Cell Motility and the Cytoskeleton 42:125–133 (1999) Bending Patterns of ATP-Reactivated Sea Urchin Sperm Flagella Following High Salt Extraction for Removal of Outer Dynein Arms Charles J. Brokaw* Division of Biology, California Institute of Technology, Pasadena Two procedures were used for extraction of demembranated sea urchin sperm flagella with increased KCl concentrations, to remove outer dynein arms. Extraction with 0.55 M KCl in the Triton-demembranation solution produced a rapid fall in average sliding velocity to 50% of its unextracted value, with extensive changes in bending behavior of the distal half of the flagellum. Extraction with 0.42 M KCl following demembranation and activation by incubation with cAMP produced a more gradual fall in sliding velocity, reaching 50% of the unextracted value after 180 sec extraction. This procedure produced somewhat more normal bending patterns. In both cases, the bending pattern of the basal region of the flagellum is altered very little by extraction, in agreement with data from Chlamydomonas mutant flagella deficient in outer arm dyneins. Cell Motil. Cytoskeleton 42:125–133, 1999. r 1999 Wiley-Liss, Inc. Key words: axoneme; image analysis; motility INTRODUCTION The movements of cilia and flagella are powered by the motor enzymes known as axonemal dyneins. Crosssection electron micrographs of flagellar axonemes typically show two projections on the A tubule of each outer doublet microtubule, extending towards the B tubule of the adjacent outer doublet. These projections, referred to as the inner and outer arms, were identified as the structural locus of the axonemal dyneins [reviewed by Gibbons, 1981]. Recent work [reviewed by Asai, 1995, 1996; Porter, 1996; Porter et al., 1996] has shown that the dyneins of inner and outer arms are biochemically, genetically, and structurally distinct. Functional differences between inner and outer arm dyneins were revealed by comparison of Chlamydomonas mutants deficient in either inner or outer arm dyneins [Brokaw and Kamiya, 1987]. The flagella of mutants lacking outer arm dyneins generate normal bending patterns at reduced frequencies. The flagella of mutants with partial inner arm deficiencies generate bending patterns with reduced amplitude, with frequencies only slightly less than normal. Because these Chlamydomonas flagella are short, they provide little opportunity for distinguishing between effects on bend initiation and bend propagation, which are more favor- r 1999 Wiley-Liss, Inc. ably studied using the long flagella of spermatozoa of sea urchins and tunicates. Reduction of beat frequency in flagella deficient in outer arm dynein was first demonstrated in sperm flagella of the Hawaiian sea urchin species Colobocentrotus atratus [Gibbons and Gibbons, 1973]. The usual procedure for membrane removal by Triton X-100 and reactivation of motility in solutions containing MgATP was modified, by increasing the KCl concentration of the Triton solution from 0.15 M to 0.50 M. Spermatozoa reactivated after extraction with 0.5 M KCl had beat frequencies about half the normal value (when reactivated with 1 mM ATP). Visual observations and photographs revealed no significant alterations in the form of the bending pattern. The decrease in frequency was correlated with disappearance of outer arms from the electron micrographs and solubilization of axonemal ATPase activity, indicating a rough proportionality between the beat frequency and the amount of dynein Contract grant sponsor: NIH; Contract grant number: GM 18711. *Correspondence to: C. J. Brokaw, Division of Biology, Caltech 156–29, Pasadena, CA 91125. E-mail: email@example.com Received 8 September 1998; accepted 3 November 1998 126 Brokaw retained in the axoneme. Later studies [Gibbons and Gibbons, 1979] demonstrated an increase in frequency following rebinding of ‘‘latent activity dynein’’ prepared from another sea urchin species (Tripneustes gratilla). Attempts to use other sea urchin species for this type of experiment have been disappointing [Ogawa et al., 1977; Gibbons and Gibbons, 1979], with the usual results including observation of a wide range of reduced beat frequencies and abnormal bending patterns following KCl extraction. However, Fox and Sale  found an improved procedure for KCl extraction of outer arm dynein from spermatozoa of the sea urchin Lytechinus pictus, which gave narrowly dispersed frequencies and ‘‘waveforms that were similar to those of intact outer arm axonemes.’’ They used an antibody specific for outer arm dynein to confirm the removal of outer arm dynein after brief KCl extraction. No quantitative analysis of bending pattern parameters has been made in any of these experiments with KCl-extracted sperm flagella. The present paper attempts to provide this analysis, using two different extraction procedures, one of which is similar to that of Fox and Sale . Although the quality of the reactivated preparations is less than optimal, some information can be obtained that reinforces the earlier conclusion that the major effect of outer arm dynein removal is on beat frequency and sliding velocity, without otherwise altering the generation of flagellar bends. MATERIALS AND METHODS Two procedures were used for demembranating and reactivating spermatozoa from the sea urchin, Lytechinus pictus. The first procedure was based on the standard procedure used in this laboratory [Brokaw, 1985, 1995]. Demembranation was carried out by diluting 1 to 2 µl of ‘‘dry’’ sperm suspension with 100 µl of solution containing 0.25 M KCl, 10 mM Tris buffer, 1 mM MgSO4, 1 mM DTT, 1 mM EGTA, and 0.04% Triton X-100, at pH 8.2. After 30 sec, this was mixed with 300 µl of activation solution containing 0.05 M KCl, 10 mM Tris buffer, 1 mM DTT, 0.5 mM MgSO4, 0.2 mM ATP, and 10 µM cAMP, and incubated for 180 sec. The standard procedure involves addition of 5 µl of 0.2 M CaCl2 during the last 20 or 30 sec of this incubation, but this was omitted in the present work. To prepare KCl-extracted spermatozoa, 50 µl of 3.0 M KCl was added at the end of the incubation in activation solution, to increase the KCl concentration to 0.42 M, and the incubation was continued for up to an additional 180 sec. A portion of the suspension was then diluted about 1:100 into reactivation solution for observation and recording. The second procedure, based on that of Fox and Sale , used the same Triton demembranation solution, except for the addition of 50 µM cAMP and 0.4 mM PMSF and an increase in Triton concentration to 0.05%. For KCl extraction, the KCl concentration of the Triton solution was increased to 0.55 M. After varying extraction times, the spermatozoa were diluted directly into reactivation solution. With both procedures, the reactivation solution contained 0.25 M K acetate, 20 mM Tris buffer, 1 mM DTT, 1 mM EDTA, 0.1 mM EGTA, 1.41 mM MgSO4, 0.135 mM ATP, and 0.5% polyethylene glycol, to obtain 0.3 mM Mg⫹⫹ and 0.1 mM MgATP at pH 8.2. All procedures were carried out at a room temperature of 18°C. Reactivated spermatozoa were photographed as described in previous work [Brokaw, 1984, 1986], while swimming at the upper surface of an open drop of reactivation solution, on a microscope stage maintained at 18° C. Multiple flash photographs, using flash rates of 50 or 120 sec⫺1, were obtained using moving film photography with a Grass C4R oscilloscope camera (Grass Instruments, Quincy, MA) and Kodak Tmax 3200 35 mm film. Portions of the transilluminated negatives containing 7 to 9 sperm images were digitally photographed as a 1,280 x 1,024 pixel image with a Pixera Pro digital camera (Pixera Corp., Los Gatos, CA) fitted with a Nikon 55 mm Micro-Nikkor lens and interfaced with a Macintosh PowerPC computer. The digital image was cropped and stored in TIFF format, without compression. It was then analyzed using a version of the public domain NIH Image program that was modified to include routines for tracing flagellar images using procedures of Brokaw . The datasets generated by this program, providing angular orientations of the flagellum at equal intervals along the length, were then analyzed by a program called FlagFitter, using procedures for parameter extraction by descriptive modelling described in previous work [Brokaw, 1984, 1996]. These programs are available at www.cco.caltech.edu/⬃brokawc/software.html. The descriptive model used for this analysis was the same one (‘‘Model 8’’) used for recent analyses of shear angle data from Ciona sperm flagella [Brokaw, 1996, 1997]. In contrast to earlier descriptive models used for sea urchin sperm flagella [Brokaw, 1984, 1991a], the newer model contains independent variables for propagation velocities of several transitions between bends, which allows modelling of variations in bending behavior along the length. However, since the angular orientation of the sperm head is not a reliable baseline for determining sliding in these sea urchin spermatozoa [Brokaw, 1991b], analysis was also performed in curvature mode [Brokaw, 1984], as in earlier work with sea urchin spermatozoa. For analyses in curvature mode, the synchronous shear amplitude parameter was fixed at 0, and there was no extra weighting of the basal region of the flagellum. The differences between the results obtained with these two fitting procedures were minor, and with the exception of the examples Bending Patterns of Sea Urchin Sperm Flagella 127 shown in Figures 3B and 4B, the results presented in this paper are from the curvature mode analyses. RESULTS Procedures for demembranation and reactivation of Lytechinus spermatozoa evolved gradually after the demonstration by Gibbons and Gibbons  that Triton X-100 was the reagent of choice for membrane removal, and have become significantly different from the early procedures used in the original outer arm extraction experiments with Colobocentrotus. The current standard procedure [Brokaw, 1985, 1995] includes incubation with cAMP and ATP to achieve full activation, exposure to millimolar Ca⫹⫹ to increase the symmetry of the reactivated movement, and reactivation in solutions containing K acetate instead of KCl [Gibbons et al., 1985]. Attempts to extract outer arm dynein using this standard procedure, with the addition of KCl to the Triton extraction solution or at the end of the incubation period produced unsatisfactory results. Somewhat better results were obtained by eliminating the exposure to millimolar Ca⫹⫹, and adding KCl at the end of the cAMP incubation step, to increase the KCl concentration to 0.42 M. Although this procedure does not give symmetric bending patterns, and the decrease in frequency and sliding velocity is slow, it gives better results than other variations that were examined. Extraction With 0.42 M KCl After Activation Results from the best experiment with this procedure are summarized in Figures 1–4. As shown in Figure 1, there are gradual decreases in frequency and computed average sliding velocity as the extraction time is increased up to 3 min, and both of these measures appear to level off at about half their initial values. The difference between the shapes of these two time courses is the result of small changes in shear amplitude. In usual experiments of this type with reactivated sperm flagella, the standard deviations for frequencies within a sample are less than 5% of the frequency. The samples usually show a high level of motility, with only small numbers of sperm beating abnormally, and these are not included in the measurements. With the KCl-extracted spermatozoa, the samples are different. There are many erratically beating spermatozoa, and only those with reasonably stable, vigorous beating are photographed for measurements. As seen in Figure 1, the standard deviations for beat frequency increase noticeably, to 10% or more of the frequency. Figure 2 shows plots of shear angle, curvature, and bending patterns representing the average values of parameters from the samples before extraction and after complete (180 sec) extraction. Figures 3 and 4 show curves for representative examples from each of these Fig. 1. Parameters obtained from descriptive modelling of reactivated sperm flagella, as a function of time of extraction with 0.42 M KCl. Sample sizes were 34 for the 0 time sample, and ranged from 20 to 24 for the extracted samples. Sample standard deviations are shown. In the propagation velocities panel, open points are for the second interbend transition and solid points are for the third interbend transition. In the attenuation panel, open points are for principal bends and solid points are for reverse bends. The attenuation parameters are defined in Brokaw [1994a]. 128 Brokaw Fig. 2. Extraction with 0.42 M KCl after demembranation and activation. Curves for shear angles and curvature as a function of position, and average bending patterns were computed using the average values of parameters obtained from descriptive modelling of the data sets. A: Obtained from analysis of the unextracted sample (n ⫽ 34). The average frequency was 19.0 sec-1 and the average sliding velocity was 111 rad sec-1. B: Obtained after 180 sec extraction with 0.42 M KCl. The average frequency was 9.9 sec-1 and the average sliding velocity was 55 rad sec-1. X-Y plots of bending patterns were obtained by integration of the shear curves, and manual adjustment of the orientations of the individual patterns to minimize overlaps. samples. More than half of the individual flagella in the unextracted sample displayed bending patterns very close to that shown by the example in Figure 3. However, for the extracted sample, there was a wide range of bending patterns, and there were only three good candidates that could be selected as having patterns close to the average pattern shown in Figure 2. The example is shown as a demonstration of the ability of the descriptive model to match the altered bending patterns obtained after extraction. The average values of shear amplitude for these two samples are almost the same: 2.92 radians before extraction (Fig. 2A) and 2.78 radians after extraction (Fig. 2B). The major difference between the bending patterns of Fig. 3. Results for a typical example of a sperm flagellum in the unextracted sample of Figure 2. A: Original data. B: Results of fitting a descriptive model in shear angle mode. C: Results of fitting a descriptive model in curvature mode. Image frequency 120 sec-1; beat frequency 20.0 sec-1. these two samples is shown more clearly by the curves of curvature vs. length in Figure 2. In the extracted sample, there is a large decrease in velocity of propagation of the third interbend transition, associated with an increase in the curvatures of both the Bending Patterns of Sea Urchin Sperm Flagella 129 appears only at the very end of the flagellum, when both the average curvature and the shear amplitude decrease (Fig. 2). Extraction also causes a gradual increase in asymmetry, which peaks in the 60-sec sample, and then declines so that the 180-sec sample shows only slightly greater asymmetry than the unextracted sample (Fig. 1). However, the large increases in standard deviations for asymmetry and attenuation (Fig. 1) attest to the diversity of bending patterns seen in the samples after extraction. Extraction With 0.55 M KCl in the Triton Extraction Solution Fig. 4. Results for an example of a sperm flagellum in the 180 sec extraction sample of Figure 2. A: Original data. B: Results of fitting a descriptive model in shear angle mode. C: Results of fitting a descriptive model in curvature mode. Image frequency 50 sec-1; beat frequency 10.5 sec-1. principal and reverse bends in the distal half of the flagellum. Since the propagation velocity of the second transition increases and dominates the calculation of average propagation velocity, or wavelength, this causes a relatively small decrease in wavelength. Attenuation Results in Figures 5 to 7 were obtained with an extraction procedure similar to that used by Fox and Sale , except that the KCl concentration was reduced to 0.55 M. Higher concentrations caused changes in the sperm head shape resulting from DNA extraction [Gibbons and Gibbons, 1973], which prevented accurate location of the head by the automated image analysis procedures. With this extraction procedure, there is a very rapid drop in frequency, which is interpreted to mean that extraction of outer arm dynein is complete within 30 sec. The decrease in frequency is less than 50%, but there is also a rapid decrease in shear amplitude, so that the decrease in computed sliding velocity is about 50%, and is similar to that obtained with 0.42 M KCl-extraction after demembranation and activation (Fig. 2). Figure 6 shows changes in average bending pattern following extraction with KCl in the Triton demembranation solution. The major change in transition propagation velocity seen with KCl-extraction after demembranation and activation (Fig. 2) also occurs here, except that the changes occur more rapidly, in association with the decreases in frequency and sliding velocity (Fig. 5). However, there is one major additional change, which is a strong attenuation of reverse bends. This already reaches a value of 1.5 (an attenuation value of 2.0 means that the reverse bend completely attenuates in 0.5 cycle) after 20 sec extraction, and increases to values near 2 for longer extraction times. A modification of this procedure was attempted, in order to reduce the asymmetry of the bending patterns. After the incubation in Triton, with or without 0.55 M KCl, the sperm suspension was mixed with two volumes of solution containing 0.10 M KCl and 2 mM CaCl2 and incubated for a further 30 sec. The results shown in Figure 7B were obtained by combining samples obtained following 30- and 50-sec extractions. Extractions for shorter or longer times gave results that were too heterogeneous to use. The Ca⫹⫹ extraction nearly eliminates the asymmetry in the basal region of the unextracted flagella, but has less effect on the asymmetry of the KCl-extracted flagella. The results are consistent with the results obtained without exposure to millimolar Ca⫹⫹, 130 Brokaw Fig. 6. Extraction with 0.55 M KCl in the Triton solution. A: Obtained from average parameters obtained from analysis of the unextracted sample (n ⫽ 43). The average frequency was 15.5 sec-1 and the average sliding velocity was 100 rad sec-1. B: Obtained from average parameters obtained from a sample after 60 sec extraction with 0.55 M KCl in the Triton solution (n ⫽ 21). The frequency was 9.6 sec-1 and the sliding velocity was 50 rad sec-1. X-Y plots of bending patterns were obtained by integration of the shear curves, and manual adjustment of the orientations of the individual patterns to minimize overlaps. and suggest that measuring asymmetric bending patterns does not give distorted results. DISCUSSION Fig. 5. Parameters obtained from descriptive modelling of reactivated sperm flagella, as a function of time of extraction with 0.55 M KCl in the Triton solution. The 0 time sample contains data from two samples extracted in Triton solution with the normal 0.25 M KCl concentration, for 30 and 180 sec, and the combined sample size was 43. Sample sizes ranged from 20 to 22 for the extracted samples. Sample standard deviations are shown. In the propagation velocities panel, open points are for the second interbend transition and solid points are for the third interbend transition. In the attenuation panel, open points are for principal bends and solid points are for reverse bends. The two extraction methods examined here are complementary. Extraction with 0.42 M KCl following demembranation and activation produces somewhat more normal bending patterns, without the strong attenuation of reverse bends that is seen after extraction with 0.55 M KCl in the Triton solution. However, extraction is gradual, and interpretation of the result obtained after 90 or 180 sec of extraction as the result of complete extraction of outer dynein arms would be tenuous without the results from extraction with 0.55 M KCl in the Triton solution, Bending Patterns of Sea Urchin Sperm Flagella 131 Control of the Bending Pattern Fig. 7. Effect of extraction with 0.55 M KCl in the Triton solution, followed by 30 sec exposure to millimollar Ca⫹⫹, to decrease asymmetry. A: Obtained from analysis of the unextracted sample. B: Obtained after 30 and 50 sec extraction with 0.55 M KCl in the Triton solution. For the unextracted sample, some parameters were: sample size, 23; frequency, 16.0 sec-1; sliding velocity, 98 rad sec-1; shear amplitude 3.07 rad. For the extracted sample, some parameters were: sample size, 45; frequency, 9.56 sec-1; sliding velocity, 46 rad/sec-1; shear amplitude 2.43 rad. X-Y plots of bending patterns were obtained by integration of the shear curves, and manual adjustment of the orientations of the individual patterns to minimize overlaps. which rapidly reach and remain at the same endpoint. In both cases, the assumption that these KCl extraction procedures selectively remove outer dynein arms is based on earlier work in other laboratories. This more detailed examination of bending patterns of reactivated sperm flagella following KCl extraction that is assumed to remove outer dynein arms reveals that, at least under the conditions utilized here, there are measurable changes in bending pattern. Although a major effect is a general degradation of the quality of movement and an increased variability in bending pattern and frequency, there are some consistent features of the extracted bending patterns. Observations on Chlamydomonas mutants deficient in either outer or inner arm dyneins led to the interpretation that outer arm dyneins increase the frequency of beating and provide much of the power for movement, while inner arm dyneins are responsible for determining the bending pattern [Brokaw and Kamiya, 1987; Brokaw, 1994b]. Is any modification of this conclusion required by the results in the present paper? The major effects of extraction on the bending pattern are seen in the distal half of the flagellum. Comparison of the bending patterns in the first 20 µm of flagellar length, in Figures 2, 6, and 7, show only minor differences. There are no consistent changes in the propagation of transitions in this region (the second transition). The shear amplitude differences may reflect differences in frequency that are constrained by the sliding velocity. The major bending pattern difference in this region is an increase in asymmetry. The increased asymmetry appears immediately when sliding velocity decreases immediately (Fig. 5) and more slowly when sliding velocity decreases gradually (Fig. 1), suggesting some correlation with outer arm dynein removal. However, there is a recovery towards more symmetric bending patterns with longer KCl extraction (Figs. 1, 5). An increase in asymmetry was also measured with Chlamydomonas mutants lacking outer arm dyneins, but not with the sup-pf-1 mutant that has a reduced frequency as a consequence of an altered ␤ heavy chain in its outer arm dyneins [Brokaw and Kamiya, 1987]. To the extent that the basal 20 to 25 µm of the sperm flagellar length can be expected to be more comparable with the flagella of Chlamydomonas, which are typically less than 15 µm in length, the results are consistent with the conclusions from observations of Chlamydomonas mutants deficient in outer arm dyneins. The regulation of bending pattern symmetry has been shown to be influenced by Ca⫹⫹ concentration and there is evidence for multiple Ca⫹⫹ sensors, only one of which appears to be calmodulin-like [Brokaw, 1991a]. Many observations on bending pattern symmetry can be equally well explained by two hypotheses: one, that Ca⫹⫹ has differential effects on dynein activity, the other that dynein activity is always symmetric and is superimposed on a ‘‘biased baseline’’ curvature that is determined by an independent contractile system [Eshel and Brokaw, 1987]. The latter hypothesis is supported by observations that Ca⫹⫹ can alter the shape of vanadate-inhibited flagella [Gibbons and Gibbons, 1980; Okuno and Brokaw, 1981a], but Lindemann and Kanous  have described how these observations might be rationalized with the former hypothesis. In the sup-pf-2 mutant of Chlamydomonas, a major asymmetry in outer dynein arm presence has no influence on the symmetry of the bending pattern [Rupp 132 Brokaw et al., 1996; Brokaw and Luck, 1985]. Consequently, at this time, it is not possible to relate an increase in asymmetry caused by outer dynein arm extraction to any viable model for regulation of bending pattern asymmetry. The changes in bending pattern in the distal region of the flagellum cannot be compared directly with observations on the short flagella of Chlamydomonas. The attenuation of reverse bends seen in the samples extracted with KCl in the Triton solution resembles attenuation seen with Lytechinus sperm flagella under other less than optimal conditions that did not involve outer arm dynein extraction [Okuno and Brokaw, 1981b]. Gibbons and Gibbons  described attenuation of bending waves following extraction with KCl in the Triton solution for times greater than 1 min. They noted the similarity to abnormalities seen under other conditions, notably those involving oxidation of sulfhydryl groups. There is no basis for deciding whether the effects seen in the present experiments are direct results of removal of outer arm dyneins, or an unrelated effect of exposure to high KCl concentrations. However, these changes do not alter the fundamental conclusion that the inner dynein arms are sufficient to initiate bending waves with near-normal parameters, except for reduced sliding velocity. Reduction of Sliding Velocity Since dynein is known to be a generator of microtubule sliding, sliding velocity is expected to be a fundamental parameter of its operation. Within an axoneme, the level of dynein activity might regulate the sliding velocity, but allow multiple combinations of frequency and shear amplitude that would have the same sliding velocity. However, the frequency of flagellar operation is a quantity that can be unambiguously measured, while sliding velocity varies with time and position within the axoneme. Defining an average sliding velocity requires some model for flagellar operation. In the present work, sliding velocity is calculated as twice the product of frequency and shear amplitude, thus averaging out temporal variations in velocity within a bending cycle. Shear amplitude is computed by ignoring spatial variations associated with synchronous sliding and changes in amplitude that occur in the distal region of the flagellum. It is expressed in angular units, radians, thus avoiding the issue of differences in sliding velocity between different outer doublet microtubules. In spite of the arbitrariness of this definition of average sliding velocity, the observations on KClextracted flagella provide some support for the idea that sliding velocity is a more fundamental parameter than frequency. The percentage change in sliding velocity following KCl-extraction is ⫺50% for both extraction procedures, but the percentage change in frequency is ⫺38% following extraction with 0.55 M KCl in Triton solution and ⫺48% with 0.42 M KCl extraction following activation. This difference, of course, corresponds to a decrease in shear amplitude following extraction with 0.55 M KCl in Triton solution. Previous studies [Gibbons and Gibbons, 1973; Fox and Sale, 1987; Hard et al., 1992] have shown that the percentage change in frequency following extraction of outer arm dynein decreases as the ATP concentration of the reactivation solution is reduced. No data are available to determine whether this is also true for sliding velocity, but the methods described in the present paper should be able to answer this question. ACKNOWLEDGMENTS I thank Sandra Nagayama and the late Bibi JentoftNilsen for invaluable assistance with these experiments. LITERATURE CITED Asai DJ. 1995. Multi-dynein hypothesis. 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