Brief extraction with detergent induces the appearance of many plasma membrane-associated microtubules in hepatocytic cells.
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 0.1% 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 1.25 micron per micron2 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 micron microtubule per micron2 membrane, the effects of various conditions on microtubule occurrence were determined. The Saponin-induced appearance of microtubules in the membrane-associated network could be inhibited by: 1% and 2% glutaraldehyde, 0 degrees C, millimolar Ca2+, absence of Mg2+ (subsequent reversal of inhibition by addition of Mg2+ was shown), and 20 microM-nocodazole (but not 20 microM-colchicine). In addition to Saponin, extraction with 0.1% Nonidet P-40 or 0.1% Triton X-100 also resulted in microtubule-containing cortical networks. However, 0.1% Triton N-101 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.
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D002118:Calcium; D002462:Cell Membrane / Q000648:ultrastructure; D003078:Colchicine / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D005976:Glutaral; D007074:Immunoglobulin G; D008114:Liver Neoplasms, Experimental / Q000648:ultrastructure*; D008274:Magnesium; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure*; D015739:Nocodazole; D012503:Saponins / Q000494:pharmacology*; D013501:Surface-Active Agents / Q000494:pharmacology*; D013696:Temperature; D014404:Tubulin / Q000276:immunology
D001562:Benzimidazoles; D003902:Detergents; D007074:Immunoglobulin G; D012503:Saponins; D013501:Surface-Active Agents; D014404:Tubulin; D008274:Magnesium; D015739:Nocodazole; D003078:Colchicine; D002118:Calcium; D005976:Glutaral