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The Interplay Between Polarity Regulators, Calcium, and the Actin Cytoskeleton During Tip Growth

Plant cell growth is a meticulously regulated process whereby the cell wall is selectively loosened to allow for turgor-pressure driven expansion. The rate of expansion must equal delivery of new material, or the cell will lyse. In many plant cells, this process happens diffusely around the cell. However, a number of plant cells have anisotropic shapes that require exquisite spatial control of secretion. One simple example of anisotropic patterning is tip growth; highly polarized cell expansion utilized by pollen tubes, root hairs, and moss protonemata. Investigating the role various molecules have in tip growth sheds light on how plant cells are patterned. One such molecule is actin. A dynamic actin cytoskeleton is essential for tip growth in general and specifically for the protonemata of the moss Physcomitrella patens. In P. patens, actin is essential for cell polarity in protonemata. In this dissertation, I provide background information on tip growth research, focusing on advances in P. patens as well as introduce P. patens as a model organism. Additionally, I contributed to developing a novel method for long-term imaging of live protonemata. To investigate regulators of the actin cytoskeleton, I silenced an inhibitor of the Rho/Rac of Plants (ROP)- the ROP Guanine Dissociation Inhibitors (RopGDIs). Knocking down RopGDIs resulted in an increase in actin dynamics as well as a dramatic loss of polarized growth. In addition to ROP signaling, calcium has been implemented as a key regulator of tip growth in pollen tubes and root hairs. Using a FRET-based calcium probe, I described a previously unreported apical calcium gradient in growing protonemata. With two complementary signal-sifting analyses, I discovered that oscillations in the calcium gradient are the sum of discrete frequencies of variable influence. Additionally, I reported that calcium acts antagonistically on an apical actin structure. Finally, I described loss-of-function phenotypes for the actin-regulating protein Villin, putative stretch-activated channels Mid1-Complementing Activity, as well as both ER Calcium and Autoinhibitory Calcium ATPases. The work presented here is the foundation for developing models of calcium dynamics during tip growth in the moss P. patens.