Project Title: High Resolution Imaging and Modeling of Protein Transport in Cilia
Project Duration: May 25 – August 1, 2015 (10 weeks), 40 hours per week.
- Karl F. Lechtreck Ph.D.
University of Georgia, 635C Biological Science Building
1000 Cedar Street, Athens, GA 30602
Phone 706-542 0167 (office) 706-542 2075 (lab)
Peter Kner Ph.D.
College of Engineering
329B Driftmier Engineering Center
- Graduate Student/PostDoc mentors (Name, Affiliation and Email/Phone):
Julie Craft, firstname.lastname@example.org, phone: 706-542 2075 (lab)
Project Description: The Kner and Lechtreck labs are interested in high resolution in vivo imaging. We are collaborating to analyze the transport of proteins inside cilia. Cilia are thread-like cell projections with important roles in motility and signaling; defective cilia cause numerous diseases ranging from blindness to polycystic kidney disease. Ciliary precursor proteins are transported by motor-driven carriers (“IFT trains”) from the cell body to the ciliary tip. At the tip the protein cargoes are unloaded and the IFT trains are remodeled so that they can return to the cell body. Because IFT trains arrive with a frequency of 1 Hz at the tip, the tip is usually crowded with proteins making a detailed analysis of cargo unloading and train remodeling difficult.
REU Student Role and Responsibility: How is cargo unloading linked to the remodeling of IFT trains at the ciliary tip? We hypothesize that cargo unloading is mechanistically linked to the brake-up and remodeling of the IFT trains. Heather Bomberger (Virginia Tech, Biological Systems Engineering), REU NanoBio student in the Summer of 2014, has developed and installed a ‘Laser gate’ for the TIRF microscope which allows for an improved imaging of individual IFT trains, their cargoes, and the fate of these proteins inside cilia. A strong laser beam is used to first bleach the cilium making the fluorescent protein-tagged IFT trains and their cargoes invisible. The spot-like laser beam is then placed at the ciliary base to bleach the fluorescence signal of all incoming trains. The gate will be briefly opened to allow for the entry of one or a few unbleached trains which can be tracked as they pass through the cilium; all subsequent trains will be bleached again as they enter the cilium.
The REU student 2015 is expected to continue work on the laser gate and measurements of IFT and IFT cargo using the gate. The student will continue the project by developing software to control the gate by monitoring transport in the cilium and by developing software to programmatically move the gate and bleach the cilium. As the project proceeds, the student will collect and analyze data on ciliary transport using the laser gate system. The student will also be involved in developing monte carlo simulations of one-dimensional ciliary transport in order to test different hypotheses of the length control mechanism in cilia.
Expected Outcome for REU student:
The student will be engaged in preparing a manuscript for the book METHODS IN MOLECULAR BIOLOGY: CILIA in the series Methods in Molecular Biology (Springer). Manuscripts are due in August 2015 and we have been invited to contribute a chapter. Depending on the quality of the data obtained by the student an original publication of the data will be considered.