the plasmids bearing the CAT gene are vehiculated by the sperm into the eggs and the bacterial gene is expressed in the embryos.

Respiratory burst oxidase of fertilization.

Partially reduced oxygen species are toxic, yet sea urchin eggs synthesize H2O2 in a "respiratory burst" at fertilization, as an extracellular oxidant to crosslink their protective surface envelopes. To study the biochemical mechanism for H2O2 production, we have isolated an NADPH-specific oxidase fraction from homogenates of unfertilized Strongylocentrotus purpuratus eggs that produces H2O2 when stimulated with Ca2+ and MgATP2-. Concentrations of free Ca2+ previously implicated in regulation of egg activation modulate the activity of the oxidase. Inhibitors were used to test the relevance of this oxidase to the respiratory burst of fertilization. Procaine, two phenothiazines, and N-ethylmaleimide (but not iodoacetamide) inhibited H2O2 production by the oxidase fraction and oxygen consumption by activated eggs. The ATP requirement suggested that protein kinase activity might regulate the respiratory burst of fertilization; consonant with this hypothesis, H-7 and staurosporine were inhibitory. The respiratory burst oxidase of fertilization is an NADPH:O2 oxidoreductase that appears to be regulated by a protein kinase; although it bears a remarkable resemblance to the neutrophil oxidase, unlike the latter it does not form O2- as its initial product.

Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus.

To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.

Immunocytochemical localization of the 35-kDa sea urchin egg trypsin-like protease and its effects upon the egg surface

Trypsin-like protease in sea urchin eggs is thought to reside in cortical granules since it is secreted at fertilization and has been isolated with cortical granule fractions from unfertilized eggs. A 35-kDa serine protease has been purified from Strongylocentrotus purpuratus eggs by soybean trypsin inhibitor-affinity chromatography. For this report the protease was localized by immunocytochemistry before and after fertilization, and its potential biological activity was examined by application of the isolated enzyme to the unfertilized egg surface. The protease was localized on sections by immunofluorescence and immunoelectron microscopy, and was found to reside in the spiral lamellae of S. purpuratus cortical granules and in the electron-dense stellate core of Arbacia punctulata granules. At fertilization the enzyme is secreted into the perivitelline space and accumulates only very briefly between the hyaline layer and the nascent fertilization envelope. Shortly thereafter the enzyme is lost from the perivitelline space and immunological reactivity is no longer associated with the egg surface. The 35-kDa cortical granule protease has vitelline delaminase activity but does not appear to destroy vitelline envelope sperm receptors as judged by the fertility of protease-treated eggs.

Dispersal of sperm surface antigens in the plasma membranes of polyspermically fertilized sea urchin eggs

Polyspermically fertilized Strongylocentrotus purpuratus eggs were fixed at varying times after insemination and exposed to a monoclonal antibody (mAb J18/29) directed against a group of sperm surface antigens. Indirect immunofluorescence microscopy reveals that the sperm surface components recognized by mAb J18/29 are quickly incorporated into the egg plasma membrane and begin to disperse as early as 1.5 min after insemination. At subsequent times after insemination, they undergo further dispersal so that by 45 min they are distributed evenly over the entire surface of the egg. These results provide evidence for the free lateral mobility of sperm membrane components in the fertilized egg.

Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs

Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH 4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.

Isolation and characterization of sea urchin egg spectrin: calcium modulation of the spectrin-actin interaction.

Sea urchin egg spectrin has been purified from a homogenate of unfertilized Strongylocentrotus purpuratus eggs using standard biochemical procedures. SDS-PAGE analysis of the molecule revealed a closely spaced, high molecular weight doublet at 237/234 kDa (present in an equimolar ratio). Rotary shadowed images of egg spectrin revealed a double-stranded, elongate, flexible rod-shaped contour, measuring 210 nm in length and approximately 4-8 nm in width. Additionally, this molecule is shown to be immunologically related to avian erythroid spectrin, since it crossreacts with antibodies prepared against the chicken erythrocyte alpha-spectrin/240 kDa subunit. The interaction of egg spectrin with actin was examined by sedimentation and falling-ball viscometry assays. The binding and cross linking properties of spectrin to actin demonstrate a unique Ca++-sensitive regulation at micromolar Ca++ concentrations. This observation provides new insight into the way Ca++ may regulate spectrin-actin interactions in vitro and further suggests possible structural and modulatory roles for egg spectrin in the developing sea urchin embryo.