The research goal of my 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 macromolecular structure essential for the correct distribution of chromosomes to each daughter cell.  The spindle is composed of microtubule polymers and many other factors that generate an antiparallel bipolar array.  Duplicated sister chromosomes attached to microtubules by their kinetochores are segregated to opposite spindle poles during anaphase.  The spindle also dictates the position of the cleavage plane, where an actin/myosin-based contraction pinches the two daughter cells apart during cytokinesis.

The primary assay that we have used to study spindle assembly is an in vitro system derived from eggs of the frog Xenopus laevis.  The cell cycle state of these cytoplasmic extracts can be controlled, and they can recapitulate many of the detailed morphological changes associated with mitosis.  The real power of this system comes from its biochemical accessibility and the microscopy approaches that can be utilized to follow dynamic events in real time.  Individual components of the extract can be specifically manipulated, and mitotic sub-processes such as microtubule dynamics and chromosome condensation can also be dissected and imaged, allowing a detailed structural and functional analysis of the complex and dynamic macromolecular cell division apparatus.

In collaboration with the laboratory of Karsten Weis, we are studying one chromatin-driven pathway of 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, which include both protein and RNA components.

One approach we have taken to identify novel cell division factors was to combine phenotypic screening of chemical libraries with affinity chromatography in Xenopus egg extracts to identify compounds that disrupt spindle assembly and their molecular targets.  We have also utilized proteomic techniques by isolating subcellular structures from dividing mammalian cells, including the cell division remnant (midbody) and mitotic chromosomes to identify constituent proteins, whose function is then evaluated by RNA interference and phenotypic analysis.