Katherine I. Swenson

The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes

Fertilized clam embryos synthesize several new cell-cycle-related proteins. The cloned cDNA and derived amino acid sequences of one of these, cyclin A, are presented here. Immunoblots with an anti-cyclin A antibody reveal that cyclin A is undetectable in oocytes, appears within 15 min of fertilization, and is destroyed near the end of each meiosis and mitosis. We directly tested the ability of cyclin A to induce M phase by injecting SP6 cyclin A mRNA into Xenopus oocytes, which are arrested at the G2/M border of first meiosis. The injected mRNA was translated, with the result that the Xenopus oocytes entered meiosis. These findings indicate that the rise in cyclin A plays a direct and natural role in driving cells into M phase.

Formation of gap junctions by expression of connexins in Xenopus oocyte pairs

RNAs coding for connexins 32, 43, and the putative lens gap junction protein MP26 were tested for their ability to induce cell-cell coupling in Xenopus oocyte pairs. Large, voltage-insensitive conductances developed when connexin32 and 43 RNA-injected oocytes were paired both with themselves and with each other. Oocyte pairs injected with water manifested small conductances, which were symmetrically voltage-dependent. MP26 RNA-injected pairs displayed no conductances above control values. Unexpectedly, connexin43/water oocyte pairs developed high, asymmetrically voltage-dependent conductances, a property not displayed by the connexin32/water pairs. In single oocytes, these proteins remained intracellular until pairing, at which time the connexins, but not MP26, concentrated at the appositional areas.

Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to “myosin II” has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.

Three translationally regulated mRNAs are stored in the cytoplasm of clam oocytes

In situ hybridization was used to examine the spatial distributions of three translationally controlled maternal RNAs in oocytes and two-cell embryos of the clam Spisula. 3H-labeled single-stranded RNA probes were generated from SP6 recombinant clones containing DNA inserts encoding portions of histone H3 (the DNA sequence which is presented here), cyclin A, and the small subunit of ribonucleotide reductase. Hybridization of these probes to oocytes, in which the mRNAs are translationally inactive, shows that these mRNAs are stored in the cytoplasm. There is no evidence for sequestration of any of the RNAs within the nucleus or any other discrete structure. Instead they appear to be evenly distributed throughout the cytoplasm.