Biology Scholarship Proposal Example 1
Objective: I will investigate the relationship between the Drosophila WD40 protein Fritz and the actin cytoskeleton. I will stain Drosophila embryos expressing a Fritz-GFP fusion protein for actin localization and determine the degree of co-localization of Fritz and actin using confocal microscopy. I will optimize the detection of the Fritz-GFP fusion protein in embryo extracts by Western blot using an anti-GFP antibody and will assess the relative ability of different anti-actin and anti-GFP antibodies to immunoprecipitate actin and Fritz-GFP respectively from embryo extracts. Finally I will develop a co-immunoprecipitation assay to test whether an anti-actin antibody can co-immunoprecipitate the Fritz-GFP fusion protein from embryo extracts and conversely whether an anti-GFP antibody can co-immunoprecipitate actin.
Significance: Much of animal development involves the coordinated growth, patterning and differentiation of epithelial cell layers. As epithelial cells develop they acquire specific properties that are vital for epithelial function. One property is cell polarity in which cells develop molecular and morphological asymmetries. Epithelial cell polarity is conventionally defined in two axes. One is apical-basal polarity which distinguishes the top and bottom of the cell. The other is planar cell polarity (PCP) which determines the orientation of the cell within the epithelial plane. Drosophila epithelial PCP is under the control of the Frizzled PCP signaling pathway. The Frizzled pathway consists of a group of ‘Core’ PCP proteins (including Frizzled and Disheveled) that function upstream of a group of PCP ‘Effector’ proteins (including Fritz and Fuzzy). The Core PCP proteins show a polarized localization within developing Drosophila wing epithelial cells and this localization appears critical for correct actin cytoskeleton polarization. The PCP Effector proteins’ role is to link actin polarization with Core PCP protein localization. Recent studies have shown that the PCP Effector proteins also adopt a polarized localization within developing wing cells.
Our lab has adopted the ventral epidermis of the Drosophila as a model system for planar cell polarization as an alternative to the conventional wing model. We specifically study the development of rows of actin-rich structures called predenticles which are the precursors of the ventral denticle teeth used by larvae when crawling. The normal organization of predenticles requires the PCP Effector proteins and our lab has begun to investigate how the PCP Effector proteins behave within developing denticle cells. One unexpected preliminary result is that a Fritz-GFP fusion protein co-localizes with actin in predenticles.
Approach: I will express a Fritz-GFP fusion protein in specific sets of embryonic cells using the Drosophila Gal4-UAS system. Specifically I will cross a UAS-fritz-gfp line with Gal4 lines (e.g. NP5432) that express within the denticle cells of the ventral embryo. I will collect the Fritz-GFP expressing embryos and use conventional procedures to stain with an anti-actin primary antibody and a fluorescent secondary antibody. I will then use confocal microscopy to determine the degree of co-localization of Fritz-GFP with actin particularly within the denticle cells. I will make embryo extracts from Fritz-GFP expressing embryos, run then on denaturing acrylamide gels and Western blot by conventional procedure. I will optimize detection of Fritz-GFP on Western blots by an anti-GFP antibody. I will also investigate the effectiveness anti-GFP and anti-actin antibodies in an immunoprecipitation assay and develop a co-immunoprecipitation assay to test for physical interaction between Fritz-GFP and actin.
Outcomes: I expect to determine the degree of co-localization of a Fritz-GFP fusion protein with actin in the developing Drosophila embryo. I also expect to develop a co-immunoprecipitation assay to test for direct or indirect physical interactions between Fritz-GFP and actin proteins. The work should increase our understanding of the relationship between Fritz and the actin cytoskeleton. It will also establish the use of specific reagents (e.g. anti-GFP antibody) and techniques (e.g. immunoprecipitation and co-immunoprecipitation).
Budget: Undergraduate Student Stipend: $1,000