VISUALIZATION OF THE PEROXISOMAL COMPARTMENT IN LIVING MAMMALIAN-CELLS - DYNAMIC BEHAVIOR AND ASSOCIATION WITH MICROTUBULES

Peroxisomes in living CVI cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP-PTS1) . The organelle dynamics were examined and analyzed using time-lapse c onfocal laser scanning microscopy. Two types of movement could be dist inguished: a relatively slow, random, vibration-like movement displaye d by the majority (similar to 95 %) of the peroxisomes, and a saltator y, fast directional movement displayed by a small subset (similar to 5 %) Of the peroxisomes. In the latter instance, peak velocities up to 0.75 mu m/s and sustained directional velocities up to 0.45 mu m/s ove r 11.5 mu m were recorded. Only the directional type of motion appeare d to be energy dependent, whereas the vibrational movement continued e ven after the cells were depleted of energy. Treatment of cells, trans iently expressing GFP-PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisom e morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel , or the microfilament-destabilizing drugs cytochalasin B or D, did no t exert these effects. High resolution confocal analysis of cells expr essing GFP-PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP-PTS1-labe led peroxisomes were found to distribute themselves in a stochastic, r ather than ordered, manner to daughter cells at the time of mitosis.