Research Summary
Motor proteins in axonal transport, brain developmental disease, and synaptic function.

Dr. Vallee's Laboratory

Cytoplasmic dynein is a large microtubule motor protein which is responsible for retrograde axonal transport. It is also involved in neuronal migration, mitosis, secretion, endocytosis, nuclear migration, viral transport, and many other basic cellular processes.

Recent evidence has implicated cytoplasmic dynein in the "smooth brain" disease, lissencephaly, which arises from sporadic mutations at the LIS1 locus. This condition is thought to arise from a defect in the migration of neuronal/glial progenitor cells during early development. Neurons exhibit more-or-less normal differentiation, but are scattered randomly within an expanded cerebral cortex. We have found the LIS1 gene product to associate with cytoplasmic dynein directly. Surprisingly, the LIS1 protein colocalized with cytoplasmic dynein at mitotic kinetochores and the mitotic cell cortex. Interference with LIS1 expression of function caused dramatic changes in mitotic spindle orientation, chromosome behavior, and mitotic index. Together, these results suggested that mitotic or cell cycle defects might contribute to lissencephaly and other brain developmental abnormalities. In the case of lissencephaly, cell division defects could affect neuronal distribution indirectely by affecting the timing of neurogenesis.

We are also interested in determining whether LIS1 and, by extension, cytoplasmic dynein are directly involved in cell migration. To the end, we have begun to examine a number of neuronal and nonneuronal systems. We find that in wounded fibroblast monolayers, both cytoplasmic dynein and LIS1 redistribute prominently to the leading cell edge during migration. Inhibition of cytoplasmic dynein and LIS1 interfere with the reorientation of the microtubule network and forward cell movement. These results suggest that cytoplasmic dynein, regulated by LIS1, pulls on microtubules at the leading cell edge. We believe that one result of this activity is to ensure that the cytoskeleton and membranous organelles are carried forward. In addition, our results may reflect the operation of a novel tension-sensing mechanism, which dictates the direction of actomyosin-based lamellipodial activity.

Together with the laboratories of Drs. Arnold Kriegstein and Jim Goldman, we are setting up assays to test the role of cytoplasmic dynein and LIS1 in migration of neuronal/glial progenitor cells in brain slices and other preparations. We are also interested in defining the role of dynein and LIS1 in glioma cell migration, a serious problem in the control of brain cancer.

We are also interested in the basic mechanisms by which cytoplasmic dynein is selectively targeted to diverse subcellular organelles, how its activity is switched on and off during axonal transport and other functions, and how it produces force. The dynein motor domain is ten-fold larger than that of kinesin, and is thought to consist of a hexameric ring of six AAA ATPase units from which protrudes an unusual 10-12 nm microtubule-binding stalk. We are interested in determining the structure of the motor domain and its subdomains, in learning the role of the multiple ATPase modules, and in determining how conformational information is transmitted through the stalk to ATPase sites. Of considerable interest, the LIS1 protein associates with one of the AAA modules, suggesting that it may function by regulating motor activity.

Effect of dynactin disruption on dividing COS-7 cells. Cell at left is overexpressing the dynamitin subunit of the dynactin complex; cell at right is a non-expressing control. Blue, anti-dynamitin; green, anti-tubulin; red, CREST auto-immune anti-centromere. Dynactin overexpression inhibits mitosis during prometaphase and distorts the mitotic spindle (from Echeverri et al., 1996).

A) Neuronal progenitor cells at progressive stages of development in the vertebrate ventricular zone. Aspects of cell motility potentially affected by LIS-1 mutations are shown. Apical (ventricular) surface at bottom. B and C) Dividing polarized MDCK epithelial cells examined by confocal anti-tubulin immunofluorescence microscopy. B) Control mitotic spindle lies parallel to coverslip, as shown. C) Mitotic spindle in LIS-1 overexpressing cell is misoriented. Regulation of spindle orientation by LIS1 is proposed to control affect the timing of neurogenesis in the developing brain and the subsequent destination of migrating neurons. (from Faulkner and Vallee, 2000).