The spatial control of cytokinesis (control of the plane of cell division) is a fundamentally important process for the development of plants as for other eukaryotes. In plant cells, a cortical preprophase band (PPB) of cytoskeletal filaments appears to be involved in establishing a """"""""division site"""""""" in the cell cortex, to which the phragmoplast (a cytoskeletal structure that functions as the cytokinetic apparatus) is actively guided during cytokinesis. Mechanisms governing these processes are poorly understood. The overall goal of this research is to advance our understanding of the spatial control of cytokinesis in plant cells. During the previous grant period, we cloned the Tangled1 (Tan1) gene of maize and showed that it is required for proper orientation of cytoskeletal arrays involved in plant cell division. Tan1 encodes a basic protein related to the microtubule (MT)-binding basic domain of adenomatous polyposis coli (APC, a human tumor suppressor). We showed that TAN1 can bind directly to MTs in vitro, and that anti-TAN1 antibodies label MT-containing cytoskeletal structures in dividing plant cells that are misoriented in tan1 mutants (PPBs, spindles and phragmoplasts). These findings led to the hypotheses that MT-associated TAN1 protein mediates interactions of the PPB, spindle and phragmoplast with other cell components that are necessary for orienting these arrays. To establish a system for more in-depth analysis of TAN1 function, Specific Aim 1 of this proposal is to elucidate the function and expression pattern of an Arabidopsis TAN1-like gene, ATN, and the localization of its protein product. To test the hypothesis that ATN, like TAN1, helps to orient cytoskeletal arrays in dividing cells by mediating their interactions with other cell components, Specific Aim 2 is to analyze the function and localization of proteins with which ATN interacts in the yeast two-hybrid system, and their interaction with ATN in plant cells. In addition, analysis of two new discordia (dcd) mutants during the previous grant period led to the hypothesis that these genes are involved in an actin-dependent process needed for guidance of phragmoplasts to cortical division sites in asymmetrically dividing cells of the maize leaf epidermis. To test this hypothesis, Specific Aim 3 is to clone and analyze the Dcdl gene and its protein product. Insights gained from these studies will contribute to a better understanding of structural aspects of cell division that are essential for normal eukaryotic development, and the loss of which is associated with neoplastic diseases in humans and animals. In particular, this work has the potential to shed new light on the function of APC tumor suppressor protein, and thus on the treatment of human colon cancer.
