Biomedical subjects

D L Poccia

Publications and source records attributed to D L Poccia.

At least 19 recordsLinked to original sources

PubMed2001

Role for phosphatidylinositol in nuclear envelope formation.

PtdIns is a minor membrane phospholipid that is important in signal transduction. Recently, derivatives of PtdIns phosphorylated at the 3-position of the inositol ring have been implicated in the regulation of constitutive membrane traffic and in membrane fusion events. Assembly of the nuclear envelope (NE), a crucial step in the progress of mitosis, is also likely to involve membrane fusion reactions. We therefore investigated the role of PtdIns and phosphoinositide 3-kinase (PI-3K) activity in NE formation in vitro. GTP-induced NE formation was blocked by wortmannin and LY294002, two specific inhibitors of PI-3K, suggesting a role for PtdIns phosphorylated at the 3-position. PtdIns-specific phospholipase C mimicked GTP hydrolysis as an inducer of NE formation. This induction was dependent on a membrane vesicle subfraction (MV1) that was highly enriched in PtdIns, as determined by heteronuclear two-dimensional NMR spectroscopy. On the basis of these results, we suggest that the MV1 population serves as a source of membranes rich in PtdIns that might facilitate fusion, possibly through the production of the membrane-destabilizing lipid diacylglycerol.

Androstadienes↗

PubMed2001

Linker DNA destabilizes condensed chromatin.

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.

Animals↗

PubMed2000

Phospholipid identification and quantification of membrane vesicle subfractions by 31P-1H two-dimensional nuclear magnetic resonance.

An approach to the direct quantification of phospholipids from two-dimensional 31P-1H nuclear magnetic resonance (NMR) spectroscopy with isotropic proton mixing has been developed as a general method for phospholipid analysis of minor membrane vesicle subfractions. Membrane vesicles were subfractionated by sedimentation to density equilibrium in a sucrose gradient, and a modified Folch method was employed to extract their phospholipids. The coefficient for the NMR detection efficiency of each phospholipid and the relative mole percentage of the phospholipids present in the membrane vesicles were calculated. We demonstrate low detection limits such that relative concentrations of phospholipids in membrane subfractions may be determined even in the submicromolar range.

Animals↗

PubMed2000

Rearrangements of sea urchin egg cytoplasmic membrane domains at fertilization.

Fertilization in the sea urchin is accompanied by rapid reorganization of the egg endoplasmic reticulum (ER). ER-derived vesicles contribute to one of three classes of membranes used in assembling the male pronuclear envelope in vitro. We provide here biochemical evidence for the rearrangement of sea urchin egg cytoplasmic membrane domains at fertilization up to the first mitosis, with respect to two nuclear envelope markers, lamin B and lamin B receptor (LBR), using purified vesicles prepared from homogenates fractionated by floatation on sucrose gradients. In unfertilized eggs, immunoprecipitation data indicate that most of lamin B and LBR are localized in the same vesicles but do not interact. By 3 min post-fertilization, both proteins are more widely distributed across the gradients and by 12 min most of lamin B and LBR are localized in vesicles of different densities. This partitioning is maintained throughout S phase. At mitosis, most lamin B and LBR remain in distinct vesicles, while a small proportion of lamin B and LBR, likely derived from the disassembled nuclear envelope, associate in a minor subset of vesicles. The results illustrate a dynamic reorganization of egg cytoplasmic membranes at fertilization, and the establishment of distinct membrane domains enriched in specific nuclear envelope markers during the first cell cycle of sea urchin development. Additionally, we demonstrate that male pro-nuclear membrane assembly occurs only when both cytosol and membranes originate from fertilized but not unfertilized eggs, suggesting that fertilization-induced membrane rearrangements contribute to the ability of the egg to assemble the male pronuclear envelope.

Animals↗

PubMed1997

Protein kinase C-mediated interphase lamin B phosphorylation and solubilization.

Disassembly of the sperm nuclear envelope at fertilization is one of the earliest events in the development of the male pronucleus. We report that nuclear lamina disassembly in interphase sea urchin egg cytosol is a result of lamin B phosphorylation mediated by protein kinase C (PKC). Lamin B of permeabilized sea urchin sperm nuclei incubated in fertilized egg G1 phase cytosolic extract is phosphorylated within 1 min of incubation and solubilized prior to sperm chromatin decondensation. Phosphorylation is Ca2+-dependent. It is reversibly inhibited by the PKC-specific inhibitor chelerythrine, a PKC pseudosubstrate inhibitor peptide, and a PKC substrate peptide, but not by inhibitors of PKA, p34(cdc2) or calmodulin kinase II. Phosphorylation is inhibited by immunodepletion of cytosolic PKC and restored by addition of purified rat brain PKC. Sperm lamin B is a substrate for rat brain PKC in vitro, resulting in lamin B solubilization. Two-dimensional phosphopeptide maps of lamin B phosphorylated by the cytosolic kinase and by purified rat PKC are virtually identical. These data suggest that PKC is the major kinase required for interphase disassembly of the sperm lamina.

Animals↗

PubMed1996

Conserved binding recognition elements of sperm chromatin, sperm lipophilic structures and nuclear envelope precursor vesicles.

Detergent-resistant, lipophilic structures (LSs) at the apex and base of the conical sea urchin sperm nucleus are targets for cytoplasmic membrane vesicle (MV) binding and fuse with these vesicles to form a nuclear envelope in vitro. We report similar LSs associated with trout, frog and mammalian (mouse and bovine) sperm nuclei. The LSs are located at the implantation fossae of all species examined, as well as in the ventral hook region of mouse sperm nuclei. LSs can be removed from, and reconstituted back to, their original sites on nuclei. LS removal prevents MV binding, and reconstitution restores MV binding and GTP-induced fusion activities. Binding of LSs to chromatin or to MVs is mediated, at least in part, by proteins on each structure. Inter-specific LS-chromatin reconstitutions using sea urchin, fish, frog, and mammalian LSs indicate that site-specific binding is not dependent on species. All LSs also bind to MVs of sea urchin or bovine origin, but sea urchin MVs will only fuse with sea urchin LSs, and mammalian MVs only with mammalian LSs. These results demonstrate the conservation between echinoderms, fish, amphibians, and mammals of recognition elements for LS-chromatin and LS-MV binding. The mechanism for fusion of LSs with cytoplasmic MVs, however, is apparently not conserved, and close apposition of MVs on the chromatin surface mediated by LSs is not sufficient to permit MV-MV fusion in the presence of GTP.

Animals↗

PubMed1995

Formation of the sea urchin male pronucleus in vitro: membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling.

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of approximately 4 microns in diameter. Pronuclear swelling to approximately 7 microns requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca2+ and cytosolic factors, some of which are sensitive to heat and to the sulfhydryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations.

Adenosine Triphosphate↗

PubMed1995

Lamin dynamics during sea urchin male pronuclear formation in vitro.

The dynamics of lamin disassembly and reassembly during sea urchin male pronuclear development in vitro was investigated. Using five anti-lamin antibodies, we monitored by immunofluorescence and immunoblotting the changes in lamins during sperm chromatin decondensation, nuclear envelope (NE) formation, and male pronuclear swelling in fertilized sea urchin egg cytoplasmic extracts. We report the existence of five proteins in sperm nuclei and swollen male pronuclei (p49, p54, p65, p72, p84) which react with the antibodies. The sperm antigens resist membrane permeabilization with lysolecithin and 0.1% Triton X (TX)-100, but are removed from the lateral aspects of the nuclei by 1% TX-100. All five are completely removed from nuclei within 10 min of incubation in egg extracts. Initial chromatin decondensation and NE formation occur without reassembly of the putative lamins, but all lamins or lamin epitope-containing peptides assemble coordinately during pronuclear swelling promoted by adenosine 5'-triphosphate. Of the five pronuclear antigens, p49 and p54 appear to originate exclusively from the sperm. p65, p72, and p84 are also present in the egg cytoplasm and may be contributed to pronuclei by either source. Assembly of putative lamins and nuclear swelling, but not chromatin decondensation and nuclear envelope formation, are prevented in lamin-depleted cytoplasmic extracts. Our results indicate that p49 and p54 are not necessary for nuclear swelling, cytoplasmic p65 is required for swelling, and p72 and p84 are by themselves not sufficient but may be involved with p65 in nuclear swelling and full pronuclear development in vitro.

Animals↗

PubMed1995

Histone phosphorylation during sea urchin development.

Studies on histone phosphorylation during transitions in chromatin structure occurring in vivo during spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extracts in vitro and evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structure in vivo and provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.

Animals↗

PubMed1994

Dephosphorylation of sperm midpiece antigens initiates aster formation in rabbit oocytes.

During fertilization in most mammals, the penetrating sperm organizes an aster of microtubules. We have investigated the mechanisms underlying this function of the sperm by a series of experiments based on microinjection of isolated sperm midpieces into unfertilized oocytes. These midpieces contain antigens recognized by the MPM-2 antibody. These antigens, which are absent from the rest of the tail fraction, correspond to three phosphorylated polypeptides of 77, 81, and 85 kDa. Dephosphorylation with alkaline phosphatase abolishes antigenicity on blots and in whole sperm. Reactivity to the antibody disappears between 1 and 3 hr after calcium stimulation of oocytes, following the decline in H1 kinase activity and coincident with aster formation. In unactivated oocytes, no aster forms and the antigen remains unchanged. MPM-2 treatment of midpieces prior to injection blocks their ability to form asters in oocytes activated by calcium stimulation. The epitope also disappears in 6-methyl-aminopurine-treated oocytes, implying that maintenance of the phosphorylated state requires kinase activity. A result that confirms this view is that sperm midpieces dephosphorylated by alkaline phosphatase can be rephosphorylated after injection into oocytes or by exposure in vitro to a Xenopus oocyte cytoplasmic fraction high in H1 kinase activity. We suggest that the microtubule nucleation activity of sperm midpieces after fertilization is triggered by the calcium-induced decrease in maturation promoting factor, which results in dephosphorylation of specific sperm centrosomal proteins.

Animals↗

PubMed1994

Synthesis and processing of mammalian protamines and transition proteins.

Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [3H]-arginine, [3H]-histidine, [35S]-cysteine, or [32P]-PO4, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms.

Animals↗

PubMed1994

In vitro development of the sea urchin male pronucleus.

We have developed a cell-free extract from fertilized or unfertilized sea urchin eggs which promotes formation of male pronuclei from exogenously added permeabilized sperm nuclei. Using a buffer to simulate egg cytoplasmic conditions, three states of nuclear condensation can be distinguished: condensed (conical), partially decondensed (conical or ovoid), and decondensed (spherical). The in vitro system meets several in vivo criteria established by microinjection experiments. Decondensation is promoted at elevated pH and in activated egg cytoplasm, but does not require Ca2+. Pronuclear development is supported to > 100 male nuclei per egg-equivalents as in vivo. Pronuclear development requires addition of an ATP-generating system and is blocked by two kinase inhibitors (6-DMAP and staurosporine) at the same concentrations effective in vivo. Decondensed nuclei form by 40 min of incubation and acquire a putative nuclear envelope shown by exclusion of 150 kDa FITC-dextran by 1-2 hr. The rates of decondensation and nuclear envelope formation are accelerated by addition of GTP. Protease inhibition experiments suggest a role for nonhistone protein degradation in pronuclear progression. This system should prove useful for investigating mechanisms of the postmeiotic sea urchin male chromatin remodeling which follows fertilization, previously accessible only in vivo.

Adenine↗

PubMed1993

Phosphorylation weakens DNA binding by peptides containing multiple "SPKK" sequences.

Sea urchin testis-specific H1 and H2B histones (Sp H1 and Sp H2B) are characterized by reversibly phosphorylated N-terminal regions consisting largely of multiple clustered "SPKK" tetrapeptides (serine-proline adjacent to two basic amino acids). This report presents data showing differences in DNA affinities between peptides containing dephosphorylated and phosphorylated N-terminal regions. Sp H1 and its phosphorylated derivative (pSp H1) were purified by hydroxylapatite chromatography. Peptides containing the N-terminal regions of Sp H1 and pSp H1 (NP and pNP, respectively) were produced by digestion with Staphylococcus aureus protease. NP and two forms of pNP differing in phosphate content were purified by DNA-cellulose chromatography. The DNA affinities of the peptides were compared using several criteria. NP was bound more tightly by DNA-cellulose than pNPs. NP precipitated DNA under a broad range of NaCl concentrations; pNPs did not. Both NP and pNPs protected DNA against thermal denaturation, but NP created a more stable DNA-peptide complex. Thirty to sixty times more pNP than NP was required to obtain equivalent inhibition of Hoechst 33258 binding to DNA. NP did not behave as a competitive inhibitor of DNA binding by Hoechst 33258 binding to DNA. We conclude that during spermatogenesis, dephosphorylation of the Sp H1 N-terminal region increases its basicity and thus its affinity for DNA.

Amino Acid Sequence↗

PubMed1993

Two steps required for male pronucleus formation in the sea urchin egg.

We have developed a system for microinjection of sea urchin sperm nuclei into live eggs and oocytes in order to study conditions which are required for pronuclear development. The system bypasses sperm-initiated surface events that result in egg activation. By comparison to male nuclei of normally fertilized eggs or immature oocytes, the injected nuclei faithfully reflect the ability of the egg cytoplasm to promote male pronuclear development. Isolated nuclei injected into fertilized eggs swell and transform into male pronuclei like the endogenous male pronuclei. Nuclei injected into immature oocytes remain conical like fertilizing sperm nuclei. If sperm nuclei are injected into unfertilized, unactivated eggs, they undergo incomplete decondensation and fail to form pronuclei. This partial decondensation can be inhibited with 6-dimethylaminopurine (6-DMAP). If unfertilized eggs are first incompletely activated by raising internal pH with ammonia, injected sperm nuclei will complete pronuclear development. Increasing pH or Ca2+ does not cause sperm nuclear swelling or chromatin decondensation in immature oocytes. These experiments reveal two conditions necessary for pronuclear formation. The first condition, resulting in partial decondensation, develops during meiotic maturation and can be blocked by 6-DMAP. The second requires cytoplasmic alkalinization, but not the fertilization-induced internal Ca2+ increase or inositol triphosphate pathway activation.

Adenine↗

PubMed1992

Packaging and unpackaging the sea urchin sperm genome.

Two species of histones in sea urchin sperm (Sp H1 and Sp H2B) are chimeric molecules whose highly basic amino-terminal domains are dephosphorylated at the last stage of sperm cell differentiation, and rephosphorylated immediately following fertilization. The phosphorylated regions consist largely of repeating tetrapeptides with two basic residues flanking Ser-Pro residues ('SPKK' motifs) and are predicted to have beta-turn secondary structures. Alteration of the charge and structure of the SPKK sites may play a role in the unusually dense DNA packaging of the mature sperm chromatin. The motif resembles the target site of cell-cycle-associated cdc2 kinases and is found in several other proteins whose nucleic acid affinities may be altered during the cell cycle.

Amino Acid Sequence↗

PubMed1991

A complex pattern of H2A phosphorylation in the mouse testis.

Phosphorylation of H2A histones in mouse testis was examined using testis tubule cultures labeled with 32PO4. Histones were analyzed by two systems of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography of the gels. Of the 32PO4 detected in histones, 95% was incorporated by certain modified forms of the H2A variants H2A.1 and H2A.X. Phosphorylation sites were mapped to N- and C-terminal regions of the modified variants by SDS gel electrophoresis and autoradiography of peptides generated by cleavage of in vitro-labeled proteins with N-bromosuccinimide. Incorporation rates differed for N- and C-terminal regions from different modified forms, demonstrating a complex pattern of H2A phosphorylation in the mouse testis.

Animals↗

PubMed1990

Sperm histones and chromatin structure of the "primitive" sea urchin Eucidaris tribuloides.

The "primitive" sea urchin Eucidaris tribuloides resembles the advanced sea urchins (euechinoids) in many respects, yet some features of its biochemistry and morphogenesis are more similar to other echinoderms such as starfish or sea cucumbers. Two unique characteristics of the sperm chromatin of all known euechinoids are an extremely long average nucleosomal repeat length and the presence of two male germ-line-specific histone variants, Sp H1 and Sp H2B. Histone composition and nucleosomal repeat length of the sperm chromatin of Eucidaris were compared to those of several euechinoids and a starfish. Eucidaris sperm chromatin contained large H1 and H2B histone variants typical of euechinoids. The H1 was about nine amino acids smaller than Sp H1 of the advanced urchin Strongylocentrotus purpuratus. Its Sp H2B molecules were the same size as in the euechinoids. Peptide maps showed that N-terminal regions of Sp H1 and Sp H2B contained repeating basic amino acid motifs characteristic of euechinoids. The smaller size of Eucidaris H1 is accounted for by a smaller C-terminal region. The repeat length of Eucidaris sperm chromatin was slightly shorter than that of two euechinoids, but significantly larger than starfish, which lacks a large H2B. The Sp H2B gene of Eucidaris was expressed during spermatogenesis in the same cell types as for S. purpuratus. Thus Sp histone subtype expression and chromatin structure in this distantly related echinoid closely resemble the euechinoids. The presence of an Sp H2B and a very long repeat length appear to be characteristic of the echinoids only.

Amino Acid Sequence↗

PubMed1990

Phosphorylation of Plant H2A Histones.

Phosphorylation of wheat (Triticum aestivum) and alfalfa (Medicago sativa) H2A histone variants was examined during early seedling growth. The C-terminal regions of wheat H2A variants contain multiple S-P tetrapeptides (serine-proline adjacent to a pair of basic amino acids) which resemble known phosphorylation sites in histones from other species. Phosphorylation of nucleosomal core histones was assessed by autoradiography of proteins labeled in vivo with (32)Pi and resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylation sites were mapped by cleaving in vivo labeled H2A variants with N-bromosuccinimide. Essentially all phosphorylation of nucleosomal core histones in wheat and alfalfa seedlings occurred within the C-terminal peptides obtained from wheat and alfalfa H2A variants. A hypothesis accounting for the presence of large H2A and H2B histone variants in plants and phosphorylation of plant H2A C-terminal regions is proposed. The utility of S-P tetrapeptides for modulation of DNA-protein interactions is discussed.

Journal Article↗