VISUALIZATION OF THE GDP-DEPENDENT SWITCHING IN THE GROWTH POLARITY OF MICROTUBULES

Microtubules are filamentous polar polymers with plus and minus ends. This polarity plays a crucial role in a variety of cellular functions such as chromosome movement and organelle transport. To examine the re lationship between the growth polarity of microtubules and guanine nuc leotide dependence, we polymerized microtubules from axonemes of sea u rchin sperm flagella either with GTP or with GTP and GDP, and observed individual microtubules by dark-field microscopy. Tubulin concentrati ons were adjusted in each case to grow microtubules from only one end of each axoneme. The growth polarity of microtubules was determined us ing N-ethylmaleimide-modified tubulin (NEM-tubulin). Ln the presence o f GTP only and at low tubulin concentrations, microtubules grew from t he plus ends of axonemes. Surprisingly, in the presence of GTP and GDP , microtubules grew from the minus ends, even at high tubulin concentr ations. To confirm these results, we used a perfusion chamber to monit or the growth polarity of microtubules from the same axoneme under dif ferent conditions. Exchanging a solution containing only GTP for one c ontaining GTP and GDP elicited a switch in the growth polarity of micr otubules from the plus ends to the minus ends. These results suggest t hat GDP directly affects microtubule polymerization and inverts microt ubule growth polarity, probably by inhibiting microtubule growth at th e plus ends. (C)1998 Academic Press.