All cells display some form of polarity which manifests itself in an asymmetric distribution of specific molecules or organelles, yet the mechanisms driving cell polarity remain obscure. This research project seeks to reveal fundamental knowledge surrounding the maintenance of cell polarity, the delivery of secretory products to specific locations within the cell, and the dynamic maintenance of cell architecture. The central hypothesis of this project is that plant myosin XI motor proteins play a crucial role in these critical aspects of cell growth and development. Root hairs are an excellent experimental system to test this hypothesis since they grow only at their tip and simultaneously display tightly focused polarity and strong cytoplasmic streaming. Myosin XI mutants that lead to reduced root hair elongation without eliminating cytoplasmic streaming have been identified previously. A current working model assumes that these myosins are responsible for the delivery of specific organelles or molecules to the growing tip and are therefore involved in maintenance of cell polarity. Thus, this project investigates the specific cellular functions of myosin XI motor proteins in root hairs of the model plant Arabidopsis thaliana. The larger project falls broadly into two Specific Aims. (1) Identification of the targets of MYA1 and XI-K in root hair elongation by quantitative cell biological analysis of organelle motility and root hair architecture as well as determination of myosin localization. (2) Identification of the mechanism of cargo attachment to MYA1 by specific modification of surface residues of the cargo-binding domain and by a general screen for interacting proteins. The intellectual merit of this project lies in the new insights into fundamental cell biological processes that will be obtained as a result of the research. In particular, this project addresses the dynamic maintenance of cell architecture, the role of cytoskeletal motors in establishing cell polarity, and the delivery of secretory products to regions of growth. In addition, novel quantitative methods for the comprehensive cellular, genetic, and biochemical analysis of myosin function in a model cell type will be developed. The broader impact of the work rests predominantly on the training of young scientists, primarily two graduate students and a postdoctoral associate. The training of talented under-graduate students as well as high school students will be continued in hopes of attracting them to a future in science. Students and postdocs will collaborate with colleagues here at the University of Tennessee and present their findings at national meetings. The results of this work will be published in peer-reviewed journals and made available to a broader audience on the lab web site (www.bio.utk.edu/cellbiol).
Myosin motor proteins are nano-machines that literally walk along cables, so-called actin filaments, that stretch throughout cells. Despite their small size, these motors can rapidly deliver large cargo to distant places in the cell. This process is most easily observable in plant cells which display so-called cytoplasmic streaming. This project investigated the function of a specific myosin motor protein in cytoplasmic streaming during growth of root hairs, which are thin, finger-like projections that help plants collect water and minerals from the soil. We found that loss of one particular myosin, XIK, in a mutant plant resulted in slower growth of root hairs which were therefore shorter. We examined different parts of the root hair cells with the help of fluorescent markers and discovered that movement of small structures called peroxisomes was slower when this motor was missing. More importantly, we discovered that the actin cables that serve as the tracks for this motor also moved more slowly in the myosin mutant. This demonstrates that the motors not only move cargo along the actin filaments but also lead to displacement of these actin filaments. We were also able to repair the defect in the mutant by introducing a healthy copy of the XIK gene that was tagged with a fluorescent protein gene. This enabled us to see the distribution of the XIK myosin protein in living cells. Interestingly, the tagged motor accumulated at the tip of growing root hairs, exactly at the place where growth occurs. While secretory vesicles that are necessary for growth were still moving to the root hair tip, we found that a regulatory protein that binds to the cell surface in the tip had reduced accumulation in this area in mutant root hairs. We speculate that this effect together with the slower movement of actin filaments and some organelles is causing the reduced root hair growth. Our research therefore demonstrates that intracellular movements driven by myosin motors are necessary for normal growth of cells. In a second project, we identified a potential binding protein of a myosin motor. Such binding proteins are important to link the motors to their cargo, much like a trailer hitch allows you to pull a boat behind your car. So far, we were not able to conclusively show direct binding of the two proteins, but an indirect interaction in combination with other proteins is also possible. This work has not only allowed us to gain a better understanding of myosin motor proteins and their roles in cell growth, the research also was a crucial element in the education of four high school students, 33 undergraduate students, four graduate students, and one postdoctoral researcher. These junior scientists learned a wide variety of experimental techniques such as live-cell microscopy, quantitative image analysis, molecular biology, genetics, biochemistry, physiology. As part of this project, we also co-organized a workshop on Mathematical Modeling of Intracellular Movements which was held at the National Institute of Mathematical and Biological Synthesis.
