Axonemes isolated from the sperm of the sea urchin, Tripneustes gratilla, were briefly digested with trypsin. The digested axonemes retained their typical structure of a cylinder of nine doublet-tubules surrounding a pair of single tubules. The digestion modified the axonemes so that the subsequent addition of 0.1 mM ATP caused them to disintegrate actively into individual tubules and groups. The nucleotide specificity and divalent-cation requirements of this disintegration reaction paralleled those of flagellar motility, suggesting that the underlying mechanisms were closely related. Observations by dark-field microscopy showed that the disintegration resulted from active sliding between groups of the outer doublet-tubules, together with a tendency for the partially disintegrated axoneme to coil into a helix. Our evidence supports the hypothesis that the propagated bending waves of live-sperm tails are the result of ATP-induced shearing forces between outer tubules which, when resisted by the native structure, lead to localized sliding and generate an active bending moment.Although much is known about the detailed fine structure of the flagellar axoneme (1-5), there is relatively little evidence concerning the functional role of this stiucture in the mechanism of flagellar motility. In this paper, we report preliminary observations on the active disintegration produced when ATP is added to axonemes whose structure has been modified by brief digestion with trypsin. By studying the movements of the flagellar tubules during this disintegration, we have been able to obtain information about the nature of the mechanical forces induced by ATP. Our results strongly support the "sliding filament" model of flagellar bending (6, 7).Axonemes were isolated from sperm of the sea urchin, Tripneustes gratilla, by extraction and differential centrifugation in a solution containing 1% (w/v) Triton X-100, 0.1 M KCl, 5 mM MgSO4, 1 mM ATP, 1 mM dithiothreitol, 0.5 mM EDTA, and 10 miM Tris- Preliminary electron-microscopic examination of the digested preparations showed that the cylindrical structure of nine outer doublet tubules surrounding the two centraltubules remained largely intact in most axonemes (Fig. 1). Comparison with undigested preparations indicated that only slight structural changes resulted from digestion. The most apparent change was the disruption of the radial spokes near the point where they normally connect to the sheath surrounding the central tubules. Disruption of the nexin links that connect adjacent doublets (3, 5) was not directly apparent in the micrographs, but was revealed upon dialysis of digested axonemes against low concentrations of EDTA. The dynein arms on the doublets (3) appeared to be relatively resistant to the digestion. More detailed descriptions of the effects of digestion on the fine structure of the axoneme will be published elsewhere.Although the trypsin digestion did not itself destroy the basic structure of the axoneme, it modified it in such a way that the structure became hi...

Flagellar axonemes isolated from sea urchin sperm were digested with trypsin for various time periods . The course of digestion was monitored turbidimetrically and was found to take two different courses depending on the presence or absence of ATP in the digestion mixture . It was found that ATP induced active disintegration of the axonemes after slight digestion . Samples of the digested axonemes were examined with the electron microscope to determine the effects of trypsin digestion on the substructures of the axonemes . The rate at which trypsin sensitized the axonemes to ATP paralleled the rate at which it damaged the radial spokes and the nexin links, while the dynein arms were removed much more slowly . The results suggest that inactive dynein arms form cross bridges between the adjacent doublet tubules in digested axonemes, and that when activated by the addition of ATP, they induce an active shearing force between adjacent doublets . The radial spokes and the nexin links are not directly involved in the production of mechanical force, but they may participate in regulating the sliding between tubules to produce a propagated bending wave .

We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy . In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity of growth off cellular nucleating centers . We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca" or colchicine as measured directly by darkfield microscopy . The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell.KEY WORDS microtubules " darkfield microscopy -kinetochore " microtubule polarity During the cell cycle, microtubules are involved in many different functions. Often, several groups of microtubules will be present at one time, each with a unique time of appearance and disappearance in the cell (50,15) . In addition, microtubules of different stability have been described in several structures even within the same cell (5, 36). The mechanism by which the cell controls spatially and temporally the appearance, orientation, sta-J. CELL. BIOLOGY

Mitotic cells Iysed into solutions of polymerizable microtubule protein contain a spindle which is similar to the living spindle in two respects: it will lose and gain birefringence when cooled and warmed, and it will move anaphase chromosomes to the opposite ends of the cell. Early anaphase cells lysed into buffers containing high molecular weight polyethylene glycol and nucleotide triphosphates will continue chromosome motion and spindle elongation in the absence of exogenous spindle subunits. These results suggest that while spindle growth requires microtubule polymerization, anaphase motions do not.Considerable effort has gone into the study of cell-free preparations of the mitotic apparatus (MA). Much has been learned about the structure of the isolated MA (1-4), and something is known of its chemistry (5-12), but two interesting questions have remained unanswered: what is the character of the equilibrium between the spindle and its subunits, and what is the nature of the motors that move the chromosomes?Previous studies have isolated the MA by "stabilizing" it, i.e., making it reversibly less labile by lysing mitotic cells into buffers which are poor solvents for protein (9). In such buffers the MA will keep its birefringence and overall form for hours, but it will not shrink and regrow as a function of temperature the way the spindle does in situ (13). Since the work of Hoffman-Berling on chromosome motion in glycerol-extracted cells (14), no one has, to our knowledge, been able to study the physiology of anaphase-like processes in vitro.In this paper we describe an experimental system for studying spindle functions in vitro and present some of our early results. We have taken advantage of the recent discovery of techniques for the reassembly of isolated microtubule protein (tubulin) (15, 16) to design a procedure based upon an equilibrium between assembled and disassembled subunits. Independent investigations based upon the same experimental design have been initiated by two groups at Woods Hole, Massachusetts. Preliminary reports of their findings have recently appeared in abstract form (17, 18). We lyse mitotic Pt K1 cells with a nonionic detergent into buffers containing various concentrations of polymerizable tubulin, and we monitor the magnitude and longevity of spindle birefringence and spindle size with the light microscope. Our results show that the spindle is stable for more than 1 hr after lysis in solutions containing sufficient concentrations of tubulin. The Abbreviations: PIPES, Piperazine-N-N'-bis[2-ethane Sulfonic Acid]; EGTA, ethylene bis(oxyethylene-nitrilo) tetraacetic acid; GTP, guanosine triphosphate; GEP, GTP + EGTA + PIPES; ATP, adenosine triphosphate; Tx, Triton X-100; MA, mitotic apparatus. stabilized spindles will lose birefringence when cooled and regain at least some of it when rewarmed. Cells deprived of spindle birefringence by cold treatment and then lysed into tubulin solutions will regrow birefringence after lysis when the preparation is warmed to 370. Spindles pre...

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