Building on our development of the ultra-sensitive and high resolution, liquid crystal based polarizing microscope (LC-PolScope), we are now broadening the scope of the project to include laser microsurgery and fluorescence anisotropy measurements for the development of advanced optical methods to manipulate and analyze the dynamic cell architecture that orchestrates and sustains cell division. 1. We propose to develop a versatile laser microsurgery tool based on a solid-state laser and a liquid crystal spatial light modulator. We will use advanced beam shaping optics based on wave front phase modulation to generate ablation patterns that can be tuned to improve the focus inside a cell and to dynamically shape and position the focal region to adapt to changing cell structure and functional state. 2. Using the laser microsurgery tool we will analyze the assembly/disassembly mechanisms of microtubule (MT) arrays in actively dividing cells. In combination with the LC-PolScope we will record at high spatial and temporal resolution the structural consequences of MT loss and regrowth in the astral arrays, kinetochore bundles and interpolar spindle MTs, shedding light on the dynamic properties that distinguish these microtubule classes. 3. We propose to develop an imaging system for fast and comprehensive analysis of fluorescence anisotropy observed in cells expressing fluorescently labeled (e.g. GFP- tagged) cell division proteins. The goal will be comprehensive spectral and polarization analysis of fluorescently labeled proteins with emphasis on dynamic organization of molecular aggregates essential for cell division. 4. The instrumentation for measuring fluorescence anisotropy will be applied to two models: (1) dynamic organization of septin filaments in the hourglass and ring structures located at the bud neck in yeast cells;and (2) structure-function relationship of kinetochore proteins whose natural cycles of tension and release are important to all eukaryotic cells;for the latter, initial work will be done using yeast GFP tagged Ndc80, a ubiquitous kinetochore protein.
PROJECT NARRATIVE As in previous grant periods, the microscopy instrumentation developed under this period will have applications in medical research and clinical settings. Furthermore, the cell biological application projects that will be forthcoming under this grant will have broad implications for our understanding of cell division, a fundamental cellular process in healthy and diseased tissue.
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