Determination of Microtubule-Polarity in the Giant Amoeba Reticulomyxa Revealed by Hook-Decoration with Tubulin

RALF N. BREUKER and MANFRED HAUSER; Lehrstuhl für Zellmorphologie, Ruhr-Universität Bochum, 44780 Bochum, FRG

The giant freshwater amoeba Reticulomyxa has a multilobed cell body from which a microtubule assisted reticulopodial network extends, covering an area of several square centimetres. As reported earlier by Schliwa et al. (1), a unique dynein-like motor protein of 440 kD is obviously responsible for the bidirectional organelle transport within single filopodial strands.
There are at least two plausible explanations for the transport phenomena observed: The translocator is either capable of generating bidirectional movement or the filopodial microtubules are of mixed polarity, thus enabling anterograde and retrograde transport by a unidirectional force generator. Since no obvious microtubule-organizing centers have been found in Reticulomyxa that might specify MT-polarity, a study on MT-orientation seemed highly desirable. We have undertaken a determination of the MT-polarity in Reticulomyxa, using a modification of the tubulin hook-decoration technique according to Heidemann et al. (2).
For polarity studies, cells were lysed in 50% PHEM-buffer containing 1 mM Vanadate, 5% Hexylenglykol, 0,15% Brij 58 pH 6,9 for 1 min and subsequently rinsed several times in decoration buffer (0,5 M PIPES, 1 mM EGTA and 1 mM MgCl2) pH 6,9. After immersion in decoration buffer containing 1 mM GTP and 1,5 mg/ml pig brain tubulin at 35 0C for 20 min, preparations were fixed and epon-embedded. About 25% of all microtubules appeared decorated by single or multiple tubulin hooks. After careful examination about 10% of the decorated MTs appeared worth for further statistic evaluation of the direction of hook curvature. About 70% of the selected decorated MTs showed counterclockwise orientation when viewed towards the cell periphery. That means, polarity orientation is mostly the conventional "plus-end-distal" organization as in most other cell types. This confirms earlier results obtained by Euteneuer et al. (3) with a decoration technique using exogenous dyneins. Since under different experimental conditions tested so far, the number of decorated MTs was always rather modest we cannot exclude the possibility of a higher percentage of oppositely directed MTs.
References: [1] Schliwa et al., J. Cell Biol., 112, 1199 (1991). [2] Heidemann et al., Nature, 286, 517 (1980). [3] Euteneuer et al., Eur. J. Cell Biol. 49, 373 (1989).

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