Plant growth and development are achieved by the combined effect of two fundamental cellular processes, cell division and cell growth. This project aims at understanding an extreme form of polarized cell growth called tip growth, which occurs in root hairs, pollen tubes, and the filamentous cells of ferns and mosses. It is well established that tip growth is dependent on the actin cytoskeleton. This cytoskeleton is a cellular structure composed of nanometer size fibers. In cells, molecular motors called myosins interact with these fibers for contraction and intracellular transport. Despite the long known role of the actin cytoskeleton in tip growth, it is not known how this cytoskeleton is linked to growth. This project aims to evaluate if a plant specific myosin (myosin XI) is the link between the actin cytoskeleton and tip growth. To help understand myosin XI function, the investigators will determine its location within the cell. In addition, by preventing myosin XI expression, they will determine if myosin XI regulates the actin cytoskeleton or intracellular transport. These studies are being carried out in the emerging plant model, the moss Physcomitrella patens. This organism was chosen because of its unique genetic properties and simple development. Furthermore, a recently completed genome sequence facilitates the generation of molecular tools. As the primary producers of food and biofuel, plants are an essential component for the establishment of a sustainable future. Hence, understanding plant growth is an important endeavor of basic science, potentially becoming the basis of vital new technologies. The investigators are committed to increasing the participation of underrepresented groups in the life sciences. Throughout this project, students and postdoctoral researchers who are members of these groups will be actively recruited, trained and mentored. By coordinating with other organizations, support will be provided for undergraduate students to complete summer projects and full year internships.
Intellectual Merit The main goal of this project was to investigate the participation of myosin XI in polarized plant cell growth. Myosin XI is a member of the highly conserved myosin V subfamily of actin-dependent molecular motors. These motors have been implicated in neuronal development in animals, polarized cellular transport in fungi, and rapid intracellular movements and secretion in plant cells. An important and unresolved problem in plant cell biology is how the actin cytoskeleton and the motor myosin XI coordinate polarized cell growth and secretion. The molecular mechanisms by which actin dynamics, endomembrane traffic, and myosin XI cooperate to maintain cellular polarization are not known. Understanding this regulatory interplay is important because cellular polarization underlies many essential developmental processes in plants. To address this problem, we chose to use the moss Physcomitrella patens as a model system, because of its abundant tip growing cells and powerful molecular and cellular tools [1] (Figure 1). Initially, we used an RNA interference-based (RNAi) gene silencing approach to demonstrate that myosin XI is essential for polarized growth [2]. Because the myosin XI-RNAi plants fail to polarize, it was not possible to determine the effect of loosing myosin XI function during tip growth. To overcome this, we developed a temperature-sensitive allele of myosin XI (Figure 2). This mutant will be critical to investigate rapid loss of myosin function, which is an important tool to explore its participation in fast cellular processes. One of our main long-term goals is to obtain a general picture of how tip growing cells are organized at the cellular level, this is important in order to have a reference point to evaluate changes in cellular organization. For example, we characterized the steady state three-dimensional distribution of mitochondria, peroxisomes, chloroplast, and Golgi bodies in moss cells [3]. We found a shallow gradient in the distribution of these organelles, with increased accumulation toward the cell tip. Understanding the mechanism that maintains this gradient should be useful for future intracellular engineering or to modify plant cell size, which can result in plants with increased yields for foods, feedstocks, and biofuels. We focused on the growing tip of the moss cell to investigate the localization and dynamics of myosin XI and its relationship with the actin cytoskeleton and secretory vesicles [4]. Using fluorescent probe fusions, we discovered that filamentous actin and myosin XI, as well as secretory vesicles accumulate in this region. Interestingly, this accumulation is dynamic, showing fluctuations that are reminiscent of the periodic oscillations observed in other tip growing cells such as pollen tubes and root hairs. Using multidimensional confocal microscopy and fluctuation cross-correlation analysis, we discovered, to our surprise, that the myosin XI signal precedes F-actin’s for 18 seconds (Figure 3). In addition, when we incubated the cells with very low levels of the actin inhibitor, latrunculin B, we detected the formation of ectopic myosin XI clusters in the shank of the cell (Figure 4). These myosin XI clusters are followed by the accumulation of F-actin and actin polymerization-dependent motility, resulting in dispersion of the myosin XI cluster. Together, these data strongly support an active role of myosin XI in regulating actin polymerization and dynamics. Other outcomes include a standardized transformation protocol that provides consistent high transformation efficiencies and is simple and reproducible [5], and a standardized moss growth and morphology assay [6]. Broader Impacts As part of this project, a group of undergraduate students was recruited to help implement a social networking website for postdoctoral scholars (LectureBank.org). The goal of this network is to increase the presentation and networking skills of postdocs in the life-sciences, with emphasis on underrepresented minority groups. The website is currently under optimization by additional groups of undergraduate students. Other broader impact activities include participation as exhibitor, judge, and mentor at the Annual Conference of the Society for Advancement of Chicanos/Hispanics and Native Americans in Science and presenting research and mentoring seminars for underrepresented minority students at WPI. The temperature sensitive mutant developed from this project has been incorporated as a specimen for a very successful inquiry-based microscopy course/lab for juniors and seniors. Two postdoctoral researchers and three graduate students have participated in different aspects of this project. Several undergraduate students also participated, including two minorities. As part of a collaboration with Dr. Tüzel from the Department of Physics, all biology students closely interact with physicists, and are highly involved in a graduate level weekly Biophysics Journal Club. [1] Vidali L and Bezanilla M (2012) Current Opin. Plant Biol. 15:625-631. [2] Vidali L, et al. (2010) Plant Cell 22:1868-1882. [3] Furt F, et al. (2012) BMC Plant Biology 12:70. [4] Furt F, et al. (2013) Plant Journal 73:417-428. [5] Liu Y-C and Vidali L (2011) J. Vis. Exp. 50:2560 [6] Bibeau JP and Vidali L (2014) Plant Cell Morphogenesis. Methods in Molecular Biology. Springer pp 201-214.
