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© The Rockefeller University Press, 0021-9525/1997//71 $5.00
The Journal of Cell Biology, Volume 136, Number 1, , 1997 71-80

Article

Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules



Erik A.C. Wiemer*, Thibaut Wenzel*, Thomas J. Deerinck{ddagger}, Mark H. Ellisman{ddagger}, and Suresh Subramani*

* Department of Biology, University of California at San Diego, La Jolla, California 92093-0322; and {ddagger} National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California at San Diego, La Jolla, California 92093-0608

Peroxisomes in living CV1 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 confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (~95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (~5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 µm/s and sustained directional velocities up to 0.45 µm/s over 11.5 µm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome 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 not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

Abbreviations used in this paper: CMV, cytomegalovirus; CV1, African green monkey kidney cells; GFP, green fluorescent protein; PMP, peroxisomal membrane protein; PTS, peroxisomal targeting signal.

The authors express their gratitude to Dr. Roger Tsien for making the GFP mutant (S65T) construct available for our studies, to Dr. Jon Singer for the use of his fluorescence microscope, and to Steve Lamont for technical assistance.

Address all correspondence to Dr. Suresh Subramani, Department of Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92039-0322. Tel.: (619) 534-2327. Fax: (619) 534-0053. e-mail: ssubramani{at}ucsd.edu


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