Cyclin E in centrosome duplication and reduplication in sea urchin zygotes.

When protein synthesis is completely blocked from before fertilization, the sea urchin zygote arrests in first S phase and the paternal centrosome reduplicates multiple times. However, when protein synthesis is blocked starting in prophase of first mitosis, the zygote divides and the blastomeres arrest in a G1-like state. The centrosome inherited from this mitosis duplicates only once in each blastomere for reasons that are not understood. The late G1 rise in cyclin E/cdk2 kinase activity initiates centrosome duplication in mammalian cells and its activity is needed for centrosome duplication in Xenopus egg extracts. Since the half-time for cyclin E turnover is normally approximately 1 h in sea urchin zygotes, the different behaviors of centrosomes during G1 and S phase arrests could be due to differential losses of cyclin E and its associated kinase activities at these two arrest points. To better understand the mechanisms that limit centrosome duplication, we characterize the levels of cyclin E and its associated kinase activity at the S phase and G1 arrest points. We first demonstrate that cyclin E/cdk2 kinase activity is required for centrosome duplication and reduplication in sea urchin zygotes. Next we find that cyclin E levels and cyclin E/cdk2 kinase activities are both constitutively and equivalently elevated during both the S phase and G1 arrests. This indicates that centrosome duplication during the G1 arrest is limited by a block to reduplication under conditions permissive for duplication. The cytoplasmic conditions of S phase, however, abrogate this block to reduplication.

Characterization of a panel of six beta2-adrenergic receptor antibodies by indirect immunofluorescence microscopy.

BACKGROUND: The beta2-adrenergic receptor (beta2AR) is a primary target for medications used to treat asthma. Due to the low abundance of beta2AR, very few studies have reported its localization in tissues. However, the intracellular location of beta2AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to beta-agonist medications. Thus, a method for visualizing beta2AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant beta2AR in primary cell cultures. METHODS: A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat beta2AR expressed in HEK 293 cells. Antibodies capable of recognizing rat beta2AR were identified and used to localize native beta2AR in primary cultures of rat airway smooth muscle and epithelial cells. beta2AR expression was confirmed by performing ligand binding assays using the beta-adrenergic antagonist [3H] dihydroalprenolol ([3H]DHA). RESULTS: Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human beta2AR (Ab-Bethyl) specifically recognized human but not rat beta2AR. An antibody developed against the C-terminal domain of the mouse beta2AR (Ab-sc570) specifically recognized rat but not human beta2AR. An antibody developed against 78 amino acids of the C-terminus of the human beta2AR (Ab-13989) was capable of recognizing both rat and human beta2ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat beta2AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface. CONCLUSION: Antibodies have been identified that recognize human beta2AR, rat beta2AR or both rat and human beta2AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of beta2AR in tissues.

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos.

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.

The beta-agonist isoproterenol attenuates EGF-stimulated wound closure in human airway epithelial cells.

Asthma is a disease characterized by reversible airway obstruction. An additional hallmark of chronic asthma is altered wound healing that leads to airway remodeling. Although beta-agonists are effective in treating the bronchospasm associated with asthma, their effects on airway wound healing, which are related to airway remodeling, are unknown. It has been demonstrated that beta-agonists can alter the signaling of epidermal growth factor (EGF) receptors, which are important in timely wound healing. Therefore, we hypothesized that the beta-agonist isoproterenol would affect wound healing. Using an in vitro scrape wound assay, we demonstrated that isoproterenol attenuates EGF-stimulated wound healing in 16HBE airway epithelial cell cultures. Through experiments with forskolin and cells overexpressing beta2-adrenergic receptor-yellow fluorescent protein, we show that attenuation is due to the accumulation of cAMP and the involvement of at least one additional pathway. Furthermore, attenuation is not due to a direct effect on the EGF receptor or to an alteration of the ERK/MAPK signaling cascade. Based on these results, we propose that isoproterenol may exert its effects through other MAPK signaling pathways (JNK and/or p38) or through parallel mechanisms. These results also demonstrate a problem of potential therapeutic relevance in which a commonly prescribed medication may alter wound healing and contribute to the remodeling of asthmatic airways.

Coordinating epidermal growth factor-induced motility promotes efficient wound closure.

Wound healing is a response to injury that is initiated to reconstruct damaged tissue. In skin, reepithelialization involves both epithelial cells and fibroblasts and contributes to the reformation of a barrier between the external environment and internal milieu. Growth factors including epidermal growth factor (EGF) play important roles in promoting this process. In the present studies we employed CV-1 fibroblasts in a tissue culture model of reepithelialization to develop strategies for optimizing wound closure stimulated by EGF. We found that EGF enhanced cell motility within 6-8 h of EGF treatment in serum-free medium but wounds failed to close within 24 h. However, if medium on these cultures was exchanged for medium containing serum, cells pretreated with EGF closed new scrape wounds more rapidly than did cells that were not pretreated. These results indicate that serum factors work in concert with EGF to coordinate cell motility for efficient wound closure. Indeed, EGF enhanced the rate of wound closure in the presence of serum, and this effect also persisted for at least 24 h after EGF was removed. This coordination of EGF-induced cell motility was accompanied by an increase in the transient phosphorylation of ERK1 and ERK2. The persistent effects of EGF were blocked by transient exposure to reversible inhibitors of transcription and translation, indicating that the expression of new proteins mediated this response. We propose that EGF-stimulated CV-1 fibroblast motility is coordinated by a serum component that induces cell-cell adhesive properties consistent with an epithelial phenotype, thereby enhancing the reepithelialization process.

The sea urchin stem-loop-binding protein: a maternally expressed protein that probably functions in expression of multiple classes of histone mRNA.

Following the completion of oogenesis and oocyte maturation, histone mRNAs are synthesized and stored in the sea urchin egg pronucleus. Histone mRNAs are the only mRNAs that are not polyadenylated but instead end in a stem-loop which has been conserved in evolution. The 3' end binds the stem-loop-binding protein (SLBP), and SLBP is required for histone pre-mRNA processing as well as translation of the histone mRNAs. A cDNA encoding a 59 kDa sea urchin SLBP (suSLBP) has been cloned from an oocyte cDNA library. The suSLBP contains an RNA-binding domain that is similar to the RNA-binding domain found in SLBPs from other species, although there is no similarity between the rest of the suSLBP and other SLBPs. The suSLBP is present at constant levels in eggs and for the first 12 h of development. The levels of suSLBP then decline and remain at a low level for the rest of embryogenesis. The suSLBP is concentrated in the egg pronucleus and is released from the nucleus only when cells enter the first mitosis. SuSLBP expressed by in vitro translation does not bind the stem-loop RNA, suggesting that suSLBP is modified to activate RNA binding in sea urchin embryos.

Cyclin D and cdk4 are required for normal development beyond the blastula stage in sea urchin embryos.

cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle.

Novel localization and possible functions of cyclin E in early sea urchin development.

In somatic cells, cyclin E-cdk2 activity oscillates during the cell cycle and is required for the regulation of the G1/S transition. Cyclin E and its associated kinase activity remain constant throughout early sea urchin embryogenesis, consistent with reports from studies using several other embryonic systems. Here we have expanded these studies and show that cyclin E rapidly and selectively enters the sperm head after fertilization and remains concentrated in the male pronucleus until pronuclear fusion, at which time it disperses throughout the zygotic nucleus. We also show that cyclin E is not concentrated at the centrosomes but is associated with condensed chromosomes throughout mitosis for at least the first four cell cycles. Isolated mitotic spindles are enriched for cyclin E and cdk2, which are localized to the chromosomes. The chromosomal cyclin E is associated with active kinase during mitosis. We propose that cyclin E may play a role in the remodeling of the sperm head and re-licensing of the paternal genome after fertilization. Furthermore, cyclin E does not need to be degraded or dissociated from the chromosomes during mitosis; instead, it may be required on chromosomes during mitosis to immediately initiate the next round of DNA replication.