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Brief extraction with detergent induces the appearance of many plasma membrane-associated microtubules in hepatocytic cells

Journal of Cell Science, ISSN: 0021-9533, Vol: VOL. 68, Issue: 1, Page: 113-137
1984
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In cultured H35 hepatoma cells membrane-associated cortical networks have a microtrabecular appearance as revealed by dry-cleaving. Filaments having diameters of 15 nm can be readily distinguished within these networks and have not been described previously. Microtubules are seldom observed to be part of this structure. Extraction of cells with 0·l% Saponin in microtubule-stabilizing buffer produces holes in the membrane and reorganization of the networks resulting in the loss of microtrabecular structure, the loss of 15 nm filaments and the appearance of abundant membrane-associated microtubules (about l·25 μm per μm substrate-adherent membrane). These observations were confirmed by immunolabelling experiments with affinity-purified anti-tubulin immunoglobulin G. By both fluorescence microscopy and electron microscopy it was shown that labelled tubulin in the cortical networks became organized into microtubules upon treatment with detergent. By determination of the microtubule density, expressed as μm microtubule per μm membrane, the effects of various conditions on microtubule occurrence were determined. The Saponin-induced appearance of microtubules in the membrane-associated network coud be inhibited by: (l) l% and 2% glutaraldehyde, (2) 0°C, (3) millimolar Ca, (4) absence of Mg (subsequent reversal of inhibition by addition of Mg was shown), and (5) 20 μM-nocodazole (but not 20 μM-colchicine). In addition to Saponin, extraction with 0·l% Nonidet P-40 or 0·l% Triton X-l00 also resulted in microtubule-containing cortical networks. However, 0·l% Triton N-l0l was not effective, although holes were produced in the plasma membrane. These data provide evidence suggesting rapid polymerization of membrane-associated microtubule protein rather than detergent-induced displacement or collapse of existing microtubules. The arguments for this hypothesis and its implications are discussed.

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