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 .

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two-to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate.Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.In their pioneering work on glycosaminoglycan (GAG) ~ synthesis by cultured cells, Kraemer and Tobey (14) showed that a significant fraction of trypsin-releasable (i.e., surface) GAGs was shed during mitosis. Although the Chinese hamster ovary (CHO) cells analyzed were grown in suspension, viewed from the perspective of recent studies of cell adhesion (e.g., reference 32), it seemed likely that the shedding of GAGs was related to the familiar ease with which mitotic cells are detached from monolayers. We hypothesized that any effect of shedding would be reinforced by a coordinate reduction in GAG synthesis that would delay replacement of surface GAGs, and that a rapid resumption of GAG synthesis in early G i would be required for cell reattachment.A parallel motivation for our studies arose from the accumulating evidence that membrane vesicle-mediated transport is depressed during mitosis. This is shown clearly in work from our laboratory demonstrating depression of endocytosis ( 1, 2) and transferrin recycling (35) and from that of Warren and colleagues showing a decrease in surface transferrin receptors (41), and in depression of histamine secretion (9). These studies focus primarily on fusion and budding events at the plasma membrane. In addition, the insertion of the G t Abbreviations used in this paper. CHO, Chinese hamster ovary; GAG, glycosaminoglycan.protein of vesicular stomatitis virus is also depressed during mitosis (42), which suggests a defect in its processing through the Golgi apparatus. Since carbohydrate chains of GAGs are largely assembled within the Golgi apparatus by multiple enzymes (31), a general defect in processing should affect GAG synthesis. Moreover, the apparent localization of sulfation to the trans-Gol~ apparatus (7, 44) would serve to focus on later steps of the processing sequence.We describe here the rates of labeled sulfate incorporation into CHO cells during mitosis, in their subsequent movement into G1, and in late G2. We followed incorporat...

We studied the molecular form of tubulin in solution by ultrafiltration, nondenaturing electrophoresis, and chemical cross- linking. Our results are not consistent with the generally-held belief that tubulin in solution is a 110,000-mol-wt dimer. Rather, tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. We first compared the filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC- tubulin) with aldolase (142,000 mol wt). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperature and at a concentration of 0.5 mg/ml was only 0.12, whereas under the same conditions the filterability of aldolase was 0.60. We determined the average effective molecular weight of tubulin from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective molecular weight of approximately 300,000. This molecular weight was significantly reduced at 10 degrees C, indicating that oligomers dissociated at low temperatures. Oligomers were also demonstrated by chemical cross- linking using glutaraldehyde, dimethyl suberimidate, and bis[2- (succinimidooxycarbonyoxy)ethyl] sulfone. In addition, PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE system at alkaline pH. Quantitative examination of the mobilities of these bands and of standard proteins revealed that the bands represented a series of oligomeric forms. Similar electrophoretic patterns were observed in solutions of tubulin containing microtubule-associated proteins (MAPs) but with a shift to a greater proportion of higher oligomers. Nondenaturing PAGE at pH 8.3 showed that a shift towards higher oligomers also occurred in the absence of MAPs as the concentration of tubulin was increased. This concentration-dependence of oligomerization at room temperature was further demonstrated by ultrafiltration. When solutions of PC-tubulin at concentrations less than 0.25 mg/ml were ultrafiltered, filterability increased as concentration decreased. Quantitative studies of filterability following progressive dilution or concentration showed that this process was completely and rapidly reversible. A diffuse pattern of PC-tubulin on nondenaturing PAGE at pH 7 was observed and is consistent with a mixture of oligomers in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

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