Overall Research
Some of the most fascinating problems of biology concern spatial organization and temporal control. The embryo developing from a single egg generates a pattern of differentiation and growth that is closely controlled and, especially in vertebrate embryos, capable of remarkable regulation and recovery from injury. Many pathological conditions are the failure to regulate growth, control differentiation, or establish the proper morphological connections. In my laboratory we are concerned with three major problems of spatial and temporal control: control of the cell cycle, patterning of embryonic tissues, and control of cell morphology.
We started simple assays for the regulation of cell division in frog oocytes and embryos. But these assays, combined with an explosion of new genetic and biochemical information on the cell cycle, have quickly evolved into very specific biochemical investigations, applicable to all higher organisms. The two central players in cell division control are a set of unstable regulatory subunits called cyclins and a set of inactive catalytic subunits called cyclin-dependent kinases. Together, they govern the important cell cycle transitions that control DNA replication and mitosis. To study the activation and the inactivation of these kinases, we have developed in vitro systems using extracts from frog eggs. Activation and inactivation are under both external control by signals that regulate call proliferation and internal control by processes that maintain the fidelity of DNA replication and mitosis. Entry into mitosis is regulated not only by the accumulation of the appropriate cyclin but by kinases and phosphatases that we have studied.
The activation of DNA replication is poorly understood, but we have shown that it, too, is dependent on cyclins. Although more than one cyclin-dependent step may be required, we have begun to describe the downstream pathways by fractionating extracts that spontaneously synthesize DNA directly after mitosis. Exit from mitosis is regulated by cyclin degradation, which is initiated by the activation of a cyclin-dependent kinase. Cyclin degradation is of considerable interest, since the stability of the protein is under such exquisite control. Our data show that the regulated step is the covalent attachment of a protein called ubiquitin to the N-terminus of cyclin. The attachment is mediated by a 9 amino acid motif in the N-terminus of all mitotic cyclins. We have also shown that ubiquitination of another set of cyclins may be important in the activation of DNA replication. We would like to know whether regulated degradation is used elsewhere in developing embryos and to identify the mechanism of recognition.
As embryos develop the regulation of cell division and cell differentiation becomes increasingly reliant on intercellular communication, rather than on cell-autonomous mechanisms. In vertebrate embryos intercellular signals organize the body axis of the embryo on the dorsal side, including the neural tube, somites, and notochord. We have delineated the role of one of the signaling molecules, fibroblast growth factor (FGF), in frog embryonic development by expressing dominant inhibitors comprising the extracellular domain of the FGF receptor. FGF interacts with other signaling pathways, and we are attempting to understand how these signals lead to a well proportioned elaboration of embryonic tissues. In addition, we are looking for other ligand-receptor pairs in the embryo. The establishment of the body plan in the trunk of the embryo is recapitulated in the tadpole tail. But, in the tail, the pattern of cell division and cell movements is very different from that in the trunk. We hope, by studying cell specification in the tail, to determine the signals and responses most critical for pattern formation.
One of the most refractory and important problems in cell biology is spatial organization of cells, which is manifest embryonically in the organization of the egg and somatically in the differentiation and mitotic division of all cell types. We have focused our attention on the organization of the cytoskeleton, principally microtubules. Microtubules are particularly important in the mitotic spindle and in elongating cellular processes, especially those of neurons. We have been interested in the control of microtubule dynamics, the nucleation of microtubules from centrosomes in mammalian cells, and the role of microtubules in morphogenetic processes, such as axon growth and cell movement. Using low-light video microscopy of a fluorescently tagged subunit of microtubules in neurons, we have shown that the placement of microtubules is a critical early step in the choice of direction in nerve cell growth. We are determining the signals outside the cell to which microtubules respond as well as the intervening biochemical steps that result in microtubule polymerization or stabilization, and ultimately, polarized cell growth. A combination of real-time visual observation of cell biological mechanisms with reconstruction of functions by biochemical assays in cell-free systems, I believe, offers the best opportunity for understanding these difficult problems of cell biology.