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D G Capco

Publications and source records attributed to D G Capco.

At least 37 records · Page 2Linked to original sources
PubMed1992

Role of the cytoskeleton during early development.

Oocytes, eggs, and embryos from a diverse array of species have evolved cytoskeletal specializations which allow them to meet the needs of early embryogenesis. While each species studied possesses one or more specializations which are unique, several cytoskeletal features are widely conserved across different animal phyla. These features include highly-developed cortical cytoskeletal domains associated with developmental information, microtubule-mediated pronuclear transport, and rapid intracellular signal-regulated control of cytoskeletal organization.

Animals↗
PubMed1991

Parallel pathways of cell cycle control during Xenopus egg activation.

Transit from M phase into interphase in many eukaryotic cells is preceded by an increase in intracellular free calcium ([Ca2+]i), which may act via calcium-dependent enzymes to trigger the M-phase/interphase transition. To test the role of the calcium- and phospholipid-dependent enzyme protein kinase C (PKC) in the M-phase/interphase transition, PKC was activated in M-phase-arrested Xenopus eggs by treatment with the phorbol ester phorbol 12-myristate 13-acetate under conditions that prevent a rise in [Ca2+]i and activation of other calcium-dependent enzymes. Under these conditions, several cellular events characteristic of transit into interphase occur: sperm chromatin decondenses, the Golgi and the nuclear envelope reassemble, and endocytosis resumes. These events are also triggered by treatment of eggs with the diacylglycerol 1,2-dioctanoyl-sn-glycerol. Surprisingly, the activity of M-phase-promoting factor (MPF), a universal regulator of M phase, remains high under these conditions. If [Ca2+]i is subsequently raised, MPF activity is rapidly destroyed. Similarly, lysates made from eggs treated with phorbol 12-myristate 13-acetate support sperm chromatin decondensation in vitro and yet retain high MPF activity, measured either as the ability to induce meiotic resumption in oocytes or as histone H1 kinase activity. These effects are not triggered by the 4 alpha-phorbol ester isomer, which does not activate PKC, and are sensitive to the PKC "pseudosubstrate" peptide. The results suggest that two, parallel signals are generated by the rise in [Ca2+]i both of which contribute to cell cycle regulation. One pathway inactivates MPF; the other pathway activates PKC.

Animals↗
PubMed1991

Cytoskeleton of the mouse egg and embryo: reorganization of planar elements.

Examination of detergent-extracted mouse eggs and embryos reveals the existence of two cytoskeletal networks. One network is the typical thin filament network observed in somatic cells while the other is composed of large planar elements. These latter cytoskeletal structures, with individual widths of 60.0 +/- 6.8 nm, alter their spatial organization in a developmental stage-specific manner. The planar elements are composed of filaments with a diameter of 10 nm aligned side-by-side with these filaments exhibiting a linear periodicity of 20.0 +/- 1.6 nm. A biochemical fraction containing components of the planar elements has been prepared from different stages of development and disappearance of prominent polypeptides from this fraction correlates with the altered spatial organization of the planar elements. Ultrastructure and biochemistry of cytoskeletal planar elements in eggs and embryos of the mouse are comparable with cytoskeletal sheets of Syrian hamster eggs and embryos, suggesting these cytoskeletal components may have a functional role in mammalian embryogenesis. Because such structures have not been identified in eggs or embryos of species other than mammals, their function may be unique to mammalian embryogenesis.

Animals↗
PubMed1991

Analysis of inducible contractile rings suggests a role for protein kinase C in embryonic cytokinesis and wound healing.

A semi-in vitro system derived from Xenopus oocytes which allows induction of contractile ring (CR) formation and closure is described and exploited to elucidate regulatory and structural features of cytokinesis. The inducible CRs (ICRs) are composed of actin filaments and closure is actin filament-dependent as is cytokinesis in vivo. ICR closure in this system is calcium-dependent and pH-sensitive, as is cytokinesis in permeabilized cells (Cande: Journal of Cell Biology 87:326, 1980). Closure of ICRs proceeds at a rate and with a kinetic pattern similar to embryonic cytokinesis. Collectively, these data demonstrate that this system is a faithful mimic of cytokinesis in vivo. ICR formation and closure is protein kinase C (PKC)-dependent and neomycin-sensitive, indicating that the PKC branch of the polyphosphoinositide pathway regulates formation of the actomyosin ring which is the effector of cytokinesis. Kinetic measurements show that the rate of ICR closure reaches a peak of 4-8 microns/sec. Since the maximum measured velocity of actin filament translocation by vertebrate, non-muscle myosins is 0.04 micron/sec, the later observations support a model in which the CR is segmented, containing multiple sites where filaments overlap in a "sliding filament" fashion. Because the rate decreases after reaching a peak, the results also suggest that the number of overlap sites decrease with time.

Alkaloids↗
PubMed1991

Cortical membrane-trafficking during the meiotic resumption of Xenopus laevis oocytes.

Changes in the organization of membranous structures in the amphibian oocyte cortex were studied during the process of progesterone-induced meiotic resumption. Progesterone treatment of Xenopus laevis oocytes induced short term and longer term changes in the cortical membranes. In the short term, progesterone induced a burst of endocytosis mediated through coated pits and coated vesicles. Immuno-electron-microscopic localization of progesterone suggested that the progesterone receptor, bound to its ligand, is endocytosed during progesterone-induced endocytosis. Also demonstrated was the existence of a cisternal membrane network, referred to as the primordial cortical endoplasmic reticulum, which surrounds portions of the cortical granules in oocytes. The primordial cortical endoplasmic reticulum is more highly developed in the animal hemisphere than the vegetal hemisphere. Over the long term, during the meiotic resumption, more membrane is recruited into this network to form the cortical endoplasmic reticulum observed by others in the metaphase II egg. This evidence demonstrates that the cortex serves as a site for dynamic changes in membrane organization and that the most extensive changes occur in the animal hemisphere. These data support previous observations that the animal hemisphere is better structured for sperm penetration than is the vegetal hemisphere.

Animals↗
PubMed1990

Transformation of the amphibian oocyte into the egg: structural and biochemical events.

Amphibian oocytes, arrested in prophase I, are stimulated to progress to metaphase II by progesterone. This process is referred to as meiotic maturation and transforms the oocyte, which cannot support the early events of embryogenesis, into the egg, which can. Meiotic maturation entails global reorganization of cell ultrastructure: In the cell cortex, the plasma membrane flattens and the cortical granules undergo redistribution. In the cell periphery, the annulate lamellae disassemble and the mitochondria become dispersed. In the cell interior, the germinal vesicle becomes disassembled and the meiotic spindles form. Marked changes in the cytoskeleton and mRNA distribution also occur throughout the cell. All of these events are temporally correlated with intracellular signalling events: Fluctuations in cAMP levels, changes in pH, phosphorylation and dephosphorylation, and ion flux changes. Evidence suggests that specific intracellular signals are responsible for specific reorganizations of ultrastructure and mRNA distribution.

Animals↗
PubMed1990

Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Nonspecific effects of oligodeoxynucleotide injection in Xenopus oocytes: a reevaluation of previous D7 mRNA ablation experiments.

Microinjection of oligodeoxynucleotides (ODNs) complementary to cellular mRNAs has been advanced as an experimental approach to degrade target mRNAs in vivo and thereby obtain information as to the function of their cognate proteins. It is shown here that ODNs can induce a variety of aberrations in cell metabolism and structure when injected into Xenopus oocytes. Examination of histological sections of ODN-injected oocytes revealed the frequent abnormal accumulation of heavily staining basophilic material in the area of the germinal vesicle (gv). Ultrastructural analysis detected further abnormalities including blebbing of the plasma membrane, anomalous cytoskeletal structures, hyperorganised annulate lamellae, hyperinvagination of the gv, and formation of irregular nucleoli within the gv. Analysis of newly synthesised proteins by [35S]methionine radiolabelling of oocytes demonstrated that ODN injection can trigger a general decrease in both label uptake and protein synthesis. Qualitative effects on protein synthesis could also be observed, particularly a decrease in synthesis of high molecular weight proteins. The severity of ODN-induced effects is dose-dependent and highly variable from ODN to ODN. The previously reported delay in progesterone-induced maturation observed in oocytes depleted of the maternal mRNA D7 by ODN-directed degradation (Smith R. C., Dworkin M. B. and Dworkin-Rastl E. (1988) Genes and Devpt. 2, 1296-1306) is most likely a result of nonspecific ODN effects in the oocyte. Oocytes injected with effective antisense D7 ODNs that do not display detectable side effects matured with normal kinetics.

Animals↗
PubMed1990

Synthesis, assembly and organization of the cytoskeleton during early amphibian development.

All eukaryotic cells undergo changes in three-dimensional organization which are controlled by the cytoskeleton. Early embryos execute cytoskeletal-mediated changes in a rapid and dramatic fashion and are therefore useful cytoskeletal model systems. This review highlights similarities and differences between somatic cell cytoskeletons and those of amphibian oocytes, eggs and zygotes.

Animals↗
PubMed1989

Specialized cytoskeletal elements in mammalian eggs: structural and biochemical evidence for their composition.

Mammalian eggs and embryos contain an extensive detergent-resistant cytoskeletal network, including many elements which have been referred to as sheets in hamster eggs. In this study we examined the structure of the sheet-like components by using embedment-free sections and freeze-fracture electron microscopy and found that the sheets are composed of both filamentous and particulate components. In addition, exposure to a high salt extraction medium resulted in the disappearance of the sheets at the ultrastructural level. SDS-polyacrylamide gel electrophoresis of the cell fractions revealed four stainable proteins solubilized by the high salt extraction with one of the proteins being greatly enriched. Because these cytoskeletal sheets undergo an extensive reorganization coincident with key events during early development they serve as internal markers for the establishment of polarity and subsequent differentiation of the first embryonic epithelium, the trophectoderm.

Animals↗
PubMed1989

Intracellular signals trigger ultrastructural events characteristic of meiotic maturation in oocytes of Xenopus laevis.

Oocytes of Xenopus laevis were treated with agents which induce individual intracellular signals normally evoked during the process of meiotic maturation. Ultrastructural analysis of these oocytes allowed identification of specific second messengers that individually trigger single ultrastructural changes characteristic of the meiotic maturation process: Manipulation of intracellular cAMP levels induced changes in cortical granule position. Cytoplasmic alkalinization triggered a disruption of the annulate lamellae, a specialized organelle in the periphery of oocytes. Activation of protein kinase C caused rapid formation of a cortical endoplasmic reticulum and subsequent disruption of cortical granules. Manipulation of transmembrane calcium flux had varied results dependent upon the agent employed. Two of the treatments, Verapamil and zero external calcium, induced a reorganization in the oocyte periphery. The results indicate that these ultrastructural events are under the control of specific intracellular signals known to be elicited during meiotic maturation.

Animals↗
PubMed1989

Activators of protein kinase C trigger cortical granule exocytosis, cortical contraction, and cleavage furrow formation in Xenopus laevis oocytes and eggs.

Prophase I oocytes, free of follicle cells, and metaphase II eggs of the amphibian Xenopus laevis were subjected to transient treatments with the protein kinase C activators, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 1-olyeoyl-2-acetyl-sn-glycerol. In both oocytes and eggs, these treatments triggered early events of amphibian development: cortical granule exocytosis, cortical contraction, and cleavage furrow formation. Surprisingly, activation of oocytes occurred in the absence of meiotic resumption, resulting in cells with an oocytelike nucleus and interior cytoplasm, but with a zygotelike cortex. PMA-induced activation of oocytes and eggs did not require external calcium, a prerequisite for normal activation of eggs. PMA-induced activation of eggs was inhibited by retinoic acid, a known inhibitor of protein kinase C. In addition, pretreatment of eggs with retinoic acid prevented activation by mechanical stimulation and inhibited activation by calcium ionophore A23187. The results suggest that protein kinase C activation is an integral component of the Xenopus fertilization pathway.

Animals↗
PubMed1988

Analysis of proteins in the peripheral and central regions of amphibian oocytes and eggs.

Proteins in oocytes, meiotically mature eggs and zygotes of Xenopus laevis were examined to determine whether proteins in the peripheral region differ from those in the central region of these cells. We show that different regions contain different amounts of newly synthesized proteins and that during meiotic maturation and fertilization the periphery of the animal hemisphere becomes the site where most newly synthesized proteins are found. Examination of two-dimensional gels indicates that most of these proteins are found in all parts of the egg, but certain proteins demonstrate patterns of distribution which are indicative of (1) polarity, (2) developmental stage, and (3) the position within the hemisphere (central or peripheral). These results suggest that the periphery of oocytes, eggs, and zygotes is a site of greater metabolic activity compared with the central region.

Animals↗
PubMed1988

Spatial reorganization of actin, tubulin and histone mRNAs during meiotic maturation and fertilization in Xenopus oocytes.

The distribution of actin, tubulin and histone mRNAs is examined in full grown oocytes, meiotically mature eggs, and unicellular zygotes. For this analysis, oocytes, eggs and embryos were spatially divided into peripheral and central regions of both the animal and vegetal hemispheres, and the relative amounts and concentrations of these RNAs in each region were then determined. The concentration of actin and tubulin mRNAs is greatest in the periphery, whereas histone mRNA exhibits a uniform concentration throughout the oocyte. In the meiotically mature egg, actin mRNA is still concentrated in the periphery and histone mRNA still exhibits a relatively uniform concentration, but the tubulin mRNAs are more concentrated in the central regions. Following fertilization, however, the greatest concentration of mRNAs for actin, histone and tubulin is in the periphery of the zygote. The results demonstrate the existence of a system capable of altering the distributions of these mRNAs as well as a system which distinguishes between different types of mRNA.

Actins↗
PubMed1987

A method for analysis of the detergent-resistant cytoskeleton of cells within organs.

We describe a method for the preparation of the detergent-resistant cytoskeleton and nuclear matrix of cells within organs and tissues. Such cells were previously inaccessible to study because the three-dimensional organization of cells in organs prevented uniform distribution of the detergent throughout the multiple cell layers. We use the method presented here to compare the proteins present in the cytoskeleton, nuclear matrix and soluble fractions of cells from different histotypes. SDS-gel analysis demonstrates that soluble and nuclear matrix proteins differ greatly between histotypes while cytoskeletal proteins are relatively similar. Immunocytochemical analysis of tissue prepared using this procedure also demonstrates that the intracellular structure of cells within organs differs from that of in vitro cultured cells.

Animals↗
PubMed1986

Cytoskeletal reorganization during early mammalian development: analysis using embedment-free sections.

We have examined cytoskeletal reorganization during early embryonic development in the hamster by employing detergent extraction to remove soluble components of the embryos and reveal the underlying structural network. This procedure allows examination of both the cortical cytoskeleton and the cytoskeleton of the egg interior. Sections of eggs and embryos were prepared for transmission electron microscopy with the removable embedding medium, diethylene glycol disterate which allows thicker sections than conventional embedment procedures thereby providing more spatial cues for studying organization. The cytoskeleton reorganizes after fertilization, at the time of compaction and again at the blastocyst stage. These cytoskeletal reorganizations are considered in terms of the blastomere polarity hypothesis and the involvement of the cytoskeleton with early embryonic development.

Animals↗
PubMed1985

Shape-dependent regulation of cytoskeletal protein synthesis in anchorage-dependent and anchorage-independent cells.

We examine changes in protein synthesis that accompany suspension (i.e. shape alteration) of anchorage-dependent and anchorage-independent cells using a newly developed cell fractionation procedure based on detergent extraction. Using this procedure, a cell can be divided into four distinct and independent fractions: soluble, cytoskeleton, chromatin and nuclear matrix-intermediate filament. This fractionation procedure is used to investigate protein synthetic events associated with the release from anchorage-dependent growth, characteristic of transformed cells. Suspension results in several unexpected events in both anchorage-dependent (3T3) cells and anchorage-independent (SVPy 3T3) cells. Suspension of 3T3 cells results in a reduction of total protein synthesis; however, two proteins are enhanced in their amount of synthesis. Suspension of SVPy 3T3 results in about 20 proteins proceeding through short and long-term alterations in the rate of synthesis. The synthesis of some of these proteins is inhibited, others undergo a transient decrease in synthesis upon suspension and then increase above their rate in the anchored state, while the synthesis of others steadily increases after suspension. Suspension of anchorage-dependent cells results in a fraction specific shift in the rates of protein synthesis. Possible roles for these fraction-specific proteins are considered.

Animals↗