Rapid Increase in Plasma Membrane Chloride Permeability during Wound Resealing in Starfish Oocytes

Plasma membrane wound repair is an important but poorly understood process. We used femtosecond pulses from a Ti-Sapphire laser to make multiphoton excitation–induced disruptions of the plasma membrane while monitoring the membrane potential and resistance. We observed two types of wounds that depolarized the plasma membrane. At threshold light levels, the membrane potential and resistance returned to prewound values within seconds; these wounds were not easily observed by light microscopy and resealed in the absence of extracellular Ca2+. Higher light intensities create wounds that are easily visible by light microscopy and require extracellular Ca2+ to reseal. Within a few seconds the membrane resistance is ∼100-fold lower, while the membrane potential has depolarized from −80 to −30 mV and is now sensitive to the Cl− concentration but not to that of Na+, K+, or H+. We suggest that the chloride sensitivity of the membrane potential, after wound resealing, is due to the fusion of chloride-permeable intracellular membranes with the plasma membrane

Immunolocalization of the Heterotrimeric Kinesin-Related Protein KRP(85/95) in the Mitotic Apparatus of Sea Urchin Embryos

AbstractWe have used monoclonal antibodies to perform confocal light microscopic immunolocalization of KRP(85/95), a heterotrimeric plus-end-directed microtubule motor protein, in dividing cells of sea urchin embryos. Embryos were stained during the first division cycle, and dissociated blastomeres were stained at the 32- to 64-cell stages. Double labeling of the dividing cells with anti-tubulin and anti-KRP(85/95) showed a clear concentration of the motor protein in the mitotic apparatus; KRP(85/95) appeared to associate with pericentriolar regions during prophase, with kinetochore-to-pole microtubules during metaphase, and, in a striking fashion, with the spindle interzone during anaphase. KRP(85/95) began to accumulate in the interzone immediately following chromosome separation and the area of concentration expanded with the lengthening of the interzonal region during anaphase. During telophase KRP(85/95) appeared to disperse with the establishment of the cleavage furrow and did not concentrate in the midbody. KRP(85/95) staining in the mitotic apparatus was punctate and detergent-sensitive, suggesting an association with membranous vesicles, but unlike kinesin, KRP(85/95) did not appear to codistribute with calsequestrin-containing endoplasmic reticulum. Finally, KRP(85/95) appears to be present in dividing blastomeres up to at least the blastula stage, but, unlike kinesin, it is not expressed in terminally differentiated, nonmitotic coelomocytes of the adult animal. These results suggest that the expression and targeting of KRP(85/95) and kinesin differ and that KRP(85/95) may play a role in vesicle transport during embryonic cell division

Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes

Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to ∼40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to ∼100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina

Identification of PLCγ-Dependent and -Independent Events during Fertilization of Sea Urchin Eggs

AbstractAt 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 Cγ (PLCγ), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCγ as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCγ 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 PLCγ 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 PLCγSH2 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 PLCγSH2 domains was further indicated by the finding that microinjection of PLCγSH2 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 PLCγSH2 fusion protein. These results indicate that PLCγ 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 PLCγ activation or Ca2+increase

Enantiomerically enriched, polycrystalline molecular sieves

Zeolite and zeolite-like molecular sieves are being used in a large number of applications such as adsorption and catalysis. Achievement of the long-standing goal of creating a chiral, polycrystalline molecular sieve with bulk enantioenrichment would enable these materials to perform enantioselective functions. Here, we report the synthesis of enantiomerically enriched samples of a molecular sieve. Enantiopure organic structure directing agents are designed with the assistance of computational methods and used to synthesize enantioenriched, polycrystalline molecular sieve samples of either enantiomer. Computational results correctly predicted which enantiomer is obtained, and enantiomeric enrichment is proven by high-resolution transmission electron microscopy. The enantioenriched and racemic samples of the molecular sieves are tested as adsorbents and heterogeneous catalysts. The enantioenriched molecular sieves show enantioselectivity for the ring opening reaction of epoxides and enantioselective adsorption of 2-butanol (the R enantiomer of the molecular sieve shows opposite and approximately equal enantioselectivity compared with the S enantiomer of the molecular sieve, whereas the racemic sample of the molecular sieve shows no enantioselectivity)

Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution

We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called “dorsal.” On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other.</p