Cilia and flagella are cellular extensions that function in motility and sensing. Human sperm cells use cilia for locomotion and the multiple cilia of epithelial cells in the airways move foreign particles outward. Non-motile cilia in the eyes and nose function in the perception of light and chemical signals. Because cilia lack ribosomes, the RNA-protein particles responsible for protein synthesis, all proteins needed for cilia function or growth must be transported from the cell body into the protruding organelle. Intraflagellar transport (IFT) is one pathway for proteins transport from the base of the cilia, to the tip, and back again. Important steps of cilia assembly are thought to happen at the ciliary tip; cilia grow by the addition of subunits at the tip and IFT complexes are remodeled at the tip to allow their return to the cell body. The goal of my work is to develop an improved technique to image individual protein particles at the ciliary tip. Proteins of the IFT particles are made visible under the microscope via florescent protein (i.e. GFP) tagging. However, crowding of the particles at the ciliary tip largely obscures the visibility of individual proteins. A focused laser beam is used to bleach the fluorescence of some IFT particles at the tip. This increases the clarity for the remaining particles, but the tip is quickly refilled with florescent particles. We used a laser gate to control the bleaching laser; the laser blinks on and off at the base of the cilium in a pattern that will bleach most IFT particles but allows a few particles to pass unbleached through the gate, enter the cilium, and be imaged as individual particles while they complete their journey through the cilium. Further, we controlled the camera to not record while the bleaching laser is on; this will facilitate data analysis as it avoids recording of overexposed frames. This concept is carried out using a program written in Micro Manager and is communicated through an Arduino to the laser shutter. This program controls the laser shutter and the camera to capture movies of single IFT particles and their cargoes inside the cilia.
Heather Bomberger, Virginia Tech