Pattern formation of cortical microtubules and cellulose microfibrils

In this thesis we study the roles of microtubules at the plasma membrane and the cellulose microfibrils in the cell wall and how they are organized. This topic is introduces in chapter 1. In chapter 2 we study the formation of the transverse cortical microtubule array that is characteristic for elongating plant cells. We found that the cortical microtubule array starts ordered, and that the first direction of microtubule order is not transverse to the axis of cell elongation but have a diagonal bias. Quantification of the orientation of microtubule nucleations revealed a significant diagonal bias, which we confirmed by simulations to be sufficient to explain the initial diagonal order. We found that during disassembly the microtubules also showed a diagonal bias and a significant amount of early microtubule nucleations were not generated from γ-TuRC microtubule nucleating complexes. This led to the idea that a proportion of the initial nucleations stem from small microtubule fragments of preexisting microtubules that remained at the cell cortex during cytokinesis or drug induced microtubule disassembly. We showed with simulations that this type of nucleation has the capacity to increase the speed with which the cortical microtubule array is reformed. In chapter 3 we investigate the trafficking of cellulose synthase complexes from assembly in the Golgi system to their insertion into the plasma membrane. We find that the actin cytoskeleton is important for the global distribution of Golgi bodies, which in turn is important for the global distribution of cellulose synthase complexes in the plasma membrane. Cellulose synthase complexes were inserted into the membrane preferentially at locations where cortical microtubules were present. We showed that osmotic stress and a number of cellulose synthesis inhibitors blocked cellulose synthase insertion into the plasma membrane. The cellulose synthase complex containing compartments were seemingly still being delivered to the cortical microtubules where they accumulated. These compartments tracked depolymerizing microtubule ends. When the osmotic stress was relieved, cellulose sythase complex insertion was resumed from these compartments. Rapid movement of proteins, organelles and metabolites in the cytoplasm of plant cells depends on the actin cytoskeleton, whereas microtubules are important in regulating the location of proteins and cellular processes. In chapter 4 we found physical interactions between cortical microtubules and actin. We also found that the formation of the actin cytoskeleton after washing out the actin depolymerizing drug latrunculin B was dependent on the presence of microtubules. In the presence of cortical microtubules, new actin filaments initiated on and in the direction of cortical microtubules. In chapter 5 we investigate the mechanism reorientation of the cortical microtubule array from transverse to longitudinal in response to light signaling. We found that cortical microtubule array reorientation in dark grown hypocotyl cells was regulated by phototropin a blue light photoreceptor. We found that microtubule reorientation was delayed in phot1 phot2 mutants. We also found that ktn1-1, a null mutant of KATANIN P60, and spr3, a GCP2 allele with impaired function, severely retarded microtubule array reorientation in response to light. spry has altered angles of microtubule nucleation relative to the mother polymer, and ktn1-1 abolishes liberation of microtubules form their nucleation complexes to yield treadmilling polymers and microtubule severing at microtubule crossovers. We found that in response to blue light, the proportion of microtubule nucleations branching at 40 degrees from the mother microtubule to nucleation at 0 degrees from the mother microtubule was higher in wild type plant than in the phot1 phot2 mutant. We also found that the chance of microtubule severing at microtubule crossovers was significanlty higher in wild type than in the phot1 phot2 mutant. We propose that upregulation of the branching nucleations is needed to create a number of longitudinally oriented microtubules. These microtubules make a large number of crossovers with the existing transverse array and have an increased chance of being severed. Severing events that result in a stable new tip contribute to the increase in longitudinal microtubule order and ultimately lead to complete reorientation from transverse to longitudinal. The spatial organization of cortical microtubules and cellulose microfibrils are essential for plant morphogenesis, but the mechanism by which is unclear. Chapter 6 discusses the process of cell elongation and offers possible additional roles for cortical microtubules beyond guiding cellulose microfibril deposition in this process.