This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
The intellectual merit of this project is to understand cellular asymmetry and membrane adhesion, two processes that are not well understood and which are crucial for eukaryotic cells. Many structures, including the mitotic spindle, nuclear pore complexes, and Golgi must be assembled asymmetrically at every cell division. Regulated adhesion between membranes is not fully understood in many cellular systems including vesicle trafficking, synaptogenesis, and fertilization. This project is focused on understanding the asymmetric arrangement of the microtubule cytoskeleton, and the asymmetric placement and complex assembly of the eyespot in the simple eukaryotic alga, Chlamydomonas. The eyespot senses light intensity and direction and sends signals to the paired flagella, which change their breast stroke wave form to turn the cell toward or away from light. As viewed by light microscopy, the eyespot is an orange-red spot at the equator of the cell with the pole of the cell marked by the location of the flagella. The eyespot is made up of a layered array of four membranes and carotenoid-filled granules. The plasma membrane contains rhodopsin photoreceptors. The envelope membranes of the single, large chloroplast are tightly apposed to the plasma membrane in the region of the eyespot. The carotenoid granules are packed against the envelope membrane in an ordered array and are subtended by thylakoid membranes. There are multiple layers of granules and thylakoids in the Chlamydomonas reinhardtii eyespot. The eyespot is completely reassembled anew at every cell division and is placed uniquely in the cell relative to the microtubule cytoskeleton. A cruciate structure of microtubule rootlets emanates from the region of the basal bodies at the base of the flagella and extends down to the equator of the cell. A two-membered and a four-membered rootlet are paired with the parental and with the daughter basal body. The eyespot is always placed adjacent to the daughter four-membered rootlet as viewed from the flagellar pole. Four genes have been identified that affect assembly and placement of the eyespot and are required for wild-type phototaxis. eye2 and eye3 mutant cells do not have eyespots, min1 mutants have miniature eyespots and mlt1mutants have two eyespots. The EYE2 protein has thioredoxin domain and LysM domains. The MIN1 protein contains a C2 domain, which, in other proteins is responsible for phospholipid binding that can be calcium-dependent, and a LysM domain. The EYE3 protein sequence encodes a homolog of a serine-threonine kinase that regulates ubiquinone biosynthesis. The MLT1 protein codes for a protein with low sequence complexity. The objectives of this project are to: 1) determine whether the microtubule rootlet system is acetylated asymmetrically, if that asymmetry is fixed relative to the cleavage furrow and mother flagellum, and how the eyespot is positioned relative to the rootlets; 2) to alter rootlet acetylation genetically or pharmaceutically and observe the affect on rootlet and eyespot positioning and function; 3) to investigate the dependence/independence of eyespot granule placement on photoreceptor and rootlet localization; and 4) to investigate the function of EYE2, MIN1, EYE3 and MLT1 proteins in eyespot assembly and placement.
The broader impacts of this work are: 1) to include the participation of a small college professor, who will share her experiences with undergraduates in her laboratory during the academic year and alternate summers, 2) to participate in the NSF REU and other undergraduate research programs at the University of Arizona, and 3) to continue outreach lab tours and high school projects in K-12.
