UC Riverside

Proper development of a tissue or organism occurs through the growth, division, and differentiation of individual cells. The plane in which these divisions occur can control many developmental processes and changes to this division plane can alter growth and development. Specification of the division site may also depend of factors such as mechanical tissue stress, individual cell shape, and cytoskeletal dynamics. The maize tangled1 (tan1) mutant displays shorter overall growth, delayed mitotic timing, and altered cell patterning. TAN1-YFP localizes to the cortical division site throughout mitosis as well as mitotic microtubule structures. Altered cell shapes in the tan1 mutant make it difficult to assess the accuracy of the symmetric divisions. A computational approach was taken to determine symmetric division planes of any cell shape based on a soap-film model. This model quantitatively determined that symmetric division planes can be accurately determined by soap-film minimization and highlighted cases where a cell deviates from the geometrically determined optimal plane. When applied to tan1 mutant cells we see that cells on average have slightly misplaced future division sites compared to wild-type, however these differences are due to the abundant proportion of altered cell shapes in the mutant. A live-cell imaging approach was taken to better characterize the tan1 mutant phenotype in later stages of mitosis and we determined that 37% of symmetric divisions in maize epidermal leaf cells were misplaced during telophase according to the initial placement of the division site. Mitotic progression was also delayed particularly during metaphase and telophase. Recombinantly expressed HIS-TAN1 was tested for its ability to bind to microtubules using a microtubule co-sedimentation assay. We determined that HIS-TAN1 can bind to in-vitro taxol stabilized microtubules with an affinity similar to other microtubule associated proteins. Addition of HIS-TAN1 to dynamically unstable microtubules displayed a microtubule crosslinking activity. Microtubules that encounter each other at low angles (below 35 °) are likely to be “zippered” together whereas microtubules that encounter each other at large crossover angles (above 55 °) are linked at a single crossover point leading to a “pulling” effect. These data indicate that TAN1 is a microtubule bundling protein which helps generate proper cell shape by maintaining division site information throughout mitosis.