Our lab is interested in the regulation of complex
cellular processes, including entry into mitosis and
apoptosis (programmed cell death). We are particularly
interested in understanding cellular mechanisms which
prevent the onset of mitosis prior to the completion
of DNA replication and in the tyrosine kinase signaling
pathways which impinge upon the decision to apoptose.
To address these problems, we use cell-free extracts
prepared from eggs of the frog Xenopus laevis which
can recapitulate cell cycle events and apoptotic processes
in vitro. For the study of cell cycle events,
extracts are prepared which can undergo multiple rounds
of DNA replication and mitosis in vitro. Progression
through the cell cycle can be monitored by microscopic
observation of nuclear morphology and by biochemically
assaying the activity of serine/threonine kinases
which control cell cycle transitions.
For the study of apoptosis, modifications in extract
preparation have allowed us to produce extracts which
can apoptotically fragment nuclei and can accurately
reproduce the biochemical events of apoptosis, including
internucleosomal DNA cleavage and activation of apoptotic
proteases, the caspases. These powerful in vitro
extracts allow us to examine and dissect critical
signaling events by:
- immunodepletion of cellular components from the
extracts,
- addition of exogenously produced recombinant proteins,
and
- direct biochemical purification of regulatory
proteins.
Using these molecular and biochemical approaches, we
are working towards the elucidation of signaling pathways
required for cell proliferation and cell death.
