Dan Szymanski
Assistant Professor of Agronomy
Ph.D., University of Illinois, 1990

The organization of the microtubule and microfilament cytoskeletal arrays plays an important role in defining cell shape and tissue development in multicellular organisms.  However, there is very little mechanistic understanding of how cytoskeletal organization is regulated.  For example, how do hormonal signals lead to cytoskeletal re-organization and a polarized growth response?  What cellular intermediaries regulate cytoskeletal organization?  It has become increasingly clear that a molecular genetic approach is essential to fully characterize this complex and important signal transduction cascade.  I am using leaf epidermal development in Arabidopsis as a powerful experimental system to analyze genes that are involved in cytoskeletal organization and polarized growth.

Arabidopsis has been established as a model plant species for using genetic, molecular, and genomic tools to study important biological questions.  Leaf epidermal development is being used to study cytoskeletal control during cell and tissue morphogenesis.  The leaf epidermis contains several unique and highly polarized cell types.  Based on cytoskeleton immunolocalization and pharmacological experiments, the microtubule cytoskeleton is essential for the establishment of cell polarity and the actin cytoskeleton is required for the maintenance of cell growth patterns (Szymanski et al. 1999).  Because of the specific contributions of microtubule and microfilament arrays to cell shape it has been possible to selectively screen for mutations that may be directly involved in cytoskeletal organization.

For example, the recessive spike mutation was identified after screening 2,000 T-DNA tagged mutant lines for leaf and cell shape defects similar to actin microfilament-defective cells.  Mutant plants display defects in actin organization, cell shape, epidermal cell adherence, stomatal control, and fertility.  The SPIKE gene has been cloned, and encodes a predicted membrane spanning protein.  A predicted cytoplasmic domain of SPIKE shares strong amino acid identity with the DOCK180 family of proteins.  In humans, drosophila, and C. elegans DOCK180 proteins are essential for transmitting extracellular signals to proteins that locally regulate actin organization. DOCK180 proteins bind to the G-protein RAC; however, the mechanism of DOCK180 function remains uncertain.  The SPIKE gene represents the first cloned plant gene that may link extracellular information to cytoskeletal reorganization.  Initial molecular genetic and biochemical experiments that begin to dissect the cell shape control pathway and address the function of the SPIKE gene have been initiated.  My research also involves the use of in vivo imaging techniques.  Backskattered (reflected light) and fluorescence confocal imaging methods have been developed to simultaneously visualize rapid vesicle dynamics and cell shape changes at high resolution in reflection mode and the movement of a GFP-tagged molecule or organelle in fluorescence mode.  Future experiments will examine vesicle dynamics, growth patterns, and GFP-actin organization in wild type and mutant lines.

The long-term goal of my research is to develop a mechanistic understanding of the relationship between extracellular information and cytoplasmic organization in the context of tissue and organ development. Preliminary data on the SPIKE gene has established the feasibility of using Arabidopsis genetics and an interdisciplinary approach to study candidate genes in this important but uncharacterized pathway.  Clearly the SPIKE gene is only one component of this regulatory circuit.  Genetic screens for additional components in the pathway are underway, and several candidate genes have already been identified.  The use of modern molecular genetic, biochemical, and imaging tools in this experimental system will make fundamental contributions to the field of cytoskeletal control and morphogenesis.

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