Regulation of nuclear position and migration: D-nudC and D-Lis1
Most differentiated cells have an inherent polarity that is apparent in the uneven distribution of a subset of macromolecules and organelles. This asymmetry extends to the nucleus that is usually present at a defined subcellular position and maintained there through an active process. In differentiated cells the asymmetric position of the nucleus is likely to facilitate cellular communication, while intracellular nuclear migration is known to be essential for developmental events that range from the union of the male and female pronuclei during fertilization to later stages of organ and tissue differentiation. Although nuclear migration is ubiquitous, relatively little is known about its mechanistic basis in higher eukaryotes. Classical studies using specific inhibitors and antibodies have indicated that nuclear movement is microtubule-dependent. However, the particular motors involved have not been unambiguously identified, and the genetic and cellular means by which nuclear migration is regulated are still unknown
Work in my laboratory centers on two genes, Drosophila nudC (DnudC), and Drosophila Lissencephaly1 (DLis1), that are critical components of an evolutionarily conserved pathway regulating nuclear migration. These genes act by affecting the activity of cytoplasmic dynein, a minus-end directed microtubule motor. Consistent with this, we have recently been able to directly implicate dynein in nuclear migration by disrupting motor function using a dominant negative approach. These experiments lead to two unexpected findings - that dynein activity is essential to anchor the nucleus after migration and that activity of the plus-end directed kinesin motor is also necessary for nuclear migration and anchoring. Work on nuclear migration is likely to have clinical significance since mutations in the human homolog of DLis1 cause Miller-Dieker Lissencephaly, an inherited human birth defect associated with severe mental retardation. This devastating syndrome results from a failure of neuronal migration during embryogenesis, suggesting a link between nuclear and neuronal movement. Questions we are currently focusing on include, (i) understanding the requirement for DLis1 in coupling the motor complex with nuclear migration, (ii) using genetic interaction screens with DLis1 and DnudC to identify other genes in the nuclear migration pathway that may play regulatory rather than structural roles, and (iii) determining the extent to which the functional relationship between the NudC and Lis1 proteins has been conserved.
Proteoglycan-mediated effects on Growth-Factor Signalling: D-EXT2
A second area of research is based on our isolation of mutations in the Drosophila homolog of Ext2, a gene that causes Hereditary multiple exostoses (HME), a bone overgrowth syndrome in humans. Biochemical and cell culture studies suggest that human Ext2 and the closely related Ext1 gene encode co-polymerases that synthesize glycosaminoglycans, polysaccharide chains attached to extracellular proteins such as heparan sulfate. Thus it would be predicted that mutations in Ext1 and Ext2 would have similar phenotypes. Surprisingly, our studies indicate that ext2 mutants show defects in multiple growth factor signaling pathways in contrast to the Ext1 ortholog tout velou (ttv) that is known to only affect diffusion of the growth factor Hedgehog (Hh).
Our initial objectives are to characterize the phenotype of mutations in ext2, to establish the role of ext2 in signaling by growth factors of the Wnt, Hh and TGFß family, and to investigate the basis for the differential requirement for ttv and ext2. This work is relevant to birth defects at two levels. In a direct sense, it is pertinent since mutations in the human Ext2 gene cause HME, an inherited autosomal dominant disorder that results from bone overgrowth initiated during childhood. Affected individuals have limbs that may be shorter than normal or develop unevenly in addition to showing characteristic bony projections (exostoses). At another level, the fact that Ext1 and Ext2 are likely to modulate multiple growth factor pathways emphasizes the need to understand their roles in patterning and development during embryogenesis.