The giant freshwater amoeba Reticulomyxa has a central
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 unique 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, therefore, undertaken a determination of the MT-polarity in Reticulomyxa, using a modification of the tubulin hook-decoration technique according to Heidemann and McIntosh (2).
For polarity studies, cells were lysed in PHEM-buffer containing 1 mM Vanadate and up to 10% Hexylenglykol, 5% DMSO, 0,5% 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:  Schliwa et al., Nucleotide Specificities of Anterograde and Reterograde Organelle Transport in Reticulomyxa Are Indistinguishable. J. Cell Biol., 112, 1199 (1991).  Heidemann et al., Visualization of the structural polarity of microtubules. Nature, 286, 517 (1980).  Euteneuer et al., Microtubule bundles of Reticulomyxa networks are of uniform polarity. Eur. J. Cell Biol. 49, 373 (1989).