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K R Foltz

Publications and source records attributed to K R Foltz.

13 recordsLinked to original sources
PubMed1999

Evidence that a starfish egg Src family tyrosine kinase associates with PLC-gamma1 SH2 domains at fertilization.

The initiation of calcium release at fertilization in the eggs of most animals relies on the production of IP3, implicating the activation of phospholipase C. Recent work has demonstrated that injection of PLC-gamma SH2 domain fusion proteins into starfish eggs specifically inhibits the initiation of calcium release in response to sperm, indicating that PLC-gamma is necessary for Ca2+ release at fertilization [Carroll et al. (1997) J. Cell Biol. 138, 1303-1311]. Here we investigate how PLC-gamma may be activated, by using the PLC-gamma SH2 domain fusion protein as an affinity matrix to identify interacting proteins. A tyrosine kinase activity and an egg protein of ca. Mr 58 K that is recognized by an antibody directed against Src family tyrosine kinases associate with PLC-gamma SH2 domains in a fertilization-dependent manner. These associations are detected by 15 s postfertilization, consistent with a function in releasing Ca2+. Calcium ionophore treatment of eggs did not cause association of the kinase activity or of the Src family protein with the PLC-gamma SH2 domains. These data identify an egg Src family tyrosine kinase as a potential upstream regulator of PLC-gamma in the activation of starfish eggs.

Animals↗
PubMed1999

Identification of PLCgamma-dependent and -independent events during fertilization of sea urchin eggs.

At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.

Action Potentials↗
PubMed1997

Surface localization of the sea urchin egg receptor for sperm.

The sea urchin egg receptor for sperm is thought to be involved in species-specific sperm-egg interactions at the egg surface. Recent revisions in the deduced amino acid sequence of the cloned cDNAs indicate that the protein encoded does not possess the common structural hallmarks of a membrane protein. Thus, investigation of the localization and association of the protein with the egg surface is crucial. We describe and characterize a new monoclonal antibody raised against recombinant sperm receptor protein. This antibody, in conjunction with several polyclonal antibodies, was used to study the receptor protein in eggs. Immunoprecipitation studies indicated that the antibodies recognize the high Mr (ca. 350 K) sperm receptor protein which copurified with egg plasma membrane-vitelline layer complexes. The sperm receptor protein was solubilized only by detergents and not by treatments designed to solubilize peripherally associated or lipid-anchored membrane proteins, suggesting a tight association with the membrane fraction. Confocal immunofluorescence microscopy of live eggs indicated surface staining. Finally, lysylendoproteinase C treatment of live eggs resulted in a loss of the high Mr receptor protein epitopes, and the concomitant release of a 70-kDa proteolytic fragment, which correlated with a reduced ability of the eggs to be fertilized. Taken together, these data indicate that at least some fraction of the sperm receptor protein is present on the egg surface, a requisite locale for a sperm binding protein.

Animals↗
PubMed1995

Gamete and immune cell recognition revisited.

Fertilization is the result of a series of successful recognition and binding events mediated by gamete surface molecules. Recent advances in the identification and characterization of some of these recognition molecules provide extremely valuable information necessary to understand sperm-egg recognition and subsequent egg activation. We discuss these new data in the context of the model of gamete recognition first proposed by F.R. Lillie in the early part of the 20th century, and revisited periodically in the subsequent literature, which relates fertilization events to those of immune cell recognition and activation events. Here we discuss the principles underlying the molecular recognition and activation mechanisms of gametes and immune cells.

Animals↗
PubMed1995

Sperm-binding proteins.

Gamete recognition and binding are mediated by specific proteins on the surface of the sperm and egg. Identification and characterization of some of these proteins from several model systems, particularly mouse and sea urchin, have focused interest on the general properties and functions of gamete recognition proteins. Sperm-binding proteins located in egg extracellular coats as well as sperm-binding proteins that are localized to the egg plasma membrane are presented in the context of their structure and function in gamete binding. Unifying and disparate characteristics are discussed in light of the diverse biology of fertilization among species. Outstanding questions, alternative mechanisms and models, and strategies for future work are presented.

Animals↗
PubMed1994

Tyrosine phosphorylation of the egg receptor for sperm at fertilization.

Gamete interaction triggers a variety of responses within the egg, collectively referred to as egg activation. In addition to the hallmarks of calcium release and fertilization envelope elevation, there are cytoskeletal rearrangements, protein tyrosine phosphorylation, and an increase in pH, among others. The ultimate goal of these concerted activation events is entry of the newly fertilized egg into the cell cycle. However, the molecular mechanisms which promote downstream cell activation events remain poorly understood. One model suggests that sperm deliver an "activating factor" upon fusion with the egg plasma membrane, while a second model proposes that the egg receptor for sperm transduces a signal that mediates a cascade of subsequent events. It also is possible that multiple pathways are activated. As a first step toward testing the hypothesis of receptor-mediated signal transduction, we have investigated the tyrosine phosphorylation state of the sea urchin egg receptor for sperm using specific antibodies. The present work indicates that the sperm receptor is phosphorylated by an egg cortical tyrosine kinase in response to sperm or purified ligand (bindin) binding. Maximal phosphorylation was reached within 20 sec. These data support the hypothesis that the sperm receptor is a gamete recognition protein which responds to ligand binding and focus attention on the question of the role of this tyrosine phosphorylation signal in egg activation.

Acrosome↗
PubMed1993

Sea urchin egg receptor for sperm: sequence similarity of binding domain and hsp70.

Fertilization depends on cell surface recognition proteins that interact and thereby mediate binding and subsequent fusion of the sperm and egg. Overlapping complementary DNA's encoding the egg plasma membrane receptor for sperm from the sea urchin Strongylocentrotus purpuratus were cloned and sequenced. Analysis of the deduced primary structure suggests that the receptor is a transmembrane protein with a short cytoplasmic domain. This domain showed no sequence similarity to known protein sequences. In contrast, the extracellular, sperm binding domain of the receptor did show sequence similarity to the heat shock protein 70 (hsp70) family of proteins. Recombinant protein representing this portion of the receptor bound to the sperm protein, binding, and also inhibited fertilization in a species-specific manner; beads coated with the protein became specifically bound to acrosome-reacted sperm. These data provide a basis for detailed investigations of molecular interactions that occur in gamete recognition and egg activation.

Amino Acid Sequence↗
PubMed1993

The molecular basis of sea urchin gamete interactions at the egg plasma membrane.

Fertilization is the result of a series of well-choreographed interactions between molecules located on the surfaces of the egg and the sperm. The recent molecular characterizations of several of these surface molecules has led to a greater understanding of their roles in gamete recognition and binding. We present a review of these recent advances with emphasis on the sea urchin, a classic system for the study of fertilization. In particular, the structure and function of the sea urchin egg plasma membrane receptor for sperm, a novel cell recognition molecule, is discussed.

Amino Acid Sequence↗
PubMed1992

Identification of the sea urchin egg receptor for sperm using an antiserum raised against a fragment of its extracellular domain.

Sea urchin egg fertilization requires the species-specific interaction of molecules on the sperm and egg surfaces. Previously, we isolated an extracellular, 70-kD glycosylated fragment of the S. purpuratus egg receptor for sperm by treating the eggs with lysylendoproteinase C (Foltz, K. R., and W. J. Lennarz. 1990. J. Cell Biol. 111:2951-2959). To characterize the receptor further, we have generated a polyclonal antiserum (anti-70KL) against the purified 70-kD fragment. Anti-70KL was found to react with a single polypeptide of approximately 350 kD on Western blots, presumed to be the intact receptor, in an egg cell surface preparation. This polypeptide appeared to be tightly associated with the plasma membrane/vitelline layer complex, as it was released from these preparations only by detergent treatment. Immunofluorescence microscopy revealed that the receptor was distributed evenly over the egg surface. The anti-70KL was species specific both in its ability to recognize the egg surface protein and to inhibit sperm binding. Fab fragments generated from affinity-purified anti-70KL also bound to the egg surface and inhibited sperm binding in a concentration-dependent manner. Interestingly, treatment with Fabs caused a small percentage of eggs to undergo cortical granule exocytosis, even in the absence of external Ca2+. These results confirm earlier findings indicating that the receptor is a cell surface glycoprotein of high molecular weight that species specifically binds sperm. This antiserum provides a powerful tool for further investigation of gamete interactions and the structure of the sperm receptor.

Animals↗
PubMed1990

Molecular cloning and expression of sea urchin embryonic ciliary dynein beta heavy chain.

The determination of the structure and the expression of dynein during embryonic development are central to the understanding of dynein function. As an important first step toward these objectives, cDNAs encoding portions of sea urchin ciliary dynein were identified by antibody screening of a sea urchin cDNA expression library. Because of the complete lack of protein sequence data, it was first necessary to prove the identity of the dynein cDNAs. Of the five cDNA inserts initially cloned, one, designated P72A1, was characterized extensively. Four independent criteria demonstrated that P72A1 encoded a portion of a dynein heavy chain. (1) The beta-galactosidase-P72A1 fusion protein affinity-purified dynein-specific antibodies from crude antiserum. (2) Two other antisera to dynein, raised independently of the antiserum used to screen the cDNA library, reacted with the fusion protein. (3) A new antiserum raised against the fusion protein reacted with authentic dynein heavy chain on Western blots and stained embryonic cilia by indirect immunofluorescence microscopy. (4) Two new antisera, elicited against opposite ends of the P72A1 open reading frame, each reacted with authentic dynein heavy chain protein. Western blot analyses of dissociated dynein heavy chains revealed that P72A1 encoded a portion of the beta heavy chain. Epitope mapping experiments confirmed the identity of P72A1 as part of the beta heavy chain and also demonstrated that P72A1 encoded epitopes of the carboxyl-terminal fragment B domain of the dynein beta heavy chain. Northern blot analyses of poly(A)+ RNA revealed that P72A1 hybridized with a large RNA species ca. 12.5 kb in length. The dynein mRNA concentration increased during embryonic development. Dot blot analyses of RNA isolated at various times after embryo deciliation demonstrated that the dynein beta heavy chain mRNA accumulated rapidly in response to deciliation. The accumulation was similar to but not identical with the induction of tubulin mRNA in response to the same stimulus.

Adenosine Triphosphatases↗
PubMed1990

Purification and characterization of an extracellular fragment of the sea urchin egg receptor for sperm.

Fertilization in the sea urchin involves species-specific interaction between the ligand bindin on the surface of acrosome-reacted sperm and a receptor of high molecular weight on the surface of the egg. Efforts to understand this interaction and the resultant signal transduction events leading to egg activation have been limited because of the large size and extreme insolubility of the intact receptor on the egg surface. Earlier work suggested that an alternative strategy would be to isolate proteolytic fragments of the extracellular domain of this receptor. Consequently, we have treated S. purpuratus eggs with a specific protease, lysylendoproteinase C. This enzyme treatment abolished the ability of eggs to bind sperm and resulted in the release of proteolytic fragments that bound to sperm and showed inhibitory activity in a fertilization bioassay. One of these fragments, presumed to be a fragment of the extracellular domain of the receptor, was purified to homogeneity by gel filtration and anion exchange chromatography and shown to be a 70-kD glycosylated protein. Several lines of evidence support the contention that this fragment is derived from the receptor. First, the fragment inhibited fertilization species specifically. Second, species specific binding of the 70-kD glycoprotein to acrosome-reacted sperm was directly demonstrated by using 125I-labeled receptor fragment. Third, the fragment exhibited the same species specificity in binding to isolated bindin particles. Species specificity was abolished by Pronase digestion of the fragment. This observation supports the hypothesis that although binding is mediated by the carbohydrate moieties, species specificity is dependent on the polypeptide backbone. The availability of a structurally defined fragment of the receptor will facilitate further studies of the molecular basis of gamete interaction.

Animals↗
PubMed1988

Ionic strength-dependent isoforms of sea urchin egg dynein.

Unfertilized sea urchin eggs provide a reservoir of molecules which later are involved in microtubule-mediated movements during embryonic development. Among these molecules is egg dynein, which has been isolated in two forms, 20 S and 12 S. Evidence obtained previously from our laboratory indicates that 20 S dynein is a latent activity precursor of ciliary dynein. In contrast, others have suggested that 12 S egg dynein functions in the mitotic apparatus. It is therefore important to determine the relationship between these egg dyneins. Here we demonstrate that the sedimentation velocity of the egg dynein is dependent on the ionic strength of the extraction conditions. The 20 S dynein is obtained with low ionic strength extraction, and the 12 S form is obtained in high salt (0.6 M KCl). The 20 S dynein, after collection from a sucrose gradient, can be converted quantitatively to the 12 S form by exposure to salt, and this conversion can be followed over time. Further, the 20 S dynein can be converted entirely to 12 S dynein and then partially reconstituted to a faster sedimenting species. During these conversions, the dynein high Mr heavy chains are always coincident with the MgATPase activity, and antibodies show that the dynein heavy chains of the 20 S, 12 S, and converted species are indistinguishable immunologically. These data suggest that 12 S dynein is an ionic strength-dependent isoform of 20 S dynein that results from a partial dissociation of the 20 S polypeptide complex, similar to the relationship between 12 and 21 S sperm flagellar dynein. If the 20 and 12 S enzymes are isoforms of the same dynein, then there is compelling evidence for only a single dynein in the unfertilized egg, and that dynein is probably a ciliary precursor.

Adenosine Triphosphatases↗