Stability of Neuronal Microtubules to High Pressure In Vivo and In Vitro

Abstract

Neuronal microtubules in a variety of nerve cell types are unaffected by high hydrostatic pressures over a range of 1400-10,000 pounds/inch2 and periods of 10-45 min. Similarly, purified tubulin polymerized to form microtubules in vitro were not depolymerized by the same range of pressures. The depolymerization of microtubules in several types of non-neuronal cells, which has been reported, may have been over-generalized with regard to the direct action of pressure on microtubule stability.Within the past several years the knowledge of the structure and biochemistry of microtubules has advanced rapidly (1-3). In view of their ubiquity and their functional importance to the cell, a better understanding of the forces of interactions between tubulin subunits that result in polymerization-depolymerization will improve our concept of the physiological mechanisms of the control and action of microtubules.The commonly held view is that microtubules are sensitive to hydrostatic pressure (3)(4)(5)(6). This view is supported by reports that the microtubules of the mitotic apparatus are depolymerized by hydrostatic pressure in the range of 3000-3800 lbs./inch2 (7). Further, it was shown that the microtubules of the heliozoan axopodium are sensitive to high hydrostatic pressure (5). That is, pressures ranging from 4000 to 8000 lbs./inch2 caused a rapid disintegration of the microtubules of the axopodia and cytosome of Actinosphaerium nucleofilum. A wide variety of cells tend to become rounded and lose their motility when exposed to pressure at 4000-8000 lbs./inch2, including amoebae, human fibroblasts in tissue culture, various gate the direct effects of pressure on microtubule assembly. It was found that high hydrostatic pressure did not depolymerize either the microtubules of vertebrate neurons or microtubules in vitro, prepared from purified beef brain tubulin. MATERIALS AND METHODSThe pressure chamber used in this study was similar to the one designed by Landau and Thibodeau (8). This chamber permits tissues to be fixed under high pressure without decompression. Basically, it is two chambers in one, with the inner assembly consisting of two compartments separated by a thin cover glass. Living tissue is placed in one of the compartments. This compartment is separated from the compartment containing the fixative by the cover glass. The ends of the two compartments are covered with rubber diaphragms which allow the pressure to be transmitted equally to the tissue and fixative. The inner assembly, containing the tissue and fixative, is placed in a large outer pressure cylinder and the latter is then filled with water.The pressure cylinder was connected to a hand-operated hydraulic pump (American Instrument Co.). This assembly allowed compressions up to 10,000 lbs./inch2, with the pressures reached in less than 5 sec. After compression, the valve to the pressure cylinder was closed and the cylinder was disconnected from the pump. The cylinder with "sealed in" pressure was, after a period of time, inverted and shake...

High hydrostatic pressure effects in vivo: Changes in cell morphology, microtubule assembly, and actin organization

“…In vitro, at 37"C, 680 atm produces depolymerization of less than 15% of the tubulin assembled in the presence of a variety of microtubule-associated proteins (MAPs) [O'Conner et al, 1974;Salmon, 1975b;Engelborghs et al, 19761. In contrast, 320 atm produces nearly complete depolymerization of the CMTC in vivo.…”

Quantification and Properties of Tubulin Polymerization in Crude Brain Extracts and Preparations of Microtubular and Purified Tubulin

“…The increase in pressure produced by centrifugation at 100000 x g in the conditions described in Fig. 1 has been shown not to cause depolymerization of microtubules formed at 37 "C [16,17].…”

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