The research goal of our laboratory is to understand intracellular morphogenesis at a molecular level; in particular the complex events that underlie cell division. Our major focus is the mitotic spindle, the dynamic microtubule-based machine essential for the correct distribution of chromosomes to each daughter cell. We would like to understand how the spindle forms and functions, and how its size is determined. We apply diverse and interdisciplinary techniques, including an in vitro celluar extract system, as well as chemical, proteomic and biophysical approaches.
Developing simplified assays to study spindle assembly. Utilizing extracts prepared from eggs of the African frog Xenopus laevis and "artificial chromosomes" consisting of plasmid DNA-coated magnetic beads, we can generate bipolar spindles in vitro in the absence of focal microtubule nucleation centers (centrosomes) or specialized microtubule-chromosome attachment sites (kinetochores). DNA on the beads assembles into chromatin that is sufficient to induce microtubule polymerization and organization, but the mechanisms behind this process are unclear. We are using a molecular reconstitution approach to evaluate the roles of candidate chromatin factors by coupling them to beads individually and in combination, and in the long term, aim to reconstitute the spindle from pure components.
Elucidating the role of Ran. In collaboration with the laboratory of Karsten Weis, we are studying one pathway important for spindle assembly that depends on the small GTPase Ran. Analogous to its role in interphase nucleocytoplasmic transport, RanGTP generated by the chromatin-bound guanine nucleotide exchange factor RCC1 in mitosis functions to locally discharge cargoes from transport factors in the vicinity of chromosomes that promote spindle assembly. We have used fluorescence energy transfer (FRET) probes to demonstrate a physical gradient of RanGTP and a released cargo surrounding mitotic chromosomes, and are identifying and functionally characterizing the many downstream effectors of this pathway, as well as a small molecule inhibitor we can use to dissect its functions in living cells.
Mechanisms of intracellular scaling. We are investigating the question of how cells determine the size of their constituent structures. We compare Xenopus laevis and the related, smaller frog Xenopus tropicalis, which possesses smaller eggs, spindles and nuclei. Mixing egg extracts from the two frogs together has revealed a dynamic, dose-dependent regulation of spindle and nuclear size by cytoplasmic factors, which is independent of the amount of DNA. A variety of assays are being designed to identify the responsible scaling factors, and to test and whether organelle scaling activities are also present in smaller cells of developing embryos.
Chromosome architecture and cell division. Chromosomes play a critical role in their own transmission, yet the mechanisms determining their higher order organization and mitotic functions are not understood. We are evaluating the roles of several factors including condensin, cohesin, and histone H1 in chromosome condensation and segregation, using microscopy and biophysical approaches.
In the News
The size of a cell's nucleus varies by species, by cell type, and with disease: many cancer cells develop larger nuclei as they become more malignant. Working with the African clawed frog, Professor Rebecca Heald and post-doc Daniel Levy have discovered two proteins that control the size of the nucleus.