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The axonemal axis and Ca2+-induced asymmetry of active microtubule sliding in sea urchin sperm tails.

Structural studies of stationary principal bends and of definitive patterns of spontaneous microtubule sliding disruption permitted description of the bending axis in sea urchin sperm tail axonemes. Lytechinus pictus sperm were demembranated in a buffer containing Triton X-100 and EGTA. Subsequent resuspension in a reactivation buffer containing 0.4 mM CaCl2 and 1.0 mM MgATP2- resulted in quiescent, rather than motile, cells and each sperm tail axoneme took on an extreme, basal principal bend of 5.2 rad. Thereafter, such flagellar axonemes began to disrupt spontaneously into two subsets of microtubules by active sliding requiring ATP. Darkfield light microscopy demonstrated that subset "1" is composed of microtubules from the inside edge of the principal bend. Subset "2" is composed of microtubules from the outside edge of the principal bend and always scatters less light in darkfield than subset 1. Subset 2, which always slides in the proximal direction, relative to subset 1, results in a basal loop of microtubules, and the subset 2 loop is restricted to the bend plane during sliding disruption. Electron microscopy revealed that doublets 8, 9, 1, 2, 3 and the central pair comprise subset 1, and doublets 4, 5, the bridge, 6, and 7 comprise subset 2. The microtubules of isolated subset 2 are maintained in a circular arc in the absence of spoke-central pair interaction. Longitudinal sections show that the bending plane bisects the central pair. We therefore conclude that the bend plane passes through doublet 1 and the 5-6 bridge and that doublet 1 is at the inside edge of the principal bend. Experimental definition of the axis permits explicit discussion of the location of active axonemal components which result in Ca2+-induced stationary basal bends and explicit description of components responsible for alternating basal principal and reverse bends.

which always slides in the proximal direction, relative to subset 1, results in a basal loop of microtubules, and the subset 2 loop is restricted to the bend plane during sliding disruption. Electron microscopy revealed that doublets 8, 9, 1, 2, 3 and the central pair comprise subset 1, and doublets 4, 5, the bridge, 6, and 7 comprise subset 2. The microtubules of isolated subset 2 are maintained in a circular arc in the absence of spoke-central pair interaction. Longitudinal sections show that the bending plane bisects the central pair. We therefore conclude that the bend plane passes through doublet 1 and the 5-6 bridge and that doublet 1 is at the inside edge of the principal bend. Experimental definition of the axis permits explicit discussion of the location of active axonemal components which result in Ca2+-induced stationary basal bends and explicit description of components responsible for alternating basal principal and reverse bends.

WE
these studies to investigate a radial "switch-began ing model for regulation of active microtubule sliding in the formation of alternating basal bends in cilia and eukaryotic flagella (for review see reference 39). The hypothesis is that alternating principal and reverse basal bends are caused by the alternate activation of active microtubule sliding on opposite sides of the axoneme (40,45,47). This proposal is a consequence, in part, of the radial symmetry of dynein arms around ciliary and flagellar 9 + 2 axonemes and of the belief that the force generation of all dynein arms is unidirectional (37). Although the hypothesis is an oversimplification of the complexity of both radial and longitudinal patterns of active microtubule sliding that can be predicted for steady state bending waves in flagellar axonemes (18,(20)(21)(22)44), it is a useful model in analysis of regulation of patterns of active microtubule sliding in the formation of basal bends and in analysis of modulated patterns of microtubule sliding in Ca2+-modified waveforms (2,6,8,12,18,21,39). However, patterns of active microtubule sliding can be discussed in precise relationship to axonemal structures only if the axonemal axis is defined relative to the bending axis and bending direction.
Structural asymmetry, based upon the "5-6 bridge" and the central pair, has long been recognized for the axoneme in sea urchin sperm tails (1,14,15). This asymmetry in structure is the original basis for numbering doublet microtubules in this axoneme, but the axis of the axoneme in relationship to the bending plane and direction has not been determined. Rather, in the absence of data it has been assumed that the axis in the axoneme of the sea urchin sperm tail is the same as that in epithelial cilia which contain a 5-6 bridge and in which the axonemal axis, bending plane, and bending direction had been correlated (13,38). Considering the prominent role the sea urchin sperm cell continues to play in experimental determination of the mechanism of regulation of microtubule sliding in the formation of normal bending waves of the axoneme (3,4,17,22,42) and in determination of the mechanisms of calcium-induced modulation of normal bending (2,8,9,11,12,18,19,(27)(28)(29), it is important that the structural axis of this axoneme be precisely defined in relationship to the bending axis.
In the work reported here we have taken advantage of both structural markers in the axoneme of the sea urchin sperm tail and properties of the spontaneous, planar pattern of Ca 2+and ATP-induced sliding disruption of the axoneme to (a) determine the axonemal axis with respect to the bending axis of the flagellum, and (b) investigate the capability of the axoneme to restrict active sliding to one side of the axis in the formation of uniform, stationary basal bends. As assumed in earlier reports, we have found that the bending plane bisects the central pair and passes through doublets 1 and the 5-6 bridge, and that doublet 1 is located at the inside edge of the principal bend. A similar conclusion has been reached for the axis of the axoneme in starfish sperm (26). The patterns of axonemal fracture observed after spontaneous microtubule sliding are comparable to those described by Tamm and Tamm (45) in study of the sliding disintegration of the macrocitium from the ctenophore Bero~ and to those described by Olsen and Linck (30) of the sliding disintegration of rat sperm axonemes. These data are generally consistent with a radial "switching model" of basal bend formation in which opposite sides of the axoneme are alternatively active in formation of principal and reverse bends (39,40,47). The results were presented at the 25th Annual Meeting of the American Society for Cell Biology (35).

Materials and Methods
Lytechinus pictus sperm were obtained by injection with KCI, and undiluted semen was stored on ice until used. All subsequent procedures were carried out at 19-20"C. In a few cases sperm from Strongylocentrotus purpuratus were used.

Light Microscopy
Light microscopy was carried out using either a 100-W halogen or xenon strobe (Chadwick Helmuth Co., Inc., El Monte, CA) light source, green interference filter, Zeiss dark field ultracondensor (1.2/1.4 numerical aperture), and a water immersion objective lens (either 25x or 40x Plan Neofluar immersion lens, Carl Zeiss, Inc., Thornwood, NY). Cells were studied in suspension near the slide surface. However, precautions were taken in the assessment of cells attached to the slide surface since attachment often induced random patterns of ATP-induced sliding disruption.
For photomicroscopy, Tri-X film was exposed by single or multiple flashes of the xenon strobe and developed at 34"C in Diafine developer (Acufine, Inc., Chicago, IL). Video recordings made use of an MTI camera equipped with a newvicon tube or an MTI S.I.T.-66 camera (DAGE-MTI, Inc., Wabash, MI). Recordings were on l/2-in, video tape on the Panasonic NV 8050 time lapse recorder, and noise-free field by field analysis of motility was carried out with a Panasonic NV 8950 recorder (Panasonic Co., Secaucus, NY). In some cases image contrast from video recordings was enhanced using the Hughes Model 794 image processor (Hughes Aircraft Co., El Segundo, CA). Cell size, shape, and microtubule lengths were determined from tracings made directly from the video monitor at a final magnification of 3,000. (All size and length measurements listed in Table I were repeated from other experiments using prints made from 35-ram film to verify the accuracy of measurements made from the video monitor). Rates of sliding were determined from video recordings by timing the duration of sliding for measured averaged lengths of microtubule. Patterns of sliding disruption were determined by reconstruction from video recordings.

Electron Microscopy
Samples of demembranated cells were prepared for electron microscopy by diluting such a suspension with an equal volume of 2% glutaraldehyde made up in a buffer identical to that used for reactivation. After a 10-min incubation in fixative, cell samples were pelleted at 10,000 rpm for 15 rain in the Sorvall HB-4 swinging bucket rotor (DuPont Co., Wilmington, DE). (To obtain pellets of convenient size, the standard number of cells and volume of reactivation buffer was scaled up fourfold). Thereafter, pellets were overlaid with 1% glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.4), and fixed overnight. Samples were postfixed in 1% OsO4 in cacodylate buffer, dehydrated in a graded ethanol seres, and embedded in Epon-araldite. Uniform silver-grey sections were collected on formvar-coated, carbon-stabilized copper grids, and stained with uranyl acetate and lead citrate. Photomicrographs of cross-sections were taken by an unbiased investigator, and micrographs of the cross-sections were analyzed and scored by two different observers unaware of the sample treatment before fixation.

Breakage Assay
We developed a light microscopic assay for rapid, reproducible measurement of the degree of integrity of sperm tail axonemes vs. the deg~e of ATP-induced sliding disruption. This was necessary to facilitate large numbers of measurements for the variety of reactivation buffer conditions displayed in Table Il and Fig. 4. Demembranated cells were suspended as usual in petri dishes containing reactivation buffer of prescribed composition. After incubation for a predetermined length of time, the dishes were gently flooded with an equal volume of 2% glutaraldehyde in a buffer identical in composition to the reactivation buffer. After 5-rain fixation, aliquots of the supernate were transferred to a slide and a coverslip was then added. Using the ultracondensor and a Zeiss 25x Plan Apochromatic objective lens, 100 randomly selected cells were scored as "intact" (as in Fig. I a or Fig. I b) or "broken ~ (as in Fig. t, c-f). Each cell counted was carefully observed in three dimensions by fine focusing to be certain of the integrity of the axoneme. Cells were rarely damaged and distorted by adhesion to the substrate after fixation. Nevertheless, headless axonemes, transversely fractured axonemes, and excessively splayed axonemes were sometimes found and were not counted. Otherwise, all cells in each field were included in counts. Counts were carried out by two individuals who were unaware ofsample treatment before fixation, and all experiments were repeated at least four times. Data are displayed as the mean percentage broken. The assay is convenient and reproducible and the degree of breakage is comparable to that found by electron microscopic analyses of similarly treated samples.

Characterization of Stationary Principal Bends and the Spontaneous Microtubule Sliding Disruption
When sperm from L. pictus are demembranated and reactivated by the standard conditions described in Materials and Methods the cells become quiescent and take on uniform basal principal bends (Fig. 1 a). The principal bend has an average angle of 5.19 rad and a radius of curvature of -1.7 um (Table I). The distal axoneme has a slight reverse bend midway along the axoneme which gradually, over 1-5 min, increases in bend angle (iii, Fig. 1 b). The generation of the basal principal bend requires Ca 2÷ and ATP and can be relaxed by micromolar vanadate as reported earlier (12,34).
After incubation of-5 min, the sperm tail axonemes begin to break into two subsets of microtubules by sliding disruption (1 and 2, Fig. 1). Results of such disruption are illustrated in Fig. 1, c-f in which active sliding leads to partial separation ofaxonemes into subset 1 from the inside edge of the principal bend and subset 2, which slides from the periphery or outside Sale Axonemal Axis in Sea Urchin Sperm Figure 1. Darkfieid light micrographs of demembranated sperm ( L. pictus) cells suspended in the standard reactivation buffer, a is the characteristic shape of the Ca 2÷-and ATP-induced quiescent sea urchin sperm with a sharp basal principal bend and a relatively gentle reverse bend (iii in a) midway along the axoneme. After incubation the reverse bend increases in angle as illustrated in b. After ~5 rain (see time course Fig. 4) many of the sperm tails begin to break into two microtubule subsets (I and 2, in c-f) by active sliding. Subset 2, which scatters less light in darkfield, slides out from site x on the outside edge of the principal bend (c and f ) and always slides in the proximal direction relative to subset I. The symbols i, ii, iii, and iv refer to the approximate locations of the basal body, distal end of the principal bend, center of the reverse bend, and axonemal distal tip. The pattern of disruption is illustrated in Fig. 2. Bar, l0 vm.
edge of the principal bend as a loop. Subset 2 always slides proximally (large arrow in Fig. l, c and f ) and scatters less light in darkfield relative to subset 1. Subsets 1 and 2 usually remain attached at both the basal body and more distally at site "x", an approximate point within the basal bend where subsets 1 and 2 initially separate.
From videotape recordings we have reconstructed several features of the process and pattern of the spontaneous sliding disruption of the axoneme. Sequences of the two typical patterns are shown in Fig. 2. Sliding disruption is preceded by an increase in the angle of the reverse bend as described above (Fig. 1 b). This increase in angle lasts for various lengths of time before disruption. Initiation of sliding of subset 2 is immediately preceded by a "twitch" of the axoneme in which the angle of the principal bend fleetingly and slightly increases and correspondingly the reverse bend straightens (Fig. 2a, sequence :39 [5] to :40 [4]). Subset 2 then begins to slide and separates from subset 1 at site x: about mid-length along the arc of the principal bend (see Table I). Subset 2 slides at -12 #m/s forming a loop (curved arrows, Fig. 2). The remaining principal bend relaxes during transit of subset 2 resulting in the C-shaped arc of subset i bent in the principal bend direction (Figs. l c and 2a). The loop formed by subset 2 averaged 35 urn, whereas the axoneme averaged 40 um (Table  I): thus the subsets overlap by ~5 um after movement. The subset 2 loop often became twisted after sliding ( Fig. I d), or slid entirely off subset 1 and straightened (Fig, I e). Importantly, edge-on or end-on views revealed that subset 2 slides in the same plane as the principal bend during transit. Attachment of sperm tail axonemes to the slide surface often resulted in unrestricted sliding disruption of the axonemes, as illustrated in Fig. 3.
In many examples spontaneous sliding was accompanied by an apparent firm attachment of subsets 1 and 2 at the distal end of the axoneme (Figs. l f a n d 2b). In such cases, subset 2 slides proximally forming a loop at site x as usual but concomitantly compresses subset i, in "accordion fashion," at the principal and reverse bends (Fig. 2b). In these cases sliding also remains restricted to the bending plane, and the shear forces required for sliding are presumably restricted to the contact region just distal to site x. We have recorded several cases of this form of sliding disruption in which subsets 1 and 2 remain fused distally, but then, after proximal sliding, separate at site x. In such cases the axoneme elastically straightens after separation of subsets 1 and 2 at site x and then apparently re-anneal and the axoneme repeatedly undergoes the identical pattern of unipolar sliding each time recompressing the axoneme at the principal and reverse bends. In such repetitive sliding events, the relative polarity of active sliding between subsets 1 and 2 is maintained: under our standard conditions we have never witnessed subset 2 actively slide in the distal direction relative to subset 1 (>>1,000 examples observed).
The same patterns of disruption take place in sperm tails without heads. However, it appears that the basal body is necessary since the principal bend and breakage of headless tails require full length axonemes with a distinct bright "dot" at the proximal end of the axoneme as determined by darkfield light microscopy. We interpret the dot as the basal body and associated cellular structures.

Factors That Affect the Degree of Spontaneous Axonemal Sliding Disruption
Using the breakage assay described in Materials and Methods, we studied the time course and factors that affect the restricted pattern of breakage of the sperm tail axonemes. Under standard conditions breakage begins at ~5 min and reaches a plateau at ~15 min of 60-70% of the axonemes which undergo active sliding disruption ( Fig. 4; Table II). Thereafter, there is no significant increase in spontaneous sliding breakage. Therefore, 16 min was chosen as the stop point to test buffer conditions and other factors that affect the pattern or extent of sliding disruption. Breakage requires that the reactivation buffer contain ATP, dithiothreitol, and Ca 2+ (Table II). Without anyone of these ingredients the axonemes remain intact. Addition of 0.4 mM EGTA or reduction of added Ca 2+ to 0.1 mM also inhibited breakage. Interestingly, deletion of Mg 2+ and the addition of 0.1 mM EDTA (to chelate Mg 2+ transferred from the seawater) had no effect on the extent of ATP-induced sliding disruption. Furthermore, the removal of Mg 2÷ resulted in no apparent difference from the controls in the form of sliding disruption as revealed by direct observation and by video recordings. This suggested that CaATP 2-can support the spontaneous sliding separation of subsets I and 2. Reduction of ATP to 50 #M resulted in an increase of intact axonemes,   as revealed by the breakage assays, and such reduced concentrations of A T P resulted in renewed motility rather than quiescence as described earlier (4,12,34). The potential role of proteases in breakage was examined. Soybean trypsin inhibitor, antipapain, and leupeptin (at 25 and 50 gg/ml) had no effect on the form or extent of breakage under standard conditions. However, 25 or 50 ~g/ml of chick ovoinhibitor (Sigma T 1886) reduced breakage to 30% of control levels. This result suggested that an elastase-like protease may help provide for sliding disruption (3,45).
For suspended sperm tail axonemes in the standard reacti-   vation buffer containing 1 m M M g A T P 2-, addition ofelastase (8 or 16 #g/ml) promoted sliding disruption but did not change the pattern of sliding disruption described above or induce further unrestricted sliding of microtubules in subsets 1 and 2. However, microtubules of each subset will undergo further random unrestricted sliding disruption as illustrated in Fig. 3 if they become attached to the slide surface. Evidently restriction of sliding microtubules in subsets 1 and 2 can be overcome by the mechanical perturbation of adhesion. This result suggested that the restriction of sliding of doublets in subset 1 or 2 even after elastase treatment is not due to permanent impairment of dynein. At lowered MgATP 2-(5-50 uM), addition of elastase resulted in random, unrestricted disruption (similar to that illustrated in Fig. 3) of both suspended sperm tails and those stuck to the surface.

Electron Microscopy o f Demembranated Sea Urchin Sperm Cells
Cross-sections of sea urchin sperm tails confirm that the 5-6 bridge has a constant structural relationship to the central pair and therefore a constant relationship to all doublets (Fig.  5). Based upon study of stereo pair micrographs of replicas of quick-frozen, demembranated sea urchin sperm tails, we also confirmed that the dynein arms are oriented in a clockwise direction when cross-sections are viewed from the basal body toward axonemal tip (data not shown; see references 15 and 36). Other notable structural features of cross-sections are described in the legend of Fig. 5.
Longitudinal sections of the basal principal bend of quiescent demembranated sperm cells have revealed new details of the sperm head-tail attachment. The basal body terminates proximally in a dense staining cap-like structure (large arrow, Fig. 6) which, when viewed in the plane of the bend, is asymmetrically disposed and is skewed toward the inside of the principal bend. The basal body is connected to the sperm head by a prominent stamen-like array of filaments (small black-on-white arrowhead, Fig. 6 a). The basal body may also be connected to the sperm head by a less prominent collar of filaments. The region of the basal body-axoneme transition contains a plate-like structure (thin arrow, Fig. 6a) and an amorphic "necklace" of material presumed to be remnants of mitochondrial and plasma membrane attachments to the axoneme. Figure 6. Longitudinal sections of glutaraldehyde fixed, nondisrupted stationary principal bends (produced in the absence of calcium as described in reference 34). The plane which can be artificially constructed through the central microtubule pair is always perpendicular to the plane of bend. This is illustrated in a in which a profile of a single central microtubule is located midway in such longitudinal views, b is a longitudinal section passing tangentially to the bend and perpendicular to that of a (approximately in the plane described by the thin lines of a). As predicted, such orientations reveal the central pair in parallel (arrows in b). Notably the dense staining proximal end of the basal body (large arrow) is attached to the head by a prominent, stamen-like structure (arrowhead in a). The same prominent, stamen-like connection is seen in intact, nondemembranated sperm cells from both L. pictus and S. purpuratus whether prepared by conventional fixation procedures described in Materials and Methods or by quick freeze and freeze substitution methods (data not shown). Based upon serial section reconstruction of three axonemes, in longitudinal view, we have no evidence for twist of either the central pair or doublet microtubules in the region of the bend. Bar, 0.5 um.  (Table III) Table IlI). b is a pellet of demembranated, elastase-treated (8 ug/ml) sperm (S. purpuratus) suspended in the standard reactivation buffer. In this case subset 1 is predominantly comprised of doublets 8-4 and the central pair (87%, n = 132 cross-sections, two experiments). Fig. 8 c is a control sample of intact axonemes in which the reactivation buffer was devoid of calcium (L. pictus). A summary of frequencies and patterns of axonemal fracture is illustrated in Table   III. Bar, 0.2 urn.
The central microtubule pair sit side by side in the plane of the bend. This is illustrated in Fig. 6a in which a midlongitudinal section reveals the profile of only one of the central microtubules. Glancing longitudinal sections through bends and perpendicular to the plane of bend reveal paired central microtubules sitting side by side (Fig. 6 b). In examples studied so far prepared by conventional glutaraldehyde fixation, there is no evidence of twisting of either the axoneme or the central pair in the region of the basal bend.

Microtubule Patterns in Subsets 1 and 2
There is close correspondence of the percentage of spontaneously disrupted axonemes as determined by the "breakage assay" and by electron microscopy of cross sections (Table  II). The axonemes break into two complementary sets of microtubules: one set is usually comprised of doublets 8, 9, 1, 2, 3 and the central pair (1, Fig. 7a); a second complementary set is comprised of doublets 4, 5, the bridge, 6, and 7 (2, Fig. 7 a). Deviations from this pattern of breakage were generally limited to shifts of one doublet number such that subset t contained doublets 7-4, 8-4, 9-4, or 9-3 and the central pair (patterns and frequencies shown in Table III and Scheme I).
The frequency of patterns varied on occasion for unknown reasons (compare Exps. 1, 2, and 3 with Exp. 4 in Table III).
Since subset 1 scatters the most light when viewed by darkfield, we conclude that it is comprised of doublets 8-3 and the central pair. This conclusion is confirmed by longitudinal sections in the plane of bend of fractured axonemes and in whole mounted, negative-stained axonemes in which the central pair remained with the microtubules on the inside edge of the principal bend (data not shown). The spokes remain attached to the doublet of origin after fracture, and the orientation of central pair and 5-6 bridge relative to doublet positions does not change ( Fig. 7 and Scheme I). As described for the "breakage assay" electron microscopy revealed that Ca z÷ and ATP were required for axonemai breakage, and vanadate-treated samples remained intact (Fig. 7 c; Table II).
A restricted pattern of active sliding disruption similar to that described in Figs. 1 and 2 was found for the sperm of S.
purpuratus. However, under standard reactivation conditions spontaneous breakage was less frequent. We therefore treated the S. purpuratus sperm with elastase (8 or 16 #g/ml) which resulted in rapid sliding disruption into subsets 1 and 2. In this case subset 1 was uniformly comprised of doublets 8, 9, l, 2, 3, 4 and the central pair (Fig. 7 b), and subset 2 was variable and comprised of one, two, or three doublets. Presumably variability in subset 2 was due to continued disruption in the presence of elastase and ATP. Subset 2 * Experiments averaged because there was no significant difference in pattern frequency. * A small percentage of 9-3 and 9-4 subset 1 patterns were also found. This suggests the active dynein arms of doublet 8 can induce proximal sliding of subset 2.

The Axonemal Axis in Sea Urchin Sperm Tails
The results described here establish that the bending plane of sea urchin sperm tails bisects the central pair and passes through doublet 1 and the 5-6 bridge (Fig. 8). This conclusion is based upon longitudinal sections of basal bends parallel to the bend plane, upon longitudinal sections tangential to the bend and normal to the bend plane (Fig. 6), and upon the constant relationship of the central pair to the 5-6 bridge and therefore to the doublet microtubules (Figs. 5 and 8). Unlike the axonemes of certain cilia and flagella (24,32), we provide no evidence for the rotation of the central pair or twist in the axoneme in the region of the basal bend for the sea urchin sperm tails. In structural studies of stationary bends in Ciona sperm tails, Omoto and Brokaw (31) also conclude there is no axonemal twist in the region of the bend. However, there is other evidence for axonemal twist associated with bends in sea urchin sperm tails (16). It is possible that twists are associated with bend propagation and therefore not revealed in this study. The definitive, spontaneous sliding disruption of the axoneme in conjunction with structural markers in the axoneme and knowledge that the basal bend is the principal bend (2,12,19,20,22) has permitted determination of the relationship of axonemal organization to bending direction. High resolution darkfield light microscopy revealed that the larger set, subset 1, is located at the inside edge of the principal bend and the smaller set, subset 2, slides proximally on the convex, peripheral edge of the bend (Figs. 1 and 2). Subset 1 contains doublets 8, 9, 1, 2, 3 (or sometimes "8-4") and the central (arrows). Furthermore, given that microtubule subset 1 is toward the inside edge of the principal bend (based on darkfield observations and longitudinal sections of bends) then doublet 1 leads the inside edge of the principal bend. Therefore, the 5-6 bridge sits in the leading edge of the axoneme for the reverse bend. b shows the typical pattern of axonemal fracture due to sliding apart of subsets 1 and 2 and illustrates the maintenance of structural organization of isolated subsets 1 and 2. The stippled regions define the components predicted to actively generate the basal principal bend. (Fig. 8a, after Fig. 6 in reference 47).
pair (Figs. 7 and 8; Table III). Therefore, based upon the constant orientation of the central pair and the definitive pattern of microtubule sliding relative to the uniform, planar principal bend, doublet 1 is the inside edge of the principal bend. The 5-6 bridge is therefore the inside edge of the reverse bend (Fig. 8). Mohri et al. (26), using a different structural approach, determined that doublet 1 is the inside edge of the principal bend in starfish sperm. These conclusions for the sea urchin sperm have depended upon: the enantiomorphic structure of the axoneme; the Ca 2÷induced uniform and stationary principal bend; the identification, by high resolution darkfield light microscopy, of the inside and outside edges of the principal bend with subset 1 and subset 2, respectively; and the maintenance of structural relationships of the central pair, 5-6 bridge, and doublets of both intact and fractured axonemes. Of potential functional significance is the observation that the microtubules of isolated subset 2 are maintained in a semi-circle configuration (Fig. 7). Therefore, spoke-central pair interaction is not required for maintenance of the circular profile. The arch-like arrangement of the subset 2 may contribute to the maintenance of planarity and lack of torsion of subset 2 during active extrusion. (In experiments not reported here we have observed the dramatic helical coiling of doublet subsets after exposure to proteases and glutaraldehyde as described earlier. See references 25 and 48.) The axonemal axis described here for sea urchin sperm is virtually identical to that of cilia in the lateral epithelial cells of molluscan gills (l 3, 38). However, in sea urchin sperm the 5-6 bridge is the inside edge of the reverse bend direction, whereas in the epithelial cilia the 5-6 bridge is the inside edge of the effective bend direction. Furthermore, Ca2÷-induced arrest of sea urchin sperm tails results in a trapping of the axoneme at the end of the principal bend with doublet 1 on the inside edge of the bend. Similarly, the Ca2+-induced arrest of cilia of the lateral cells results in the trapping of cilia at the end of the reverse bend with doublet 1 at the inside edge (47). Therefore, relative to the axis and asymmetry of the axoneme, calcium stops the sperm tails and the lateral cell cilia in the same bend position with doublet 1 at the inside edge of the axoneme. This observation suggests a similar mechanism by which Ca 2÷ effects arrest for both the flagellum and cilium.
The axonemal axis of sea urchin sperm flagella can also be compared to the axis in Chlamydomonas flagella axonemes. The axoneme from Chlamydomonas flagella contains a "1-2 bridge" which is located at the inside edge of the reverse bend (23). Similarly, the 5-6 bridge of the sea urchin sperm tail is located at the inside edge of its reverse bend.

Formation of the Basal Principal Bend
As proposed by Gibbons and Gibbons (12) it is likely that the characteristic sharp stationary basal principal bend is a consequence of Ca2+-induced asymmetry of active dynein crossbridges around the 9 + 2 axoneme. Evidently Ca 2÷ and appropriate axonemal structures, possibly the radial spokes (see references 7 and 41, and further discussion below), induce a relaxed state in dynein arms responsible for reverse bend formation. (We have not addressed directly the molecular mechanisms through which Ca2+-induced asymmetry of bending and quiescence are attained. The biochemical pathway may involve CaATP 2-and asymmetrically distributed ATPases [28], calmodulin [8,33], or alteration of phosphoproteins [41,43,46].) Presumably, dynein arms on doublets of one side of the axonemal axis generate unidirectional shear forces that accumulate at the basal body and result in a corresponding unidirectional bend. Consistent with this proposal for stationary basal bend formation is that isolated segments of axonemes missing the basal body cannot generate the Ca2+-induced sharp basal bend and do not undergo the definitive, spontaneous sliding disruption described in this paper.
Based on the axis of the axoneme described here and the presumed unidirectional polarity of active microtubule sliding, we can predict the doublet microtubules which actively slide in the formation of the basal principal bend, and therefore we can suggest which doublets contain Ca2+-relaxed dynein cross-bridges. Under the standard conditions of demembranation and reactivation described in Materials and Methods, subset 2 always slides proximally relative to subset 1. Based on the assumption that all dynein arms generate force with the same polarity such that they "push" the adjacent doublet tipward (37), subset 2 must slide in the proximal direction by the activity of the dynein arms of doublet 7: that is subset 2 "walks" as a group along doublet 8 of subset 1. Since the subset 2 loop never actively slides in the reverse direction under our standard conditions we assume that the dynein arms of doublet 3 of subset 1 (or doublet 4 when subset 1 is comprised of doublets 8-4) are functionally quiescent. Therefore, based on identification of the axis of the axoneme (Fig. 8) and the unidirectional polarity of active sliding, we predict that the activity of doublets on the 6-9 slide of the axoneme (stippled area, Fig. 8) are generally responsible for the formation of the basal principal bend. This implies that calcium causes the dynein arms of doublets 1-4 to be relatively inactive or relaxed, unable to generate a reverse bend. The observation that the basal bend is maintained through most of the transit period and only relaxes near the termination of subset 2 sliding (Fig. 2) confirms that part of the forces that generate the bend are the sliding forces that propel subset 2. Buffer conditions have not been defined which would result in stationary reverse bends. The proposal that the basal bend is generated by doublets on the 6-9 side of the axoneme is consistent with a radial switching model of regulation of active sliding (39).
Certain observations in this paper are not completely consistent with a simple radial switching model of basal bend formation. After sliding and separation of subsets, subset l only partially relaxes and a substantial portion of the principal bend is retained (Figs. 1, c-e and 2 a). Therefore, factors other than the active cross-bridges of subset 2 contribute to the residual principal bend of subset 1. In other studies, it has been shown that high concentrations of vanadate (-millimolar) are required for reduced curvature of the stationary principal bend (12,28), and that in the presence of vanadate and ATP, Ca 2÷ generates a further increase in principal bend curvature (28). Therefore, it is likely that the residual curvature of principal bend of subset 1 is maintained by active forces and that those forces are different from those that reside between subsets 1 and 2 before breakage.
It is also not clear why isolated subsets 1 and 2 do not bend or twist out of the original bend plane. If it is assumed that active cross-bridges are evenly distributed on doublets 6, 7, 8, and 9, then the isolated subset 1 should continue to contain active arms on doublets 8 and 9. Based on a simple switching model, the active arms of doublets 8 and 9 should, assuming passive sliding of doublets 1 and 2, generate a bend or twist out of the original plane toward doublet 3 in the plane of the central pair. Such twists or deviations from the original bend plane are not found. A similar question can be asked about the isolated subset 2. Presumably the arms of doublet 6 remain active and those of doublet 4 quiescent. It would, therefore, be expected that subset 2 should bend or twist out of the original plane. Such twists do occur (see Fig. I d), however, if the completely detached subset 2 deviations from the original bend plane are not detected (Fig. I e). Presumably the central pair, 5-6 bridge, and other components partially impede twists or bends. Alternatively, regions of active cross-bridges may not be evenly distributed between doublets 6-9 (see below).
To this point, discussion has focused on the relationship of these data to a radial switching model of basal bending in the axoneme. I can also partially address the issue of longitudinal localization of regions of active sliding. The patterns of active breakage described in Fig. I f and 2 b result from active shear at only a small region of contact (<5 #m), site x, between subsets 1 and 2. These observations demonstrate that only a limited longitudinal segment of active cross-bridges, on doublet 7, is sufficient to maintain the basal bend in subset 1 and overcome elastic and active forces which resist bending. That the principal bend of subset 1 is bent and compressed by a restricted region of cross-bridge activity is supported by the observations of repetitive separation of subsets 1 and 2 at site x followed by straightening of the axoneme, re-annealing of The Journal of Cell Biology, Volume 102, 1986 subsets 1 and 2, and renewed unipolar sliding at the same region, site x, concurrent with renewed basal bending of subset 1. (With experimental measurement of the bending resistance of subset 1 [see Okuno,reference 27] it would be possible to calculate the sliding force in the region of contact at site x necessary to generate and maintain the basal bend.) The generation of the basal bend by active sliding at site x is consistent with other experiments that suggest local microtubule sliding is the basis for bending (see Shingyoji et al. [42]). Furthermore, as subset 2 is extruded, the sliding velocity appears to be relatively constant. Presumably, the number of dynein arms responsible for sliding decrease as subsets 1 and 2 separate. Since sliding velocity apparently does not decrease, then either elastic resistance to extrusion of subset 2 decreases proportionally to the decrease in the number of active dynein cross-bridges or active cross-bridges only exist at site x, and elastic resistance to sliding, after extrusion begins, is negligible.
It is not immediately apparent that the dynein arms of each doublet 6-9 are equally active in generating the basal principal bend of the Ca++-induced sperm tails since the fracture is always restricted to a site between a pair of doublets on the 6-9 side ofaxoneme. However, we do find variations in the site of fracture. The predominant site is between doublets 7 and 8 but we also find fractures between doublets 6 and 7 and, to a more limited extent, between doublets 8 and 9 (Table III). Therefore, to various degrees, the arms of doublets 6, 7, and 8 are active, in extrusion of subset 2. It is possible that the frequency of the site of fracture on the 6-9 side of the axoneme reflects the degree of cross-bridge activity between pairs of doublets. However, as discussed below, the site of fracture most likely reflects the site of greatest stress due to greatest interdoublet sliding during basal bend formation.
It is reasonable that the point of minimal radius og.curvature of the basal bend is a site of great outward stress. It is predictable that doublets located on the outer surface of the sharp bend, doublets 4, 5, 6, and 7, tend toward splitting away from the axoneme at the point of greatest curvature. The region of curvature contains site x (Fig. 1): the approximate point of initial separation of subsets 1 and 2. Furthermore, as illustrated in Table III and Scheme I, subset 2 is usually comprised of doublets that are symmetrically balanced around the bending axis, in particular doublets 4-7. If it is assumed that active cross-bridges are evenly distributed between doublets 6, 7, 8, and 9 on the active side of the axoneme and that interdoublet elastic links (nexin) are evenly distributed between all doublets, then we can predict, based on the axis described in this paper, that a planar bend would be expected to generate greatest interdoublet sliding between doublets 3 and 4 and 7 and 8; doublet pairs that are oriented parallel to the bending plane and the predominant sites of fracture. We predict that these points of greatest interdoublet sliding would be initial sites of fracture of nexin interdoublet links. Fracture of one nexin leads to fracture of the next, finally leading to sliding and relief of stress (see also discussion in reference 12). In effect, initial breakage of nexin links would result in planes of lowest resistance to sliding. No further disruption need take place upon relief of stress. (In many cases subset 2 is comprised of doublets 5-7. This combination of doublets is not symmetrical around the presumed bending axis. It is possible in these cases that the bend plane is actually skewed through doublet 6 or that the site of fracture on the 1-4 side of the axoneme is random.) Endogenous proteolytic activity may be required for spontaneous breakage. Chick ovoinhibitor partially blocks the sliding disruption, and elastase treatment, under standard buffer conditions, promotes a similar pattern of sliding disruption. The limited pattern of disruption observed may be due to restricted sites of proteolysis under the conditions used (for further discussion see reference 45). However, it is more likely that elastase-like enzymes disrupt various axonemal sites randomly, perhaps including the nexin interdoublet links (see reference 3), and the restricted pattern of fracture reflects a combination of restricted pattern of active dynein crossbridges and points of great mechanical stress as discussed above. This hypothesis is supported by the observation that the pattern of disruption is somewhat variable, therefore, active arms on doublets 6, 7, or 8 can effect proximal sliding of subset 2. Further it is unlikely that dynein arms of other doublets on the 1-4 side of the axoneme have in some way been permanently impaired, but rather are quiescent, since motility for intact axonemes can be restored by lowering the MgATP 2-concentration (12,34) and since random attachment of axonemes to the substrate can induced further random unrestricted sliding disruption (see Fig. 3).

The Role of the Radial Spoke System
Brokaw et al. (7) have concluded that the radial spoke system is primarily involved in inhibition of development of large curvature of reverse bends, therefore resulting in the normal asymmetric beating patterns of Chlamydomonas flagella. The results of this paper are consistent with such a proposal for the radial spoke system. Subset 1 maintains spoke-central pair attachments and also contains the doublets, numbers l, 2, and 3, which we predict are involved in generating reverse bends. Therefore, we can speculate that through the action of Ca 2+, the radial spoke system has, through an undetermined mechanism, restricted curvature formation of reverse bends. Complete inhibition of development of reverse bend curvature could, therefore, result in a quiescent flagella with a stationary, basal principal bend. Further study of the functional and chemical properties of isolated subset 1 vs. subset 2 may reveal details of the mechanism of the radial spokecentral pair system.