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Studies on reactivated cilia
Printed in Sweden Copyright ~ 1977by Academic Press, Inc. All rights of reproduction in anyform reserved ISSN 0014-4827
Experimental Cell Research 108 (1977) 311-320
STUDIES ON REACTIVATED
CILIA
II. Reactivation of Ciliated Cortices from the Oviduct
of Anolis Cristatellus L. D. TORRES, F. L. RENAUD and C. PORTOCARRERO Department o f Biology, University o f Puerto Rico, Rio Piedras, P R 00931, U S A
SUMMARY Reactivated ciliated cortices from the oviduct of the lizard Anolis cristatellus show symplectic metachrony, and resemble reactivated sea urchin sperm flagella in several characteristics, such as pH optimum and Km~, but differ in other, such as optimal ionic strength. Cilia isolated from the same source give similar results, but show a poorer reactivation. Purified ciliated cortices have adenylate kinase activity and an ATPase activity with a Km value identical with the K ~ , suggesting a tight coupling between ATP hydrolysis and movement in this system.
The chemical fine structure of cell movements involving microtubules is one of the most intriguing problems in contemporary cell biology. It has been known for a long time that microtubular structures, such as cilia and flagella, axostyles and the mitotic spindle, utilize ATP as the source of energy for movement. Recently, the chemical ultrastructure of these organelles has begun to be elucidated [3, 14, 17, 18]. However, a lot of gaps still remain in our knowledge o f how the macromolecules that compose these organelles interact in order to translate the hydrolysis of ATP into the physical displacement of their structural elements. In addition we know very little about h o w this movement is regulated. A useful approach to the solution of these problems has been the study of reactivated models, where the cell membrane has been disrupted or solubilized by extraction with glycerol or with the non-ionic detergent
Triton X-100 [4, 13]. This permits the reinitiation of movement by making exogenous ATP accessible to the motile apparatus, and in addition allows for manipulation of the environment in which the reactivation occurs. The best results have been obtained with Triton X-100 [3, 13, 18]; sea urchin sperm treated with this detergent become reactivated with a movement very similar to that of living, free-swimming sperm [13]. This has permitted a correlation to be made between the chemical properties of the flagellar axoneme and the quality of the reactivation, and to extrapolate these results to the in vivo situation. However, this approach has been used only scantily for the study of ciliary movement [10, 21, 25, 26]. Cilia and flagella are nearly identical in terms of ultrastructure, but in general differ in length, number and beat pattern [2]. Therefore, it is of interest to determine how similar these organelles are in their Exp Cell Res 108 (1977)
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Torres, Renaud and Portocarrero
response to various chemical changes. Renaud & Ramirez [20] have studied the reactivation of ciliated Tetrahymena ghosts obtained by homogenizing the cells with Triton X-100. The movement observed in this system was not coordinated and showed great variability in the percentage of motile ghosts. We have also studied the reactivation of ciliated cortical fractions from the oviduct of the tropical lizard Anolis cristateUus. The movement observed in this system is coordinated and the results are highly reproducible. Therefore we have utilized it to study the effect on ciliary movement of changes in the ionic environment in which the reactivation takes place. In addition, we have done preliminary measurements of some of the enzymatic properties of these fractions. A comparison is made between these results and those reported previously for sea urchin sperm flagella and other systems. A preliminary account of this work had been presented before [25].
Isolation of cilia Oviduct cilia were isolated by a modification of the method of Gibbons [ 10]. The transected oviducts were resuspended in glycerination solution (60 % glycerol, 25 mM KC1, 0.1% 2-mercaptoethanol and 20 mM TrisHC1, pH 8.3 at &C) and incubated in a salt-ice mixture at - 2 & C for 10 min. The cilia were then detached from the tissue by vigorous agitation with a Vortex mixer for 30 sec, and then incubated in the salt-ice bath for 30 additional seconds. This agitationincubation alternation was repeated three times. The tissue slices were then removed from the preparation, and the supernatant containing the cilia was used in the reactivation experiments described in the following section.
Reactivation of ciliated cortices and isolated cilia The preparations were reactivated by diluting 1 : 1 with reactivating solution (0.8 mM ATP, 75 mM KC1,
MATERIAL AND METHODS
Isolation of the ciliated cortices The method reported here is a modification of the procedure reported previously by Anderson [1] for the isolation of ciliated cortices from rabbit oviduct, and by Renaud & Ramirez [20] for the isolation of ciliated Tetrahymena ghosts. Adult females of the tropical lizard Anolis cristatellus was decapitated and their oviducts dissected and transferred to a small volume of Tyrode's solution in an ice bath. The tissue was then sliced longitudinally to expose the ciliated epithelium and resuspended in SET solution (0.25 M sucrose, 1.0 mM EDTA, 25 mM KCI, 0.05% Triton X-100, 0.1% 2-mercaptoethanol and 20 mM Tris-HC1, pH 8.8 at &C). The suspension was then agitated vigorously with a Vortex mixer for 30 see and incubated in an ice bath for 30 sec. This alternation of agitation and chilling was repeated four times, and the preparation was then centrifuged for 5 rain at 1000 g. The sediment, consisting mostly of nuclei and isolated cortices, was resuspended in SE solution (identical with the SET solution, but omitting the Triton X-100), centrifuged for 5 min at 1000 g and resuspended in fresh SE solution.
Exp Cell Res 108 (1977)
Fig. 1. Cross section of the ciliated epithelium of the Anolis oviduct. Note the cilia (arrow) lining the apical surface of the cell N, nucleus, x 2 500. Fig. 2. Detached ciliated cortices from the Anolis oviduct (arrows). Note that the nuclei (N) are the main visible contaminant of the preparation, x 1600.
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Table 2. The effect o f A D P on the reactivation o f oviduct ciliated cortices ADP conc. 0.4 0.8 1.0
Beat frequency (Hz)
% of reactivation
Duration of movement (min)
0 0.2 0.3
0 15 20
0 15 15
Nucleotide concentration. The minimal concentration of ATP tested that enabled the reactivation to take place was 0.025 mM. Increasing the concentration of the nucleotide gives a concomitant increase in the parameters measured, with an optimal concentration of 0.5 mM (fig. 5). Further increases in the concentration of the nucleotide result in an inhibition of the reactivation. The changes in beat frequency at different ATP concentrations are illustrated in fig. 6. Again we can see that optimal results are obtained at a concentration of 0.5 mM, giving a beat frequency of 5 Hz. Higher concentrations of ATP decreased the amplitude of the wave, and the movement observed was more vibratory than undulatory. Notice that the values obtained from film studies coincide very nicely with those obtained by visual estimation. A double reciprocal plot of the beat frequency and the ATP concentration gives a straight line (fig. 7). Extrapolation of this line serves to measure a constant, the so called Kmy, which in this case has a value of 0.13 mM. When the cortices are diluted with a reactivating solution containing ADP instead of ATP, no movement is observed if the nucleotide concentration is 0.4 mM or lower. If the ADP concentration is increased to 0.8 mM or higher, a reactivation is observed, but only after a lag period of 10 min (table 2). The type of movement is similar to that observed previously with ATP, but Exp Cell Res 108 (1977)
the reactivation is inferior in terms of all three parameters measured. For example, the beat frequency is less than one Hz as opposed to 4-5 H z with ATP under optimal conditions.
Reactivation of isolated cilia The optimal reactivation of isolated cilia was obtained by mixing 1 vol of the glycerinated suspension with 2 vol of the reactivating solution. The movement observed consists of sinusoidal waves that move from one end to the other of the cilium. However, roughly 10% of the cilia become attached to the slide by o n e end, and their movement is of the whiplash type, as in the cortices. Variations in the components of the reactivation solution gave s o m e results t h a t closely parallel those obtained for the ciliated cortices. The optimal pH in terms of the duration of movement is 7.7, which is very close to 8.1, the optimal pH for the cortices (fig. 8). In addition, the optimal concentration of KCI is 50 mM in both cases (fig. 9). The cilia could also be reactivated with calcium and manganese, in addition to magnesium, and the optimal concentrations of these cations were very similar to the optimal ones for the reactivation of the cortices (L. D. Torres, unpublished results).
1.6"
0.8=
,e'
J
Fig. 7. Abscissa: reciprocal values of the ATP conc.; ordinate: reciprocal values of the beat frequency. Double reciprocal plot of the beat frequency of the ciliated cortices at different ATP concentrations, the extrapolated value of the K,~t is 0.13 raM.
Reactivated oviduct cilia appreciably, and resulted in an increased breakage of the latter.
Reactivation o f the ciliated cortices When the cortex suspension in SE is diluted with reactivating solution containing optimal concentrations of its components, one can observe a reactivation of 80 % of the cortices. No movement is observed if the washing with SE is omitted. All the cilia become motile in 90% of the reactivated cortices, and i n t h e remaining 10% only a few cilia move per cortex, particularly in the case of cortices that have been fragmented during the isolation procedure. The movement in the intact cortices is coordinated and shows the symplectic pattern of metachronal coordination, i.e., the plane of beat coincides with that of the propagation of the wave [2]. Effect o f several variables on the reactivation pH. The effect of changes in pH on the percentage of reactivation and the duration of novement is illustrated in fig. 3. The results ;how a sharp optimum at a pH of 8.1, where 30 % of the cortices become reactivated for m average of 250 min; an initial beat frequency of 4-5 Hz was observed. All these ~arameters decrease rapidly at higher or ower concentrations of hydrogen ions, including the beat frequency which is reduced :o about 1 Hz at both pH 6 and 9. Ionic strength. Changes in the concentraion of KCI in the reactivation mixture give he results illustrated in fig. 4; namely, a )harp optimum at a concentration of 50 mM 'or all the parameters measured. However, he decrease in the percentage of reactivaion and in the duration of movement was ess sharp at higher KC1 concentrations han at lower ones.
315
6.0-
2.0
0~2
0'.s
Fig. 6. Abscissa: final ATP conc. (mM); ordinate:
beats/sec (Hz). 0 , Values measured directly; ©, values determined from f'flm studies. Variation in the beat frequency of the ciliated cortices with changes in the concentration of ATP in the reactivation mixture.
Divalent cations. No movement is observed in the absence of divalent cations. The eiTect of the addition of various concentrations of manganese and calcium ions is illustrated in table 1. The optimal concentration of calcium and magnesium is 1.0 mM in terms of the duration of movement and the percentage of reactivation, whereas that of manganese is 0.5 mM. However, magnesium was the most effective cation since it can be seen that both parameters measured are reduced considerably with calcium and manganese even at their optimal concentrations. On the other hand, the optimal concentrations of these cations in terms of beat frequency do not necessarily coincide with the optimal values for the other parameters measured, being 1.0, 2.0 and 0.75 mM for magnesium, calcium and manganese, respectively. The best results are again obtained with magnesium ions that give a beat frequency of 5 Hz, whereas with calcium and manganese one only observes 2 Hz. Exp Cell Res 108 (1977)
314
Torres, Renaud and Portocarrero
Table 1. The effect of various divalent cations on the reactivation of oviduct ciliated cortices
Cation Mgz+ Ca~+ Mn2+
Conc. (mM)
% of reactivation
Duration of movement (min)
0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0
20 80 20 0 50 35 20 0 0
150 280 100 0 125 100 140 0 0
fuge. The nuclei sediment to the bottom of the tube, whereas the ciliated cortices can be collected at the interphase between the KEM and the sucrose solutions.
Enzymatic activity of the ciliated cortices The ATPase and adenylate kinase activity of the purified cortices was determined by measuring the release of inorganic phosphate by the technique of Fiske & Subbarow [8]. One volume of the suspension of purified cortices was mixed with 1 vol of reactivation solution in a series of test tubes in a water bath at 25°C, and the reaction stopped at different time intervals by the addition of molybdate and Fiske-Subbarow reagents. The absorbance at 660 nm was measured with a Zeiss PM Q II spectrophotometer. The protein concentration was measured by the technique of Lowry et al. [16] using bovine serum albumin as a standard. RESULTS
sist m a i n l y o f the ciliary r o w s a t t a c h e d to the basal bodies. N o ultrastructural studies w e r e p e r f o r m e d , t h e r e f o r e it is n o t k n o w n if t h e y c o n t a i n ciliary rootlets a n d r e m n a n t s o f the cell m e m b r a n e , as is the c a s e with rabbit o v i d u c t c o r t i c e s [1]. T h e m a i n visible c o n t a m i n a n t s p r e s e n t in the p r e p a r a t i o n are the nuclei (fig. 2), w h i c h are resistant to solubilization b y Triton X-100. T h e c o r t e x yield v a r i e d d e p e n d i n g o n several factors. The optimal detergent concentration was b e t w e e n 0.02 and 0.06 %. H i g h e r c o n c e n t r a tions c a u s e d a disintegration o f the cortices, and l o w e r ones r e d u c e d the n u m b e r o f cortices liberated. O m i s s i o n o f the chelating agent E D T A also r e d u c e d the c o r t e x yield, and t h o s e that w e r e p r o d u c e d s e e m e d to be c o n t a m i n a t e d with c o a g u l a t e d c y t o p l a s m . A m o r e p r o l o n g e d t r e a t m e n t with the V o r t e x m i x e r did n o t i n c r e a s e the yield o f c o r t i c e s
225"
~
a
125i 75-
Isolation of ciliated cortices A cross section o f the ciliated epithelium o f the Anolis o v i d u c t is illustrated in fig. 1. T h e cilia are a p p r o x . 10--15 /zm long; and are located at the apical end o f the epithelial cells, lining the l u m e n o f the organ, particularly in its anterior region. W h e n the o v i d u c t slices are agitated in the V o r t e x mixer, the apical b o r d e r o f the ciliated cell b e c o m e s d e t a c h e d as a discrete unit (fig. 2), T h e s e isolated b o r d e r s will be r e f e r r e d to as cortices [1]. T h e s e structures s e e m to conExp Cell Res 108 (1977)
b 25 0%
~'o
Fig. 5. Abscissa: final ATP cone. (mM); ord/nate: (a) % reactivation; (b) time (rain). The duration of movement (a) min; (b) the per, centage of reactivation of the ciliated cortices at dif ferent concentrations of ATP.
R e a c t i v a t e d o v i d u c t cilia
3!3
projected at slow motion. The movies were taken with a Beaulieu 2008-S camera, using Kodak Plus-X Super 8 film.
P u r i f i c a t i o n o f ciliated c o r t i c e s
40-
6.0
9.0
These organelles were purified by a modification of the technique developed by Forstner et al. [9] for the purification of isolated brush borders. After this procedure, the cortices could still be reactivated and were suitable for enzymatic analysis. The suspension consisting of nuclei and ciliated cortices was diluted fivefold with EM solution (2.5 mM EDTA, 0.1% 2-mercaptoethanol, pH 7.0) and then centrifuged at 1000 g for 5 min. The sediment was then resuspended in KEM solution (100 mM KCI, 0.8 mM EDTA, 0.1% 2-mercaptoethanol, pH 7.0), and incubated in an ice bath for 15 min. Under these conditions, the nuclei aggregate in small clumps, whereas the majority of the ciliated cortices are not affected. The suspension is then filtered through glass wool to remove the nuclear aggregates, and the filtrate is then chased with a small volume of KEM solution. Any remaining nuclei are then removed from the preparation by layering the filtrate on top of a buffered sucrose solution (1.8 M sucrose, 0.1 M KCI, 0.1% 2-mercaptoethanol and 20 mM imidazole-HCl, pH 7.0) and centrifuging at 53000 g for 2 h, using a Beckman L3-50 Ultracentri-
Fig. 3. Abscissa: pH; ordinate: (a) % reactivation; (b) time (rain). The effect of changes in the pH of the reactivation medium on (a) the percentage of reactivation; (b) the duration of movement of the ciliated cortices.
8 11
2.5 mM Mg SO4, 0.1% 2-mercaptoethanol and 20 mM Tris-HC1, pH 8.5). In some experiments the reactivating solution was altered in order to determine the optimal concentration of its components by observing the effect of concentration changes on the quality of the reactivation. The Tris-HC1 buffer was substituted by imidazole-HC1 for pH values below 7. Magnesium ions were sometimes substituted by calcium and man:anese, and ATP by ADP. All observations were made with a Zeiss phase-contrast microscope with Neofluar 9bjectives at a total magnification of 800×. Each experiment was repeated at least twice and the results were plotted tracing the best line by eye; only one ~aint is illustrated if the results for that particular ~oint coincided in duplicate experiments. The paratinters measured were the percentage of reactivation, '.he duration of movement and the beat frequency. We l¢fine percentage of reactivation as the percentage of solated cilia or ciliated cortices showing movement in madomly selected fields. Each sample consisted of ~ r o x . 50 cortices or 25-30 cilia. The end point of the mbetivation period was arbitrarily chosen as the time which 80 % of the isolated cilia or the ciliated corbees had ceased to move. The beat frequency was ~ u r e d by visual estimation, and in the case of the iliated cortices, by analysis of films of the reactivation
b
~ ~ s I00"
200a
50
~00
Fig. 4. Abscissa: final KCI conc. (raM); ordinate: (a) % reactivation; (b) time (rain). The effect of changes in the concentration of KCI in the reactivation medium on (a) the percentage of reactivation, and (b) the duration of movement (min).
Exp CellRes 108 (1977)
Reactivated oviduct cilia
Fig. 8. Abscissa: pH; ordinate: time (min). O, Free-
swimming; A, slide-attached cilia. The duration of the reactivation of isolated oviduct cilia at different pH values.
However, the beat frequency and the duration of movement is in all cases inferior to that obtained for the ciliated cortices. For example, under optimal conditions ciliated cortices will move for over 2 h, whereas free-swimming isolated cilia will do so for only 25 rain. It is of interest to note that when the cilia become attached to the slide the duration of the movement is duplicated (fig. 8). However, this did not result in an increased beat frequency. Isolated cilia differ from ciliated cortices in their response to changes in the concentration of ATP in the reactivation medium. No movement was observed at a nucleotide concentration of 0.025 mM, in contrast with the ciliated cortices that are capable of movement at this concentration. Reactivation was observed at ATP concentrations of 0.05 mM or higher, with optimal results at 0.2 mM. Concentrations higher than this value inhibited movement; thus, the optimal concentration for the reactivation of cortices inhibits the reactivation of isolated cilia.
317
altered by the addition of various concentrations of ouabain or oligomycin to the reactivation medium. A Lineweaver-Burk plot of the ATPase activity is illustrated in fig. 10, giving an extrapolated Km of 0.13 mM. This value coincides with that of the K,e mentioned previously. The liberation of inorganic phosphate by the ciliated cortices in the presence of 1 mM ADP is illustrated in fig. 11. Notice that the rate is slow during the first 10 min, increasing dramatically afterwards. The time period of the slow initial rate coincides with the lag period before movement is observed in the cortices. DISCUSSION There are two things worth noticing when comparing the reactivation of ciliated cortices with the observations made in situ and in other systems. First of all, the fact that the movement of the cortices is coordinated, showing symplectic metachrony, suggests that the mechanism of coordination is independent of the integrity of the cell, and is possibly due to viscous interactions through the medium among adjacent cilia. A similar conclusion has been reached by other workers [2], although this theory cannot explain the results obtained with the reactivated flagellar apparatus of Chlamydomonas [5]. Observations made in preli-
25"
Enzymatic activity of the ciliated cortices At an ATP concentration of 0.5 mM, the specific activity of the ATPase of the purified cortices was 0.25/zmoles of inorganic phosphate min -1 mg -1. The results were not 21-771802
Fig. 9. Abscissa: finalKCI conc.;ordinate: time (rain). $, Free-swimming;A, slide-attachedcilia. The duration of the reactivationof isolated oviduct cilia at various concentrationsof KCI. Exp Cell Res 108 (1977)
Torres, Renaud and Portocarrero
318 B.4
0.8
10
'~0
Fig, 10, Abscissa: reciprocal values of ATPase activity x 10-1; ordinate: reciprocal values of the ATP cone. The extrapolated Km value is 0.13 raM.
Lineweaver-Burk plot of the ATPase activity of purified ciliated cortices,
minary film studies of the ciliated oviduct (C. Portocarrero, unpublished work) show that in situ the laeoplectic pattern of metachrony predominates as is the case of the lateral gill cilia of lameUibranchs [23]. The reactivated Modiolus gill ciliated epithelium shows a predominantly "normal" laeoplectic pattern, but in this case, the tissue integrity was maintained [7]. Thus, although a coordinated movement can be obtained with an isolated cortex, the specific type of coordination obtained in vivo may depend on the degree of integrity of the cell. It is of interest to note also that the beat frequency obtained with our preparations under optimal conditions (5 Hz) was inferior to that observed in oviduct tissue slices, 10 Hz (C. Portocarrero, unpublished work), and in Paramecium and gill epithelium 16--32 Hz [2]. Reactivated sea urchin sperm flagella [13] on the other hand, beat with a frequency very close to that observed in vivo, 32 Hz, which suggests that either the cortices have been adversely affected by the isolation procedure or that the reactivation conditions are still far from optimal. The addition of polyethylene glycol to our preparations could result in an increased beat frequency, as in the case of sea urchin sperm. Keeping these possibilities in mind, we will now proceed to evaluate our results. Exp Cell Res 108 (1977)
The optimal conditions reported here for the reactivation of the ciliated cortices resemble those reported for other systems [13, 20]. In our experiments the optimal pH was found to be around 8.1 which is practically identical with that reported for sea urchin sperm flagella [13], although it differs from that reported for isolated Tetrahymena cilia [10] and hamster sperm [19]. This result is not surprising since the enzyme dynein, which seems to be the energytransducing element during ciliary movement, has been reported to have a pH optimum around 8.0, both in solution [11] and bound to the axoneme [13]. However, we still do not know how this value correlates with the pH inside a cilium or flagellum in a living cell. It should also be noted that this value coincides fairly closely with the pK of sulphydryl groups, which have been implicated in microtubule movement [3]. The addition of KC1 to the reactivation medium results in effects similar to those observed with sperm [13, 19]: no reactivation occurs in the absence of KC1, and high concentrations of this salt inhibit the process. However, the optimal concentration of KCI differs among different systems, being 50 mM for the ciliated cortices and
25"
15"
5"
I'0
'
3O
Fig. 11. Abscissa: time (min); ordinate: g,moles of in-
organic phosphate x 10-a/sample. The release of inorganic phosphate by the eiliated cortices in the presence of 1 mM ADP.
Reactivated oviduct cilia Tetrahymena cilia, and 0.1-0.25 M for sperm flagella. In addition the spectrum of optimal activity is broader for sea urchin ~perm flagella (0.05-0.25 M) than for the ¢iliated cortices; for the latter the quality of the reactivation falls dramatically with slight changes in the KC1 concentration. These differences may reflect biochemical adaptations to the ionic environment in which the organisms live and suggest that although the basic structural pattern of all cilia and flagella is basically identical, there may be important chemical differences in the axonemal components of different species. Another resemblance between the ciliated cortices and the sea urchin sperm flagella is that both systems can be reactivated with magnesium, calcium, and manganese ions, the best results being obtained with magnesium. On the other hand, Tetrahymena cilia cannot be reactivated with calcium ions, and only very poorly with manp n e s e . Thus, although ATPCa -2 and ATPMn -~ complexes are good substrates for the dynein in vitro [10], they are not as effective as ATPMg -2 in motility. The response of ciliated cortices to variations in ATP concentrations resembles that sea urchin sperm flagella in that paraeters such as beat frequency are directly proportional to ATP concentration until elateau levels are reached and that the mplitude of the wave is reduced at the igher concentrations [13]. However, conntrations higher than 0.5 mM inhibit the frequency and the duration of movement of the ciliated cortices, whereas with sea urchin sperm the frequency increases even at a concentration of 4 mM. Nevertheless, ~h¢ K,¢ of 0.13 mM obtained in our experiments is very close to the value of 0.2 mM reported for sea urchin sperm flagella. It is also of interest to note that the Kms value
~
319
reported here coincides with the Km obtained for the ATPase activity of the ciliated cortices. This would suggest that under our conditions the hydrolysis of ATP is tightly coupled to movement. The total specific activity in our system is comparable to that obtained with sea urchin sperm, but we have not made any attempt to detect if there is any non-coupled ATPase activity which in the latter amounts to 30 % of the total activity [13]. The fact that the hydrolysis of ATP in our system is not altered by addition of ouabain or oligomycin suggests that most if not all the activity is due to the dynein enzyme, and not to contaminating mitochondrial or membrane ATPases. The reactivation of the ciliated cortices with ADP suggests that, as in other systems, the enzyme adenylate kinase is present in them, and catalyses the formation of ATP from ADP. The results resemble those obtained for sperm flagella in that the reactivation is inferior to that obtained with ATP, and in that there is a lag period before movement is observed [5]. The latter may represent the time necessary for enough ADP to be converted to ATP to allow movement to occur. This correlates very well with the liberation of inorganic phosphate during the process, which occurs very slowly initially and increases rapidly after 10 min. The response of isolated cilia to variations in the reactivation medium resembled very closely those reported for the cortices. However, the fact that both the beat frequency and the duration of movement are reduced when compared with the cortices suggests that they have been damaged by the shearing forces that were applied in order to detach them from the cortex. This hypothesis is backed by two observations. First of all the isolated cilia show a narrower spectrum of ATP concentration in Exp Cell Res 108 (1977)
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Torres, Renaud and Portocarrero
which motility can be observed when compared with the ciliated cortices. This suggests that either the dynein or the mechanism that couples the hydrolysis of ATP to motility has been damaged. In addition, if cilia are isolated by treatment of the tissue slices with Triton X-100 instead of glycerol, only about 20-30 % of the cilia become reactivated (C. Portocarrero, unpublished results). Since under these conditions, a more prolonged treatment with the Vortex mixer is necessary in order to detach the cilia, this suggests that this treatment is indeed deleterious for the cilia. However, it is of interest to note that under all conditions, if the cilia become attached to the slide the duration of movement is prolonged, suggesting that attachment to a substrate is important for ciliary stability. The evidence so far indicates that ciliary movement occurs by the sliding of axonemal doublets [24]. Perhaps the shearing action among adjacent doublets results in a disorganization of the axonemal structure in an isolated cilium, unless this organelle is anchored to a substrate. In an intact cell this stabilizing role would be played by the basal body. This work was supported by NIH grant RR-8102.
Exp Cell Res 108 (1977)
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
Anderson, R G W, J cell biol 60 (1974) 393. Blake, J R & Sleigh, M A, Biol rev 49 (1974) 85. Bloodgood, R A, Cytobios 14 (1975) 101. Brokaw, C J, Exp cell res 22 (1961) 151. Brokaw, C J & Gibbons, I R, J cell sci 13 (1973) 1. Child, F M, Exp cell res 8 (1961) 47. Child, F M & Tamm, S, Biol bull 125 (1963) 373. Fiske, C H & Subbarow, Y, J biol chem 66 (1925) 375. Forstner, G G, Sabesin, S M & Isselbacher, K J, Biochemj 106 (1968) 381. Gibbons, I R, J cell biol 25 (1965) 400. - - J biol chem 241 (1966) 5590. Gibbons, I R & Fronk, E J, J cell biol 54 (1972) 365. Gibbons, B H & Gibbons, I R, J cell biol 54 (1972) 75. Gibbons, I R, Molecules and cell movement (ed S Inou6 & R E Stephens) p. 207. Raven Press, N e w York (1975). Hyams, J S & Borisy, G G, Science 189 (1975) 891. Lowry, O H, Rosebrough, N J, Farr, A L & Randall, R J, J biol chem 193 (1951) 265. Mazia, D, Petzelt, C, Williams, R O & Meza, I, Exp cell res 70 (1972) 325. Mclntosh, J R, Cande, W Z & Snyder, J A, M01ecules and cell movement (ed S Inou6 & R E Step-, hens) p. 31. Raven Press, New York (1975). Morton, B, Exp cell res 79 (1973) 106. Renaud, F L & Ramirez, T, J cell biol 63 (1974) 282a. Saavedra, S & Renaud, F, Exp cell res 90 (1975) 439. Satir, P & Child, F M, Biol bull 125 (1963) 390. Sleigh, M A, Int rev cytol 25 (1969) 31. Summers, K E & Gibbons, I R, Proc natl acad sci US 68 (1971) 3092. Torres, L D & Renaud, F L, J cell biol 67 (1975) 433a. Winicur, S, J cell biol 35 (1967) c7.
Received March 16, 1977 Accepted April 14, 1977
Experimental Cell Research 108 (1977) 311-320
STUDIES ON REACTIVATED
CILIA
II. Reactivation of Ciliated Cortices from the Oviduct
of Anolis Cristatellus L. D. TORRES, F. L. RENAUD and C. PORTOCARRERO Department o f Biology, University o f Puerto Rico, Rio Piedras, P R 00931, U S A
SUMMARY Reactivated ciliated cortices from the oviduct of the lizard Anolis cristatellus show symplectic metachrony, and resemble reactivated sea urchin sperm flagella in several characteristics, such as pH optimum and Km~, but differ in other, such as optimal ionic strength. Cilia isolated from the same source give similar results, but show a poorer reactivation. Purified ciliated cortices have adenylate kinase activity and an ATPase activity with a Km value identical with the K ~ , suggesting a tight coupling between ATP hydrolysis and movement in this system.
The chemical fine structure of cell movements involving microtubules is one of the most intriguing problems in contemporary cell biology. It has been known for a long time that microtubular structures, such as cilia and flagella, axostyles and the mitotic spindle, utilize ATP as the source of energy for movement. Recently, the chemical ultrastructure of these organelles has begun to be elucidated [3, 14, 17, 18]. However, a lot of gaps still remain in our knowledge o f how the macromolecules that compose these organelles interact in order to translate the hydrolysis of ATP into the physical displacement of their structural elements. In addition we know very little about h o w this movement is regulated. A useful approach to the solution of these problems has been the study of reactivated models, where the cell membrane has been disrupted or solubilized by extraction with glycerol or with the non-ionic detergent
Triton X-100 [4, 13]. This permits the reinitiation of movement by making exogenous ATP accessible to the motile apparatus, and in addition allows for manipulation of the environment in which the reactivation occurs. The best results have been obtained with Triton X-100 [3, 13, 18]; sea urchin sperm treated with this detergent become reactivated with a movement very similar to that of living, free-swimming sperm [13]. This has permitted a correlation to be made between the chemical properties of the flagellar axoneme and the quality of the reactivation, and to extrapolate these results to the in vivo situation. However, this approach has been used only scantily for the study of ciliary movement [10, 21, 25, 26]. Cilia and flagella are nearly identical in terms of ultrastructure, but in general differ in length, number and beat pattern [2]. Therefore, it is of interest to determine how similar these organelles are in their Exp Cell Res 108 (1977)
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response to various chemical changes. Renaud & Ramirez [20] have studied the reactivation of ciliated Tetrahymena ghosts obtained by homogenizing the cells with Triton X-100. The movement observed in this system was not coordinated and showed great variability in the percentage of motile ghosts. We have also studied the reactivation of ciliated cortical fractions from the oviduct of the tropical lizard Anolis cristateUus. The movement observed in this system is coordinated and the results are highly reproducible. Therefore we have utilized it to study the effect on ciliary movement of changes in the ionic environment in which the reactivation takes place. In addition, we have done preliminary measurements of some of the enzymatic properties of these fractions. A comparison is made between these results and those reported previously for sea urchin sperm flagella and other systems. A preliminary account of this work had been presented before [25].
Isolation of cilia Oviduct cilia were isolated by a modification of the method of Gibbons [ 10]. The transected oviducts were resuspended in glycerination solution (60 % glycerol, 25 mM KC1, 0.1% 2-mercaptoethanol and 20 mM TrisHC1, pH 8.3 at &C) and incubated in a salt-ice mixture at - 2 & C for 10 min. The cilia were then detached from the tissue by vigorous agitation with a Vortex mixer for 30 sec, and then incubated in the salt-ice bath for 30 additional seconds. This agitationincubation alternation was repeated three times. The tissue slices were then removed from the preparation, and the supernatant containing the cilia was used in the reactivation experiments described in the following section.
Reactivation of ciliated cortices and isolated cilia The preparations were reactivated by diluting 1 : 1 with reactivating solution (0.8 mM ATP, 75 mM KC1,
MATERIAL AND METHODS
Isolation of the ciliated cortices The method reported here is a modification of the procedure reported previously by Anderson [1] for the isolation of ciliated cortices from rabbit oviduct, and by Renaud & Ramirez [20] for the isolation of ciliated Tetrahymena ghosts. Adult females of the tropical lizard Anolis cristatellus was decapitated and their oviducts dissected and transferred to a small volume of Tyrode's solution in an ice bath. The tissue was then sliced longitudinally to expose the ciliated epithelium and resuspended in SET solution (0.25 M sucrose, 1.0 mM EDTA, 25 mM KCI, 0.05% Triton X-100, 0.1% 2-mercaptoethanol and 20 mM Tris-HC1, pH 8.8 at &C). The suspension was then agitated vigorously with a Vortex mixer for 30 see and incubated in an ice bath for 30 sec. This alternation of agitation and chilling was repeated four times, and the preparation was then centrifuged for 5 rain at 1000 g. The sediment, consisting mostly of nuclei and isolated cortices, was resuspended in SE solution (identical with the SET solution, but omitting the Triton X-100), centrifuged for 5 min at 1000 g and resuspended in fresh SE solution.
Exp Cell Res 108 (1977)
Fig. 1. Cross section of the ciliated epithelium of the Anolis oviduct. Note the cilia (arrow) lining the apical surface of the cell N, nucleus, x 2 500. Fig. 2. Detached ciliated cortices from the Anolis oviduct (arrows). Note that the nuclei (N) are the main visible contaminant of the preparation, x 1600.
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Table 2. The effect o f A D P on the reactivation o f oviduct ciliated cortices ADP conc. 0.4 0.8 1.0
Beat frequency (Hz)
% of reactivation
Duration of movement (min)
0 0.2 0.3
0 15 20
0 15 15
Nucleotide concentration. The minimal concentration of ATP tested that enabled the reactivation to take place was 0.025 mM. Increasing the concentration of the nucleotide gives a concomitant increase in the parameters measured, with an optimal concentration of 0.5 mM (fig. 5). Further increases in the concentration of the nucleotide result in an inhibition of the reactivation. The changes in beat frequency at different ATP concentrations are illustrated in fig. 6. Again we can see that optimal results are obtained at a concentration of 0.5 mM, giving a beat frequency of 5 Hz. Higher concentrations of ATP decreased the amplitude of the wave, and the movement observed was more vibratory than undulatory. Notice that the values obtained from film studies coincide very nicely with those obtained by visual estimation. A double reciprocal plot of the beat frequency and the ATP concentration gives a straight line (fig. 7). Extrapolation of this line serves to measure a constant, the so called Kmy, which in this case has a value of 0.13 mM. When the cortices are diluted with a reactivating solution containing ADP instead of ATP, no movement is observed if the nucleotide concentration is 0.4 mM or lower. If the ADP concentration is increased to 0.8 mM or higher, a reactivation is observed, but only after a lag period of 10 min (table 2). The type of movement is similar to that observed previously with ATP, but Exp Cell Res 108 (1977)
the reactivation is inferior in terms of all three parameters measured. For example, the beat frequency is less than one Hz as opposed to 4-5 H z with ATP under optimal conditions.
Reactivation of isolated cilia The optimal reactivation of isolated cilia was obtained by mixing 1 vol of the glycerinated suspension with 2 vol of the reactivating solution. The movement observed consists of sinusoidal waves that move from one end to the other of the cilium. However, roughly 10% of the cilia become attached to the slide by o n e end, and their movement is of the whiplash type, as in the cortices. Variations in the components of the reactivation solution gave s o m e results t h a t closely parallel those obtained for the ciliated cortices. The optimal pH in terms of the duration of movement is 7.7, which is very close to 8.1, the optimal pH for the cortices (fig. 8). In addition, the optimal concentration of KCI is 50 mM in both cases (fig. 9). The cilia could also be reactivated with calcium and manganese, in addition to magnesium, and the optimal concentrations of these cations were very similar to the optimal ones for the reactivation of the cortices (L. D. Torres, unpublished results).
1.6"
0.8=
,e'
J
Fig. 7. Abscissa: reciprocal values of the ATP conc.; ordinate: reciprocal values of the beat frequency. Double reciprocal plot of the beat frequency of the ciliated cortices at different ATP concentrations, the extrapolated value of the K,~t is 0.13 raM.
Reactivated oviduct cilia appreciably, and resulted in an increased breakage of the latter.
Reactivation o f the ciliated cortices When the cortex suspension in SE is diluted with reactivating solution containing optimal concentrations of its components, one can observe a reactivation of 80 % of the cortices. No movement is observed if the washing with SE is omitted. All the cilia become motile in 90% of the reactivated cortices, and i n t h e remaining 10% only a few cilia move per cortex, particularly in the case of cortices that have been fragmented during the isolation procedure. The movement in the intact cortices is coordinated and shows the symplectic pattern of metachronal coordination, i.e., the plane of beat coincides with that of the propagation of the wave [2]. Effect o f several variables on the reactivation pH. The effect of changes in pH on the percentage of reactivation and the duration of novement is illustrated in fig. 3. The results ;how a sharp optimum at a pH of 8.1, where 30 % of the cortices become reactivated for m average of 250 min; an initial beat frequency of 4-5 Hz was observed. All these ~arameters decrease rapidly at higher or ower concentrations of hydrogen ions, including the beat frequency which is reduced :o about 1 Hz at both pH 6 and 9. Ionic strength. Changes in the concentraion of KCI in the reactivation mixture give he results illustrated in fig. 4; namely, a )harp optimum at a concentration of 50 mM 'or all the parameters measured. However, he decrease in the percentage of reactivaion and in the duration of movement was ess sharp at higher KC1 concentrations han at lower ones.
315
6.0-
2.0
0~2
0'.s
Fig. 6. Abscissa: final ATP conc. (mM); ordinate:
beats/sec (Hz). 0 , Values measured directly; ©, values determined from f'flm studies. Variation in the beat frequency of the ciliated cortices with changes in the concentration of ATP in the reactivation mixture.
Divalent cations. No movement is observed in the absence of divalent cations. The eiTect of the addition of various concentrations of manganese and calcium ions is illustrated in table 1. The optimal concentration of calcium and magnesium is 1.0 mM in terms of the duration of movement and the percentage of reactivation, whereas that of manganese is 0.5 mM. However, magnesium was the most effective cation since it can be seen that both parameters measured are reduced considerably with calcium and manganese even at their optimal concentrations. On the other hand, the optimal concentrations of these cations in terms of beat frequency do not necessarily coincide with the optimal values for the other parameters measured, being 1.0, 2.0 and 0.75 mM for magnesium, calcium and manganese, respectively. The best results are again obtained with magnesium ions that give a beat frequency of 5 Hz, whereas with calcium and manganese one only observes 2 Hz. Exp Cell Res 108 (1977)
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Table 1. The effect of various divalent cations on the reactivation of oviduct ciliated cortices
Cation Mgz+ Ca~+ Mn2+
Conc. (mM)
% of reactivation
Duration of movement (min)
0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0
20 80 20 0 50 35 20 0 0
150 280 100 0 125 100 140 0 0
fuge. The nuclei sediment to the bottom of the tube, whereas the ciliated cortices can be collected at the interphase between the KEM and the sucrose solutions.
Enzymatic activity of the ciliated cortices The ATPase and adenylate kinase activity of the purified cortices was determined by measuring the release of inorganic phosphate by the technique of Fiske & Subbarow [8]. One volume of the suspension of purified cortices was mixed with 1 vol of reactivation solution in a series of test tubes in a water bath at 25°C, and the reaction stopped at different time intervals by the addition of molybdate and Fiske-Subbarow reagents. The absorbance at 660 nm was measured with a Zeiss PM Q II spectrophotometer. The protein concentration was measured by the technique of Lowry et al. [16] using bovine serum albumin as a standard. RESULTS
sist m a i n l y o f the ciliary r o w s a t t a c h e d to the basal bodies. N o ultrastructural studies w e r e p e r f o r m e d , t h e r e f o r e it is n o t k n o w n if t h e y c o n t a i n ciliary rootlets a n d r e m n a n t s o f the cell m e m b r a n e , as is the c a s e with rabbit o v i d u c t c o r t i c e s [1]. T h e m a i n visible c o n t a m i n a n t s p r e s e n t in the p r e p a r a t i o n are the nuclei (fig. 2), w h i c h are resistant to solubilization b y Triton X-100. T h e c o r t e x yield v a r i e d d e p e n d i n g o n several factors. The optimal detergent concentration was b e t w e e n 0.02 and 0.06 %. H i g h e r c o n c e n t r a tions c a u s e d a disintegration o f the cortices, and l o w e r ones r e d u c e d the n u m b e r o f cortices liberated. O m i s s i o n o f the chelating agent E D T A also r e d u c e d the c o r t e x yield, and t h o s e that w e r e p r o d u c e d s e e m e d to be c o n t a m i n a t e d with c o a g u l a t e d c y t o p l a s m . A m o r e p r o l o n g e d t r e a t m e n t with the V o r t e x m i x e r did n o t i n c r e a s e the yield o f c o r t i c e s
225"
~
a
125i 75-
Isolation of ciliated cortices A cross section o f the ciliated epithelium o f the Anolis o v i d u c t is illustrated in fig. 1. T h e cilia are a p p r o x . 10--15 /zm long; and are located at the apical end o f the epithelial cells, lining the l u m e n o f the organ, particularly in its anterior region. W h e n the o v i d u c t slices are agitated in the V o r t e x mixer, the apical b o r d e r o f the ciliated cell b e c o m e s d e t a c h e d as a discrete unit (fig. 2), T h e s e isolated b o r d e r s will be r e f e r r e d to as cortices [1]. T h e s e structures s e e m to conExp Cell Res 108 (1977)
b 25 0%
~'o
Fig. 5. Abscissa: final ATP cone. (mM); ord/nate: (a) % reactivation; (b) time (rain). The duration of movement (a) min; (b) the per, centage of reactivation of the ciliated cortices at dif ferent concentrations of ATP.
R e a c t i v a t e d o v i d u c t cilia
3!3
projected at slow motion. The movies were taken with a Beaulieu 2008-S camera, using Kodak Plus-X Super 8 film.
P u r i f i c a t i o n o f ciliated c o r t i c e s
40-
6.0
9.0
These organelles were purified by a modification of the technique developed by Forstner et al. [9] for the purification of isolated brush borders. After this procedure, the cortices could still be reactivated and were suitable for enzymatic analysis. The suspension consisting of nuclei and ciliated cortices was diluted fivefold with EM solution (2.5 mM EDTA, 0.1% 2-mercaptoethanol, pH 7.0) and then centrifuged at 1000 g for 5 min. The sediment was then resuspended in KEM solution (100 mM KCI, 0.8 mM EDTA, 0.1% 2-mercaptoethanol, pH 7.0), and incubated in an ice bath for 15 min. Under these conditions, the nuclei aggregate in small clumps, whereas the majority of the ciliated cortices are not affected. The suspension is then filtered through glass wool to remove the nuclear aggregates, and the filtrate is then chased with a small volume of KEM solution. Any remaining nuclei are then removed from the preparation by layering the filtrate on top of a buffered sucrose solution (1.8 M sucrose, 0.1 M KCI, 0.1% 2-mercaptoethanol and 20 mM imidazole-HCl, pH 7.0) and centrifuging at 53000 g for 2 h, using a Beckman L3-50 Ultracentri-
Fig. 3. Abscissa: pH; ordinate: (a) % reactivation; (b) time (rain). The effect of changes in the pH of the reactivation medium on (a) the percentage of reactivation; (b) the duration of movement of the ciliated cortices.
8 11
2.5 mM Mg SO4, 0.1% 2-mercaptoethanol and 20 mM Tris-HC1, pH 8.5). In some experiments the reactivating solution was altered in order to determine the optimal concentration of its components by observing the effect of concentration changes on the quality of the reactivation. The Tris-HC1 buffer was substituted by imidazole-HC1 for pH values below 7. Magnesium ions were sometimes substituted by calcium and man:anese, and ATP by ADP. All observations were made with a Zeiss phase-contrast microscope with Neofluar 9bjectives at a total magnification of 800×. Each experiment was repeated at least twice and the results were plotted tracing the best line by eye; only one ~aint is illustrated if the results for that particular ~oint coincided in duplicate experiments. The paratinters measured were the percentage of reactivation, '.he duration of movement and the beat frequency. We l¢fine percentage of reactivation as the percentage of solated cilia or ciliated cortices showing movement in madomly selected fields. Each sample consisted of ~ r o x . 50 cortices or 25-30 cilia. The end point of the mbetivation period was arbitrarily chosen as the time which 80 % of the isolated cilia or the ciliated corbees had ceased to move. The beat frequency was ~ u r e d by visual estimation, and in the case of the iliated cortices, by analysis of films of the reactivation
b
~ ~ s I00"
200a
50
~00
Fig. 4. Abscissa: final KCI conc. (raM); ordinate: (a) % reactivation; (b) time (rain). The effect of changes in the concentration of KCI in the reactivation medium on (a) the percentage of reactivation, and (b) the duration of movement (min).
Exp CellRes 108 (1977)
Reactivated oviduct cilia
Fig. 8. Abscissa: pH; ordinate: time (min). O, Free-
swimming; A, slide-attached cilia. The duration of the reactivation of isolated oviduct cilia at different pH values.
However, the beat frequency and the duration of movement is in all cases inferior to that obtained for the ciliated cortices. For example, under optimal conditions ciliated cortices will move for over 2 h, whereas free-swimming isolated cilia will do so for only 25 rain. It is of interest to note that when the cilia become attached to the slide the duration of the movement is duplicated (fig. 8). However, this did not result in an increased beat frequency. Isolated cilia differ from ciliated cortices in their response to changes in the concentration of ATP in the reactivation medium. No movement was observed at a nucleotide concentration of 0.025 mM, in contrast with the ciliated cortices that are capable of movement at this concentration. Reactivation was observed at ATP concentrations of 0.05 mM or higher, with optimal results at 0.2 mM. Concentrations higher than this value inhibited movement; thus, the optimal concentration for the reactivation of cortices inhibits the reactivation of isolated cilia.
317
altered by the addition of various concentrations of ouabain or oligomycin to the reactivation medium. A Lineweaver-Burk plot of the ATPase activity is illustrated in fig. 10, giving an extrapolated Km of 0.13 mM. This value coincides with that of the K,e mentioned previously. The liberation of inorganic phosphate by the ciliated cortices in the presence of 1 mM ADP is illustrated in fig. 11. Notice that the rate is slow during the first 10 min, increasing dramatically afterwards. The time period of the slow initial rate coincides with the lag period before movement is observed in the cortices. DISCUSSION There are two things worth noticing when comparing the reactivation of ciliated cortices with the observations made in situ and in other systems. First of all, the fact that the movement of the cortices is coordinated, showing symplectic metachrony, suggests that the mechanism of coordination is independent of the integrity of the cell, and is possibly due to viscous interactions through the medium among adjacent cilia. A similar conclusion has been reached by other workers [2], although this theory cannot explain the results obtained with the reactivated flagellar apparatus of Chlamydomonas [5]. Observations made in preli-
25"
Enzymatic activity of the ciliated cortices At an ATP concentration of 0.5 mM, the specific activity of the ATPase of the purified cortices was 0.25/zmoles of inorganic phosphate min -1 mg -1. The results were not 21-771802
Fig. 9. Abscissa: finalKCI conc.;ordinate: time (rain). $, Free-swimming;A, slide-attachedcilia. The duration of the reactivationof isolated oviduct cilia at various concentrationsof KCI. Exp Cell Res 108 (1977)
Torres, Renaud and Portocarrero
318 B.4
0.8
10
'~0
Fig, 10, Abscissa: reciprocal values of ATPase activity x 10-1; ordinate: reciprocal values of the ATP cone. The extrapolated Km value is 0.13 raM.
Lineweaver-Burk plot of the ATPase activity of purified ciliated cortices,
minary film studies of the ciliated oviduct (C. Portocarrero, unpublished work) show that in situ the laeoplectic pattern of metachrony predominates as is the case of the lateral gill cilia of lameUibranchs [23]. The reactivated Modiolus gill ciliated epithelium shows a predominantly "normal" laeoplectic pattern, but in this case, the tissue integrity was maintained [7]. Thus, although a coordinated movement can be obtained with an isolated cortex, the specific type of coordination obtained in vivo may depend on the degree of integrity of the cell. It is of interest to note also that the beat frequency obtained with our preparations under optimal conditions (5 Hz) was inferior to that observed in oviduct tissue slices, 10 Hz (C. Portocarrero, unpublished work), and in Paramecium and gill epithelium 16--32 Hz [2]. Reactivated sea urchin sperm flagella [13] on the other hand, beat with a frequency very close to that observed in vivo, 32 Hz, which suggests that either the cortices have been adversely affected by the isolation procedure or that the reactivation conditions are still far from optimal. The addition of polyethylene glycol to our preparations could result in an increased beat frequency, as in the case of sea urchin sperm. Keeping these possibilities in mind, we will now proceed to evaluate our results. Exp Cell Res 108 (1977)
The optimal conditions reported here for the reactivation of the ciliated cortices resemble those reported for other systems [13, 20]. In our experiments the optimal pH was found to be around 8.1 which is practically identical with that reported for sea urchin sperm flagella [13], although it differs from that reported for isolated Tetrahymena cilia [10] and hamster sperm [19]. This result is not surprising since the enzyme dynein, which seems to be the energytransducing element during ciliary movement, has been reported to have a pH optimum around 8.0, both in solution [11] and bound to the axoneme [13]. However, we still do not know how this value correlates with the pH inside a cilium or flagellum in a living cell. It should also be noted that this value coincides fairly closely with the pK of sulphydryl groups, which have been implicated in microtubule movement [3]. The addition of KC1 to the reactivation medium results in effects similar to those observed with sperm [13, 19]: no reactivation occurs in the absence of KC1, and high concentrations of this salt inhibit the process. However, the optimal concentration of KCI differs among different systems, being 50 mM for the ciliated cortices and
25"
15"
5"
I'0
'
3O
Fig. 11. Abscissa: time (min); ordinate: g,moles of in-
organic phosphate x 10-a/sample. The release of inorganic phosphate by the eiliated cortices in the presence of 1 mM ADP.
Reactivated oviduct cilia Tetrahymena cilia, and 0.1-0.25 M for sperm flagella. In addition the spectrum of optimal activity is broader for sea urchin ~perm flagella (0.05-0.25 M) than for the ¢iliated cortices; for the latter the quality of the reactivation falls dramatically with slight changes in the KC1 concentration. These differences may reflect biochemical adaptations to the ionic environment in which the organisms live and suggest that although the basic structural pattern of all cilia and flagella is basically identical, there may be important chemical differences in the axonemal components of different species. Another resemblance between the ciliated cortices and the sea urchin sperm flagella is that both systems can be reactivated with magnesium, calcium, and manganese ions, the best results being obtained with magnesium. On the other hand, Tetrahymena cilia cannot be reactivated with calcium ions, and only very poorly with manp n e s e . Thus, although ATPCa -2 and ATPMn -~ complexes are good substrates for the dynein in vitro [10], they are not as effective as ATPMg -2 in motility. The response of ciliated cortices to variations in ATP concentrations resembles that sea urchin sperm flagella in that paraeters such as beat frequency are directly proportional to ATP concentration until elateau levels are reached and that the mplitude of the wave is reduced at the igher concentrations [13]. However, conntrations higher than 0.5 mM inhibit the frequency and the duration of movement of the ciliated cortices, whereas with sea urchin sperm the frequency increases even at a concentration of 4 mM. Nevertheless, ~h¢ K,¢ of 0.13 mM obtained in our experiments is very close to the value of 0.2 mM reported for sea urchin sperm flagella. It is also of interest to note that the Kms value
~
319
reported here coincides with the Km obtained for the ATPase activity of the ciliated cortices. This would suggest that under our conditions the hydrolysis of ATP is tightly coupled to movement. The total specific activity in our system is comparable to that obtained with sea urchin sperm, but we have not made any attempt to detect if there is any non-coupled ATPase activity which in the latter amounts to 30 % of the total activity [13]. The fact that the hydrolysis of ATP in our system is not altered by addition of ouabain or oligomycin suggests that most if not all the activity is due to the dynein enzyme, and not to contaminating mitochondrial or membrane ATPases. The reactivation of the ciliated cortices with ADP suggests that, as in other systems, the enzyme adenylate kinase is present in them, and catalyses the formation of ATP from ADP. The results resemble those obtained for sperm flagella in that the reactivation is inferior to that obtained with ATP, and in that there is a lag period before movement is observed [5]. The latter may represent the time necessary for enough ADP to be converted to ATP to allow movement to occur. This correlates very well with the liberation of inorganic phosphate during the process, which occurs very slowly initially and increases rapidly after 10 min. The response of isolated cilia to variations in the reactivation medium resembled very closely those reported for the cortices. However, the fact that both the beat frequency and the duration of movement are reduced when compared with the cortices suggests that they have been damaged by the shearing forces that were applied in order to detach them from the cortex. This hypothesis is backed by two observations. First of all the isolated cilia show a narrower spectrum of ATP concentration in Exp Cell Res 108 (1977)
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which motility can be observed when compared with the ciliated cortices. This suggests that either the dynein or the mechanism that couples the hydrolysis of ATP to motility has been damaged. In addition, if cilia are isolated by treatment of the tissue slices with Triton X-100 instead of glycerol, only about 20-30 % of the cilia become reactivated (C. Portocarrero, unpublished results). Since under these conditions, a more prolonged treatment with the Vortex mixer is necessary in order to detach the cilia, this suggests that this treatment is indeed deleterious for the cilia. However, it is of interest to note that under all conditions, if the cilia become attached to the slide the duration of movement is prolonged, suggesting that attachment to a substrate is important for ciliary stability. The evidence so far indicates that ciliary movement occurs by the sliding of axonemal doublets [24]. Perhaps the shearing action among adjacent doublets results in a disorganization of the axonemal structure in an isolated cilium, unless this organelle is anchored to a substrate. In an intact cell this stabilizing role would be played by the basal body. This work was supported by NIH grant RR-8102.
Exp Cell Res 108 (1977)
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
Anderson, R G W, J cell biol 60 (1974) 393. Blake, J R & Sleigh, M A, Biol rev 49 (1974) 85. Bloodgood, R A, Cytobios 14 (1975) 101. Brokaw, C J, Exp cell res 22 (1961) 151. Brokaw, C J & Gibbons, I R, J cell sci 13 (1973) 1. Child, F M, Exp cell res 8 (1961) 47. Child, F M & Tamm, S, Biol bull 125 (1963) 373. Fiske, C H & Subbarow, Y, J biol chem 66 (1925) 375. Forstner, G G, Sabesin, S M & Isselbacher, K J, Biochemj 106 (1968) 381. Gibbons, I R, J cell biol 25 (1965) 400. - - J biol chem 241 (1966) 5590. Gibbons, I R & Fronk, E J, J cell biol 54 (1972) 365. Gibbons, B H & Gibbons, I R, J cell biol 54 (1972) 75. Gibbons, I R, Molecules and cell movement (ed S Inou6 & R E Stephens) p. 207. Raven Press, N e w York (1975). Hyams, J S & Borisy, G G, Science 189 (1975) 891. Lowry, O H, Rosebrough, N J, Farr, A L & Randall, R J, J biol chem 193 (1951) 265. Mazia, D, Petzelt, C, Williams, R O & Meza, I, Exp cell res 70 (1972) 325. Mclntosh, J R, Cande, W Z & Snyder, J A, M01ecules and cell movement (ed S Inou6 & R E Step-, hens) p. 31. Raven Press, New York (1975). Morton, B, Exp cell res 79 (1973) 106. Renaud, F L & Ramirez, T, J cell biol 63 (1974) 282a. Saavedra, S & Renaud, F, Exp cell res 90 (1975) 439. Satir, P & Child, F M, Biol bull 125 (1963) 390. Sleigh, M A, Int rev cytol 25 (1969) 31. Summers, K E & Gibbons, I R, Proc natl acad sci US 68 (1971) 3092. Torres, L D & Renaud, F L, J cell biol 67 (1975) 433a. Winicur, S, J cell biol 35 (1967) c7.
Received March 16, 1977 Accepted April 14, 1977