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First published online 5 August 2008
doi: 10.1242/jcs.031195
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Research Article |
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan
* Author for correspondence (e-mail: chikako{at}biol.s.u-tokyo.ac.jp)
Accepted 16 June 2008
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: Dynein, Flagella, Oscillation, Imposed bending, Sperm, Sea urchin
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Introduction |
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; Holwill and Satir, 1994; Nakano et al., 2003; Shingyoji et al., 1977) (Fig. 1A,B). The doublets of the axoneme are probably all similar in their dynein functions, but the CP is thought to act as a kind of distributor of signals to regulate the activity of dynein arms according to their position in the axoneme and the phase of beating (Bannai et al., 2000; Nakano et al., 2003; Omoto et al., 1999). Thus, the dynein arms on both sides of the CP are those that are assigned by the CP to produce the microtubule sliding necessary for bend formation. As the dynein molecule is a minus-end-directed motor protein (Sale and Satir, 1977; Vale and Toyoshima, 1988), the activity of dynein arms on the two sides of the CP should alternate cyclically in order for cyclical bending in alternate directions to occur. How such alternation of dynein activity between the two sides of the CP is regulated along the flagellum is therefore important for understanding the mechanism of oscillation.
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; Okuno and Hiramoto, 1976; Shingyoji et al., 1995). In addition, bending an immotile flagellum can stimulate its dynein to start beating (Hayashibe et al., 1997; Ishikawa and Shingyoji, 2007; Okuno and Hiramoto, 1976). Thus, it is likely that the flagellar bending itself is involved in the regulation of dynein activity, which would imply that a self-regulatory feedback mechanism underlies flagellar oscillation. This idea is strongly supported by our previous study using elastase-treated axonemes of sea urchin sperm, which showed that under certain conditions externally applied bending causes a reversal of the direction of active microtubule sliding (Morita and Shingyoji, 2004). Taken together with an ultrastructural study (Nakano et al., 2003), it is tempting to speculate that the mechanical effects of bending may have initiated `switching' of the dynein activity between the two sides of the CP, probably from doublet 7 to doublet 3 (or 4) (see below) to induce backward sliding (Fig. 1B,D; pink and blue bars in Fig. 1C). As the activity of dynein arms on the doublets other than 7 and 3 (or 4) can also be switched on by externally applied bending in elastase-treated axonemes (Fig. 1C, grey bars), although independent of the presence of the CP (Morita and Shingyoji, 2004), we predict that bending regulates the activity of all dynein arms (Fig. 1C).
In this study, as a step to confirm our self-regulatory feedback model (Fig. 1C), we aimed to demonstrate that the switching of the dynein activity between the two sides of the CP does cause backward sliding (Fig. 1D). To do this, we had to solve two problems: first, establishing a way to induce backward sliding more stably–in the previous experiments, backward sliding could be induced by externally applied bending in only about 45% of the fragmented axonemes (Morita and Shingyoji, 2004); second, finding an indicator of dynein activity that could discriminate between the side of the CP with active dynein arms and the side with inactive arms.
To overcome the first problem, we need to find the conditions for bending to induce backward sliding. Among the possible variables, the direction of bending relative to the beat plane seems to be important. In sea urchin sperm, structural relations in the formation of the principal (P-) and the reverse (R-) bends at the base of a flagellum are well established. Electron microscopy, together with the analysis of bending responses under low and high Ca2+ conditions, have shown that the dynein arms on doublets 7 and 3 (or 4), which are located on two sides of the CP, are responsible for the formation of the P- and R-bends, respectively (Nakano et al., 2003; Sale, 1986) (Fig. 1A,B). At high concentrations of Ca2+, the formation of R-bends is suppressed owing to a decrease, mediated by the central pair/radial spoke (CP/RS) system, of dynein activity on doublet 3. By contrast, the activity of dynein arms on doublet 7 is not influenced by Ca2+ (Bannai et al., 2000; Nakano et al., 2003). As a result, flagella at high Ca2+ are arrested in a so-called `quiescent' form that is characterized by a large P-bend at the base (Fig. 1E). It is interesting that the dynein activity on doublet 7 seems to be dominant and that this property is independent of Ca2+ concentration, and thus, under many conditions, the `quiescence' is commoner than the `relaxed' straight form (Sale, 1985; Yoshimura et al., 2007). This means that the oscillation may be triggered by the sliding (P-sliding) necessary for P-bend formation (Shingyoji and Takahashi, 1995). These studies suggest that if we use quiescent flagella instead of axonemal fragments for the study of the effects of imposed bending (Morita and Shingyoji, 2004), we could define the direction of imposed bending by referring to the direction of the P-bend at the base (Fig. 1E). Furthermore, the second problem mentioned above can also be solved by using quiescent flagella. The dynein activity on doublet 3 is decreased at high Ca2+. Under such a condition, the velocity of microtubule sliding on doublet 3 is lower than that on doublet 7 (Nakano et al., 2003). If this is also true in the quiescent flagella, the sliding velocity can be a good marker to determine on which side of the CP dynein arms are active.
In this study, we have examined whether bending itself or the direction of bending is important for switching the dynein activity and how the combination of proximal and distal bends along the length of the flagellum is associated with the switching mechanism. By using elastase-treated Ca2+-induced quiescent flagella of sea urchin sperm, we show that a reversal of the sliding direction is induced depending on the direction of imposed bending. During this study, we found that the presence or the absence of a P-bend at the base of flagella appears to be closely related to the regulation of the dynein activity by imposed bending. Therefore, we used three types of flagella with or without a P-bend at the base [type 2 and type 3 (with), and type 1 (without); Fig. 1E]. In the type 1 experiments, quiescent flagella were cut at the base before inducing microtubule sliding (P-sliding). In the type 2 experiments, quiescent flagella without cutting were used. In the type 3 experiments, quiescent flagella were cut at the base after microtubule sliding into a pair of bundles (P-sliding) was induced. We finally analyzed the velocity of sliding in the axonemes under imposed bending and demonstrated that the velocity of active backward sliding was lower than that of forward sliding. Our results show that the switching of the dynein activity in flagella, which is induced by the combination of the direction of bending and the conformational state of the dynein-microtubule interaction, is the basis for flagellar oscillation.
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). Successive applications of ATP by repetitive UV flashes induced further sliding of the thinner bundle in the same forward direction.
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) has demonstrated that in the elastase-treated axonemal fragments that split into two bundles, a reversal of the direction of sliding can be induced between the bundles by bending the region of overlap between the two bundles by more than 90° (>1.57 rad). We repeated this experiment under the same conditions (Fig. 1D) and confirmed that bending induced the reversal of the direction of microtubule sliding in about half (48%) of the fragmented axonemes studied (n=23). By bending for 1.6-3.3 rad (supplementary material Fig. S1A), we tested the effect of the direction of imposed bending on the direction of sliding between the doublet bundles obtained from elastase-treated quiescent sperm flagella cut at the basal P-bend (type 1 in Fig. 1E). Fig. 2 summarizes typical responses. In this study, we bent the axoneme in the plane as close to the plane of beat as possible. The bending direction can be described as follows: when the region of overlap between the two bundles is bent in the same direction as that of the original basal P-bend, it is categorized as bending in the P-bend direction (left panels in Fig. 2A,B); however, when the region of overlap is bent in the opposite direction relative to the basal bend, which makes a bend similar to a reverse bend in a more distal region, it is categorized as bending in the R-bend direction (right panels in Fig. 2A,B). When we bent the distal part of the region of overlap in the P-bend direction, subsequent application of ATP induced backward sliding of the thinner bundle (Fig. 2A, left panels); such sliding occurred in 56% of the axonemes studied (Fig. 2C, part A, white box). By contrast, when we bent the distal part of the region of overlap in the R-bend direction, photoreleasing ATP induced forward sliding of the thinner bundle without a change in the sliding direction (Fig. 2A, right panels) in most of the axonemes (82%) and backward sliding was induced in only one out of 22 axonemes (Fig. 2C, part A). Bending the proximal part of the overlap region also induced similar responses; backward sliding of the thinner bundle occurred when the proximal region of overlap was bent in the P-bend direction (Fig. 2B, left panels). This type of reversal was observed in 43% of the axonemes (Fig. 2C, part B), whereas bending the proximal region in the R-bend direction did not induce backward sliding (Fig. 2B, right panels; and Fig. 2C, part B). These results show that the backward sliding can be induced by bending in the P-bend direction those flagella that displayed the forward sliding of the thinner bundle (P-sliding) before the bending.
Detailed analysis of the backward sliding revealed that in five out of the 10 flagella bent in the distal region, backward sliding occurred along the whole length of the region of overlap between the two bundles (Fig. 2A, left), although in four of the 10 axonemes, the movement of the thinner bundle at the more distal part of the bending region was inhibited because of splitting of the thinner bundle from the thicker bundle as shown in Fig. 2D. Sliding of this latter case occurred when the distal edge of the thinner bundle was in the bending region. By contrast, when the proximal region was bent, as is shown in Fig. 2B (left) and Fig. 2E (the same flagellum as shown in Fig. 2B, left), the backward sliding occurred only in the proximal part of the bending region (open arrows), whereas the forward sliding continued in the distal part of the bending region (filled arrows). The simultaneous sliding in the opposite directions at the proximal and distal parts of the imposed bending was observed in two of the three axonemes that showed backward sliding on bending the proximal region of the flagella.
In the type 1 experiments (Fig. 1E), the frequency of occurrence of backward sliding induced by bending did not change compared with that in the experiments using fragmented axonemes (Fig. 1D). However, the rate of bent axonemes that disintegrated into many smaller bundles or individual doublets, which was about 40% in the previous study (Morita and Shingyoji, 2004), decreased to 14% (boxes with oblique lines in Fig. 2C). This shows that, in the type 1 experiments, bending induced stable sliding between the two bundles, possibly as it was applied to the axoneme in the plane of the beat.
Effects of the direction of imposed bending on the direction of microtubule sliding in elastase-treated quiescent flagella
The above results show that the directionally controlled bending is effective to induce stable sliding between the two bundles. Although the effect may involve a reversal of the direction of sliding, the rate of reversals was not high (about 50%). This may mean that a specific dynein-regulating factor related to the switching mechanism may not have been well controlled. The bending angle and the position of bending along the axoneme appeared not to be important (supplementary material Fig. S1). One strong candidate for this switching would be the conformational state of dynein-microtubule interaction under the control of the CP/RS system. In other words, the dynein-microtubule attachment states that are involved in the formation of bends would be related to the subsequent activity of dynein arms located close to or within the bends. To test whether the conformational state of dynein arms along the axoneme affects the rate of backward sliding, we examined the effects of the existence of the basal P-bend (type 2 and type 3 in Fig. 1E). When we induced sliding in the elastase-treated axonemes of quiescent flagella without cutting at the basal P-bend (type 2 in Fig. 1E), by photoreleasing ATP (Fig. 3A), 40-50% of the axonemes showed sliding in which the thinner bundles slid mostly towards the head without a large change in the shape of the basal P-bend. In these axonemes, forward sliding of the thinner bundles was induced several times by repeating the UV flashes.
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When we bent the distal part of the region of overlap in the P-bend direction (Fig. 3B), the sliding direction of the thinner bundle did not change and forward sliding was induced by UV flashes in 63% of the flagella (Fig. 3D; n=16). When we bent the distal part of the region of overlap in the R-bend direction, however, subsequent application of ATP induced backward sliding of the thinner bundles in 71% of the axonemes (Fig. 3C,D; n=31), and the forward sliding decreased to 6.5%. The backward sliding was induced along the whole region of overlap (Fig. 3C) in 19 out of the 22 axonemes; the three that were not induced showed splitting of the distal part of the thinner bundle near the bent region. These responses resembled those observed in the quiescent flagella with a cut at the P-bend described above (type 1 in Fig. 1E; Fig. 2), although the effective direction of bending to induce the backward sliding was opposite and the frequency of the occurrence of the backward sliding was different. The rate of disintegration into smaller bundles or individual doublets was also about 18% in the type 2 experiments, which was similar to the rate in the type 1 experiments.
If the presence of the basal P-bend functions to maintain the conformational state of dynein-microtubule interaction through the CP/RS system, the flagellum cut at the proximal but not at the distal part of the P-bend would be expected to preserve the conformational information and to respond to imposed bending (type 3 in Fig. 1E) (in the same way as the flagellum without cutting). Fig. 3E shows sequential tracings of one example. In this experiment P-sliding was first induced at the basal P-bend (UV1, 2) and then the flagellum was cut at the base (Cut). By manipulating only the proximal region of the flagellum, a new R-bend was formed in a more distal region of the basal P-bend (Bending). ATP application (UV3) induced backward sliding in a proximal region (open arrows). We found that in the distal part of the bending region, forward sliding (P-sliding) still occurred (filled arrows). In two other quiescent flagella cut at the base after forward sliding was induced at the original P-bend, backward sliding was induced by bending in the R-bend direction. These results indicate that, even in the flagellum cut at the base, the information provided by the dynein–microtubule interaction at the basal P-bend seems to be maintained, unless further microtubule sliding is induced after cutting.
Velocity of microtubule sliding on thinner and thicker bundles in quiescent flagella
In axonemal fragments, the activity of dynein on doublet 3 is inhibited at 10–4 M Ca2+, whereas that on doublet 7 is not (Nakano et al., 2003). To test whether this is also true in the quiescent flagella, we analyzed the behaviour of microtubules interacting with the dynein arms exposed on the thicker or the thinner bundle obtained from elastase-treated axonemes.
Quiescent flagella treated with elastase also showed splitting into two doublet bundles in the presence of 1 mM ATP and 10–4 M Ca2+. In some cases, sliding brought about splitting along the whole length of a flagellum (Fig. 4A), in which the thinner bundle slid toward the head. After the ATP was replaced with 1 mM caged-ATP, we applied singlet microtubules to the dynein arms exposed on the thinner and thicker bundles, and analyzed their movement. Photoreleasing ATP from caged-ATP induced microtubule sliding on each bundle and all the observed microtubules slid away from the head of the flagella (Fig. 4A), indicating that the polarity of dynein force generation on these bundles is towards the minus-end of the microtubule. Fig. 4B shows distribution of the velocity of microtubule sliding on the thinner (left) and thicker (right) bundles. The velocity on the thinner bundle was higher than that on the thicker bundle at 10–4 M Ca2+. The difference was statistically significant (Mann-Whitney U test, P<0.05). These results showed that in quiescent flagella, the activity of dynein arms on doublet 3 is inhibited at a high Ca2+ concentration, and the inhibition can be detected as a decrease in the velocity of microtubule sliding.
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) indicating that the imposed bending increases the dynein activity. Comparing the velocities of backward and forward sliding, we found that under imposed bending the velocity of the backward sliding was significantly lower than that of the forward sliding. The decrease of sliding velocity in backward sliding is statistically significant in any of the three types of flagella (P<0.05 for axonemal fragments and severed quiescent flagella; P<0.01 for quiescent flagella). The results support the idea that when the direction of sliding changes from the forward sliding (P-sliding) to the backward sliding, the site of dynein activity changes from the thinner to the thicker bundle and the backward sliding is caused by the activity of the dynein arms of the thicker bundle. More specifically, they indicate that imposed bending induces switching of the dynein activity from doublet 7 to doublet 3.
Time lag at the onset of backward sliding
Throughout the above experiments, we found that the forward sliding always occurred immediately after an application of a UV flash. Such immediate response in the forward sliding was independent of imposed bending, in quiescent flagella that were cut at the basal P-bend as well as those without a cut. Fig. 5A-E shows examples of time courses of the immediate response recorded in the absence (A and UV1 in B-E) and presence (UV2, 3 in B) of imposed bending. Similar immediate response to a UV flash was also observed in most of the backward sliding (Fig. 5C, UV2-4). However, we found that some of the backward sliding occurred after a short delay (0.8-1.3 seconds) (Fig. 5D, UV2) or a pause (Fig. 5E, UV2). In such flagella, immediate backward sliding was induced at the next UV flash (UV3 in Fig. 5D,E).
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Discussion |
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; Holcomb-Wygle et al., 1999; Ishikawa and Shingyoji, 2007; Morita and Shingyoji, 2004; Okuno and Hiramoto, 1976; Shingyoji et al., 1991), in order to understand the mechanism of flagellar oscillation, an analysis of the effects of bending on microtubule sliding seems indispensable. The switching hypothesis (Satir, 1985), proposed as a pioneering model to explain the regulation of dynein activity in beating flagella and cilia, postulates alternate activation of the dynein arms on either half of the axoneme beside the CP. Several ultrastructural observations demonstrating splitting of the axoneme into two doublet bundles suggest that the switching of active microtubule sliding occurs between the two sides of the CP during flagellar beating (Fig. 1A,B) (Holwill and Satir, 1994; Nakano et al., 2003; Satir and Matsuoka, 1989; Wargo et al., 2004). However, there has been no clear evidence to demonstrate which dynein arms are active and how the activity of dynein is switched on and off when flagella beat. To demonstrate this, detailed analysis of microtubule sliding is necessary in axonemes that are capable of beating.
A previous study (Morita and Shingyoji, 2004) has demonstrated that a reversal in the direction of microtubule sliding between split bundles of the elastase-treated axoneme of sea urchin sperm flagella, which retain the ability of beating, is induced by imposed bending of the region of overlap of the two bundles. As the microtubule sliding that splits the axoneme into two bundles is caused mainly by the activity of dynein on doublet 7 under high-ATP and high-Ca2+ conditions, the reversal of the sliding direction is probably due to the activity of dynein on doublet 3 (or 4) unless the polarity of dynein force generation changes. But we did not know whether this was really the case (Fig. 1D).
In the present study, we used the quiescent flagellum with the sperm head intact and microtubule sliding was induced in the quiescent axonemes after elastase treatment. This procedure enabled us to determine the direction of beating plane in addition to the longitudinal polarity, proximal and distal directions of the flagellum that correspond to the minus and plus ends of the microtubule. In this way, when the thinner of the two bundles slides towards the head, we can infer that the dynein arms on doublet 7 are the main generator of power for the sliding (Nakano et al., 2003) (Fig. 1E). If the activity of dynein arms is switched by imposed bending from doublet 7 to doublet 3 on the thicker bundle, then the direction of sliding of the thinner bundle is expected to reverse.
In sea urchin sperm, the dynein activity on both sides of the CP is differently controlled and the dynein arms on doublet 3, which are near the C2 microtubule of the CP, are less active than those on doublet 7 (Nakano et al., 2003). This study shows that this feature of lower sliding velocity is also observed in the quiescent flagella (Fig. 4B). By comparing the sliding velocities of forward and backward sliding, we demonstrated that the backward sliding was significantly slower than the forward sliding, indicating that the backward sliding was induced by the activity of dynein on doublet 3. Thus, the present results show that the reversal of the direction of microtubule sliding is caused by switching of dynein activity between the two sides of the CP. This is the basis for the alternation of microtubule sliding underlying the cyclical bending of flagella.
Factors important for switching the dynein activity
The amount of shear and the curvature of the axoneme are varied by imposed bending. Backward sliding was induced by bending with a curvature of 0.22-0.60/µm when the bending angle was larger than 1.7 rad [except in one case (0.81 rad)] in all the axonemes used in this study. However, there was no correlation between the bending angle and the occurrence of backward sliding (supplementary material Fig. S1), indicating that the absolute value of bending angle, which corresponds to the amount of shear, is not the sole determinant of the switching of dynein activity. The most important factors associated with the switching of dynein activity are the direction of bending and the conformational state of the dynein–microtubule interaction.
In the elastase-treated quiescent flagella that had been severed near the base, bending the region of overlap between the two bundles in the same direction as that of the original principal bend at the base (the P-bend direction) induced backward sliding of the thinner bundle, but bending in the R-bend direction did not induce backward sliding (Fig. 2). The results showed that bending was effective only when it was applied in a fixed direction. Thus, bending in the P-bend direction and that in the R-bend direction did not equally affect the regulation of dynein activity. The present results show that the dynein arms (mainly those on doublet 7) that generate principal sliding (Ps) in the axoneme respond to bending in the P-bend direction and that their activity can be switched off, which is followed by activation of the dynein arms on the opposite side of the CP to induce reverse sliding (Rs) (Fig. 6C1). This type of regulation, which is also explained according to curvature-controlled models (Fig. 6A) (Brokaw, 1985; Brokaw, 2001), would be important for the initiation of cyclical bending by switching of the dynein activity at the flagellar base. By contrast, the present observation showing that bending the axoneme in the R-bend direction cannot induce a reversal of the sliding direction is not consistent with the models controlled by the curvature (Fig. 6A). The present results and conclusions apply directly only to the case of a new reverse sliding following a preceding P-sliding. However, we have no reason to believe that the situation would be different for a new P-sliding in the axoneme displaying an R-sliding, if the technical limitations preventing its observation could be overcome.
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In the elastase-treated quiescent flagella (Fig. 3), the effective direction of bending to induce backward sliding was reversed. Bending in the R-bend direction in a region more distal to the original P-bend induced an R-sliding by switching of the dynein activity. In this case, the formation of a pair of bends composed of a proximal P-bend and a more distal R-bend activated the dynein arms to produce reverse sliding along the bend. The results indicate that the difference in the amount of sliding induced by a pair of opposite bends is important for the switching of dynein activity. The localized cyclical bend formation (Fig. 6B) induced by successive iontophoretic application of ATP to either a protease-untreated or an elastase-treated sea urchin sperm flagellar axonemes (Shingyoji et al., 1977; Shingyoji and Takahashi, 1995) probably involves a regulatory mechanism similar to that underlying the activation of dynein by paired bends. In these locally reactivated axonemes, microtubule sliding occurs only in the region (interbend region) between the pair of opposite bends, and a reversal of the sliding direction between the two bends leads to alternation of the direction of these bends (Fig. 6B). By contrast, the backward sliding observed in the present study was induced either along the entire flagellum, including the pair of bends (Fig. 3C) or the proximal part of the bending region (Fig. 3E), and such a change in the sliding direction caused by bending may be involved in the regulation of microtubule sliding underlying the propagation (Fig. 6C2) and growth (Fig. 6C3) of bends in normal beating. Thus, coupling of the mechanism regulating microtubule sliding in the paired bend formation (Fig. 6C2,3) with the regulation that induces a reversal of the sliding direction in the bending region (Fig. 6C1,2) may constitute a basis for the coordination of cyclical bend formation and propagation. This idea is supported by the previous finding that cyclical bend formation and propagation can be induced by imposed bending so as to form a pair of opposite bends in demembranated nonmotile sperm flagella at 2-3 µM ATP (Ishikawa and Shingyoji, 2007). The influence of the distal bend to the proximal sliding is also indicated by the previous study showing that immotile bull sperm flagellum in the presence of Ni2+ recovers its beating by bending the distal region in the direction opposite to the original basal bend (Lindemann et al., 1995). These results suggest that the conformational state of dynein-microtubule interaction in the combination of the proximal and distal bends is responsible for the continuous switching of the dynein activity along the flagellum.
In beating flagella, the regulation of the activity of all dynein arms in the bend plane as well as the switching of dynein activity in the plane of the CP in the interbend regions contribute to produce cyclical beating (Fig. 1C). To understand the flagellar oscillation, we have to elucidate the mechanism regulating the combination of these dynein functions. The disintegration of axonemes into smaller bundles or individual doublets induced by bending, which was observed frequently in the previous study (Morita and Shingyoji, 2004), was infrequent in the present study. This suggests that the activation of all dynein arms by the imposed bending requires that the force be applied to dynein arms in the direction not parallel to the beat plane. In addition to the change in the dynein attachment state resulting from the shear induced by the imposed bending, axonemal distortion would occur along the doublets in the bend plane, tilting the radial spokes and changing the geometric relationship between the doublets (Lindemann, 1994a; Lindeman and Mitchell, 2007). These factors may cause activation of the dynein arms on the doublets in the bend plane, and are probably linked to the switching of the activity of dynein on both sides of the CP in the manner predicted by the `geometric clutch' model (Lindemann, 1994a; Lindemann, 1994b). Thus, the combination of the regulation of the activity of all dynein arms in the bend plane with the mechanism of switching the dynein activity on both sides of the CP is essential for flagellar beating.
Roles of the CP/RS system and bend asymmetry
The change in the CP/RS interaction with the direction of bending probably mediates the mechanical feedback from flagellar bending (Bannai et al., 2000; Mitchell and Nakatsugawa, 2004; Warner and Satir, 1974). The regulation through chemical as well as mechanical signals may also play an important role in this process. Among the chemical signals, regulation of dynein activity by protein phosphorylation and dephosphorylation seems important (Inaba, 2002; Nakajima et al., 2005; Smith and Yang, 2004) although the exact pathways of this regulation remain unclear (Yoshimura et al., 2007). The regulation through the CP/RS system is responsible at a physiological, high-ATP condition. By contrast, at lower ATP concentrations (less than 0.1 mM), the presence of CP/RS system is not essential (Bannai et al., 2000; Nakano et al., 2003; Yoshimura et al., 2007), suggesting that other inherent asymmetries in the axoneme are required. The role of the CP/RS system at high ATP, which, as is discussed below, is probably associated with the inhibition and activation of the dynein activity by ATP and ADP, respectively (Yoshimura et al., 2007), is similar in cilia and flagella of different species (Bannai et al., 2000). The recent finding of the central pair component hydin, the gene encoding which is present broadly in organisms with the ability to assemble motile 9+2 axonemes, may indicate that switching of dynein activity through the CP/RS system is a conserved property of axonemal mechanisms for alternating bends (Lechtreck and Witman, 2007; Lechtreck et al., 2008).
In the present study, we observed a time lag at the onset of backward sliding. This may reflect a process in the transduction of chemical signals from CP1 to CP2 in the region of bending. In normal flagellar beating, bending is formed in alternate directions by rapid and smooth switching of the dynein activity without delay. In the present experiments, dynein activation was brought about by a UV-photolysis of caged-ATP, which may have modified the signal transduction process and enabled us to observe the event at the switching process. The time lag occurred more frequently in the elastase-treated quiescent flagella than in the other two types of axonemes (Fig. 5F). It is tempting to speculate that this may be related to the differences in propagation ability among the three types of axonemes. In the models proposed by Machin and Brokaw (Machin, 1958; Brokaw, 1971; Brokaw, 1985) to explain the flagellar movement, wave propagation can occur if a time lag is introduced into the relationship between the active bending moment and the curvature. In our study, only backward sliding observed in the elastase-treated quiescent flagella, which appeared to retain the regulatory mechanism related to bend propagation (Fig. 6C2), accompanied a time lag. This may indicate a close association between the time lag and wave propagation. A similar delay between the formation of bends was also reported in reactivated Ciona sperm flagella under high-LiCl (10 mM) and low-MgATP (10 µM) conditions (Brokaw, 1989b), in which a pause appears after the P-bend growth. At the end of the pause, active R-sliding is turned on throughout the P-bend and this active sliding causes the P-bend to start propagating. This also supports our idea that the backward sliding (R-sliding) induced throughout the pair of bends (Fig. 6C2) is related to the regulation of sliding that produces wave propagation.
Our recent studies have revealed that the key mechanisms of the regulation of the activity of flagellar dynein are ATP-induced inhibition and ADP-induced activation. These mechanisms are involved in the regulation of both the dynein molecules (Inoue and Shingyoji, 2007) and flagellar beating (Yoshimura et al., 2007). The inhibition and activation of dynein activity seem to be associated with binding of ATP only and both ATP and ADP, respectively, to the three noncatalytic regulatory sites of dynein (Inoue and Shingyoji, 2007). We have shown that caged-ATP behaves in the same way as a non-hydrolysable ATP analogue and binds stably to dynein (Inoue and Shingyoji, 2007). In the presence of caged-ATP, dynein is in an inhibited state and after hydrolysis of the UV-released ATP the dynein becomes active probably by binding of ADP to some of the regulatory sites. According to the protocol for reactivating the dynein by UV photolysis of caged-ATP, the dynein regulatory sites, as well as the ATP hydrolysis site, were in an inactive state before the UV flash and the dynein was forming a crossbridge between doublet microtubules, which may be important for maintaining the conformational states of dynein-microtubule interaction along the flagellum.
It has recently been shown that ADP releases the inhibition of flagellar movement by ATP, and a part of the activation of dynein through protein phosphorylation appears to be caused by the ADP binding to dynein (Yoshimura et al., 2007). Our preliminary experiments show that the induction of backward sliding by imposed bending requires the presence of ADP, suggesting that the activation of dynein by mechanical force is also induced by a mechanism involving ADP binding. Mechanical deformation of the axoneme caused by bending may induce CP/RS-mediated phosphorylation and/or dephosphorylation of some axonemal proteins. Such regulation of the dynein arms on the two sides of the CP is probably caused by some direct (chemical and mechanical) influences from the CP1 and CP2, although chemical and structural differences between the CP1 and CP2, which have been reported in Chlamydomonas (Smith and Yang, 2004), have not been demonstrated in sea urchin sperm. By contrast, the activity of dynein arms on other doublets may be regulated by the mechanical deformation but would be independent of the CP/RS system. How, for oscillatory bending, the mechanical and chemical signals modulate and coordinate the dynein activity by ADP binding to the dynein regulatory sites, and how these signals relate to each other are important points for the future study.
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) with some modifications. The demembranated sperm were labelled with 9.4 µM tetramethylrhodamine (Molecular Probes C-1171) for 8 minutes on ice for the experiments of imposed bending (for Figs 2, 3, 5; Fig. 4C) or labelled with 13 µM cy5 (Amersham Biosciences PA25001) for the experiments of sliding movement of rhodamine-labelled singlet microtubules on doublet bundles of the axonemes (Fig. 4A,B) (Nakano et al., 2003). The sliding disintegration was induced in the assay solution containing 20 mM HEPES-KOH (pH 7.8) instead of Tris-HCl (Nakano et al., 2003).
Observation of sliding and application of bending
A quiescent waveform was induced in the reactivating solution (HEPES) containing 10–4 M Ca2+ and 1 mM ATP (for A. crassispina and for P. depressus in Fig. 4A,B) or 5 mM ATP (for P. depressus in the remaining experiments). A suspension of the quiescent flagella was introduced into a 10 µl perfusion chamber (Morita and Shingyoji, 2004). ATP concentration was decreased by perfusing 40 µl of reactivating solution (HEPES) without ATP and with 20 units/ml hexokinase and 20 mM glucose more than three times. The quiescent flagella were treated with elastase and their sliding disintegration was induced by photoreleased ATP from caged-ATP (p3-[1-(2-Nitrophenyl) ethyl] ATP, Dojindo 349-05501) according to a previously published method (Morita and Shingyoji, 2004), except that 1.0 mM caged-ATP with 40 units/ml hexokinase at 10–4 M (for A. crassispina and P. depressus) or 10–3 M (for P. depressus) Ca2+ were used. Axonemes were bent with a glass microneedle according to previous methods (Morita and Shingyoji, 2004). The experiments of sliding of singlet microtubules on doublet bundles were carried out according to a previous method (Nakano et al., 2003). Recording and analysis of the microtubule sliding were also studied according to previous methods (Morita and Shingyoji, 2004).
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