Microtubules play a central role in many aspects of plant growth and development including cell expansion, division and signaling. In plants, microtubules become organized into four distinct arrays with varying functions. It has recently been shown that microtubules in the cortical array become repositioned by a treadmilling motility mechanism that is driven by frequent cycles of microtubule plus end growth, pausing, and shortening (termed dynamic instability), and intermittent episodes of minus end depolymerization. An elucidation of microtubule end behavior is essential for understanding important aspects of microtubule organization and function. In animals and fungi, dynamic instability is regulated by a collection of proteins called +TIPS (plus end tracking proteins) that are preferentially localized to microtubule plus ends, where they also participate in capturing and moving cellular components. Little is known about what +TIPS exist in plants, let alone how they regulate dynamic instability and affect microtubule function. The Principal Investigators have recently identified a novel plant specific +TIP named SPIRAL1 (SPR1) in a forward genetics screen looking for Arabidopsis mutants affected in root cell expansion. The organs of spr1 mutants exhibit both touch induced and constitutive directional cell expansion defects that are enhanced by lower temperatures and suppressed by anti-microtubule drugs. Sequence analyses show that the 12 kD SPR1 protein has five homologues in Arabidopsis, with these proteins having highly conserved direct repeat sequences at their amino and carboxy termini, separated by a region of low complexity that is predicted to form a rod-like structure. Given that a stably expressed SPR1:GFP fusion protein behaves like other GFP labeled +TIPS in that it preferentially localizes to growing microtubule plus ends and disperses upon microtubule shortening, the Principal Investigators predict that like those other +TIPS, SPR1 regulates microtubule dynamic instability, recruits proteins to microtubule plus ends, and/or links microtubules to other cellular structures. To test these hypotheses, important parameters of dynamic microtubule behavior will be analyzed in the spr1-6 null allele as well as in SPR1 over-expressing plants, using time-lapse confocal microscopy and fluorescent microtubule markers. It will be determined if SPR1 binds directly to microtubules by performing in vitro microtubule pull-down assays. A yeast two-hybrid system will be used to screen libraries for SPR1 interacting proteins and to test candidate MAPs for SPR1 interactions. Particular focus will be placed on identifying the relationship between SPR1 and Arabidopsis homologues to the well-characterized animal EB1-like proteins, given that animal EB1 proteins have many binding partners and the Arabidopsis AtEB1 proteins exhibit localization dynamics similar to SPR1. Together, these experiments will allow the Principal Investigators to determine how SPR1 and other plant +TIPS participate in microtubule plus end function. This project should also provide insights into the poorly understood connection between microtubules and directional cell expansion in plants. The knowledge gained on +TIPS in plants may provide a perspective that aids in the understanding of +TIPS function in animals and fungi.
Broader impacts: The PIs will actively recruit individuals from underrepresented groups in science to participate in this project and receive training in basic research. Project participants will include a female graduate student and a female undergraduate in Dr. Sedbrook's laboratory, and students recruited from local community colleges in Dr. Ehrhardt's laboratory. A subset of the experiments will be integrated into a biotechnology lab course run by Dr. Sedbrook that is designed to teach undergraduates and graduate students basic molecular techniques in a way that develops their critical thinking and troubleshooting skills. The Biological Sciences department at Illinois State University has recently purchased a Leica confocal microscope system with funds derived from an NSF equipment grant, so this project will allow the investigators to take full advantage of this important instrument for both research and teaching. Images and movies of dynamic microtubules in living plant cells generated from these studies will be used to enhance a variety of courses taught by the PI at Illinois State University and will be integrated into the Plant Cell Imaging web site created by the Dr. Ehrhardt. This web site features images and movies of living plant cells that reveal their fascinating inner life. This site has been profiled in Science magazine, and is used as a resource for teaching cell biology at several secondary and higher educational institutions around the country.
