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Signaling pathways from guidance receptors to the cytoskeleton during development.

My work focuses on the role of the family of RhoGTPases in neuronal development.  RhoGTPases are involved in modulating the actin cytoskeleton and promoting polarity in a number of different cells.  Although existing evidence suggests that GTPases are involved in regulating cytoskeletal dynamics required for cell movements, it is not clear how their activities are spatially and temporally regulated to carry out these functions in vivo.

We use the nematode Caenorhabditis elegans as a model for elucidating the signaling pathways involved in neuronal development and axon pathfinding.  C.elegans is a simple, easily maintained organism that has its genome completely sequenced and the cell lineage during development completely defined.  Previous work has been done characterizing a regulatory protein in C.elegans that influences the levels of functionally active RhoGTPase.  This protein (UNC-73) is a guanine nucleotide exchange factor (GEF) and activates GTPases by stimulating exchange of GDP for GTP.  UNC-73 activates the Rac GTPase (both the C.elegans and mammalian forms) and activated Rac leads to membrane ruffling caused by formation of actin structures protruding from the cell membrane.  Mutants of unc-73 have a variety of defects in axon guidance and cell motility, indicating that UNC-73 and the Rac GTPase are necessary for proper neurodevelopment.

Of relevance to human pathological conditions is the family of mammalian proteins Kalirin/Trio, which is closely related to UNC-73.  Kalirin is expressed predominately in the brain, while Trio expression occurs in most tissues. Studying these proteins can provide insights into actin microfilament regulation as it relates to guided cell and growth cone migrations and has obvious implications in mammalian neuronal development and regeneration.  

Projects will involve using previously synthesized deletion mutants of the GTPases and their regulators to perform transgenic rescue experiments with wild-type and mutant DNA.  This allows us to examine the structure/function relationships of various proteins.  Identifying effectors of the GTPases through the use of recombinant proteins and mass spectrometry technology will also be a focus of the lab.  Genetic approaches can also be used to create double mutants to determine enhancers or suppressors of the original mutant or of the transgenic animals and will help to delineate the various signaling pathways.  Finally, testing the mammalian orthologs for interactions and determining if there is conservation of the signaling pathways between species will allow us to apply what we learn in the nematode to the human condition.

Therefore, my research plan combines genetic, cell biological and biochemical approaches to investigate the cell signaling role of proteins such as UNC-73 and the Rho-family GTPases in the control of axon guidance, cell motility, and cell morphology as they relate to neuro-pathological conditions. C.elegans provides an excellent opportunity to study the signaling pathways that contribute to human diseases. 

Representative Publications:

T.J. Kubiseski, J. Culotti, and T. Pawson. Functional Analysis of the C.elegans UNC-73B PH Domain Demonstrates a Role in Activation of the Rac GTPase in vitro and Axon Guidance in vivo. Mol.Cell.Biol. 23:  6823-6855 (2003).

R. Steven, T.J. Kubiseski, H. Zheng, S. Kulkarni, J. Mancillas, A. Ruiz, C.W.V. Houge, T. Pawson, and J. Culotti.  UNC-73 activates the Rac GTPase and is required for Cell and Growth Cone Migrations in C. elegans.  Cell 92:  785-795 (1998)

B. Aghazadeh, K. Zhu, T.J. Kubiseski, G.A. Liu, T. Pawson, Y. Zheng, and M.K. Rosen.  Structure and mutagenesis of the Dbl homology domain.  Nature Struct. Biol. 5:  1098-1107 (1998).

M.K.H. McGavin, K. Badour, L. Wilson, T.J. Kubiseski, J. Zhang, and K.A. Siminovitch.  The Intersectin 2 Adaptor Links WASP-mediated actin polymerization to TCR endocytosis.  J. Exp. Med. 194:  1777-1787 (2001).

T.J. Kubiseski, Y.M. Chook, W.E. Parris, M. Rozakis-Adcock, and T. Pawson.  High Affinity binding of the Pleckstrin Homology Domain of mSos1 to Phosphatidylinositol (4,5)-bisphosphate.  J. Biol. Chem.  272:  1799-1804 (1997).