No abstract

In human peripheral blood polymorphonuclear leukocytes and lymphocytes, GSH-oxidizing agents promote the movement of surface-bound concanavalin A (Con A) into caps and inhibit the assembly of microtubules (MT) that is normally induced by Con A binding. Con A capping and inhibition of MT assembly occur when GSH levels in cell suspensions are decreased by 30-70%, and return of GSH to control levels is accompanied by the appearance of cytoplasmic MT and by inhibition of the capping response with Con A. Oxidation of GSH markedly stimulates the hexose monophosphate shunt, and regeneration of GSH occurs rapidly.The data indicate that MT cannot be assembled or maintained in the face of decreased GSH levels. Thus, GSH homeostasis becomes critical during physiological events such as phagocytosis which simultaneously induce the assembly of MT and the production of agents like H202 that can oxidize GSH.Although a great deal of information is presently available about the conditions required for microtubule (MT) assembly in vitro, there is still no clear understanding of how assembly is regulated in vivo. Nath and Rebhun (21) have recently shown that oxidation of GSH in fertilized sea urchin eggs inhibits mitotic spindle formation and causes dissolution of preformed spindles. We proposed that intracellular GSH may also influence the organization and function of cytoplasmic MT. To test this hypothesis, we examined the effects of GSH oxidation on the assembly of MT in human peripheral blood polymorphonuclear leukocytes (PMN). Two relatively specific oxidizing agents were employed: diamide (diazene dicarboxylic and bis-N,N-dimethylamide) (15, 16) and BHP (tertiary butylhydroperoxide) (30).PMN were chosen for study first because MT assembly can be reproducibly induced by brief exposure of a variety of ligands including the plant lectin concanavalin A (Con A) (8,10,25), and second because the presence or absence of MT in Con A-treated PMN can be inferred from the distribution of surface-bound lectin. Thus, Con A shows a homogeneous surface distribution on cells that are competent to assemble MT but moves into a single aggregate-a cap-on the surface of cells treated with agents that bind with tubulin subunits and inhibit their polymerization (colchicine and the new carbamate anti-MT agent, R17934) (22-27).

The topographical distributions of concanavalin A-binding sites on the surfaces of 3T3, proteasetreated 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts appear to be different, as shown by a shadow-cast replica technique using concanavalin A and a hemocyanin marker, or as shown previously on isolated membranes with concanavalin A coupled to ferritin. However, chemical fixation of cells before labeling with concanavalin A and hemocyanin, or labeling exclusively at 40, allows one to distinguish between inherent concanavalin A-binding-site topography and potential rearrangement of sites induced by the action of the multivalent concanavalin A molecule itself. The inherent distribution of binding sites on 3T3, protease-treated 3T3, and transformed cells is actually the same on all cells, i.e., dispersed and random. Treatment of unfixed transformed or protease-treated 3T3 cells, but not normal 3T3 cells, with concanavalin A and hemocyanin at 370 (or at 40 with subsequent warming to 37°), however, results in clustering of binding sites, presumably due to crosslinking of neighboring lectin-binding sites by the quadrivalent concanavalin A. Thus, the underlying difference between concanavalin A-binding sites on normal as compared with transformed or protease-treated normal cells lies not in the inherent topography of binding sites, but rather in the susceptibility of the sites to aggregation by concanavalin A. The latter may reflect an increased mobility of lectin-binding sites on transformed or protease-treated cells.

The activities of specific transport systems were determined before and after large portions of the surface membrane had been interiorized by phagocytosis of inert particles. In five separate transport systems in rabbit polymorphonuclear leukocytes (adenosine and two adenine transport systems) and alveolar macrophages (adenosine and lysine transport systems), the rate of transport was unaffected even after an estimated 35–50% of the membrane had been internalized. Studies of the kinetics of lysine and adenosine transport, exchange diffusion of lysine transport in alveolar macrophages, and the specificities of adenine transport in polymorphonuclear leukocytes indicate that the nature of the membrane transport systems is not altered by phagocytosis. Therefore the constancy of transport indicates that the number of carriers remains the same before and after phagocytosis. It was also shown that this constancy of transport did not depend on the introduction into the surface of new transport sites during phagocytosis. Therefore transport sites are preserved on the surface during the internalization of membrane which accompanies phagocytosis. The results are best explained by the concept that the membrane is mosaic in character with geographically separate transport and phagocytic sites.

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentary. We have studied the assembly/ disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a steady state at pH <7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH-dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubuhn is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules . A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH .The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents) . However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable . Such preparations are readily disassembled by cold and podophyllotoxin (PLN) . Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of "treadmilling."The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo .