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Address correspondence to D.R. Mitchell, Dept. of Cell and Developmental Biology, State University of New York Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210. Tel.: (315) 464-8575. Fax: (315) 464-8535. email: mitcheld{at}upstate.edu
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Abstract |
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Key Words: cilia; radial spoke; dynein; motility; microtubule
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Introduction |
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Regulation of eukaryotic ciliary and flagellar motility is essential for a wide range of biological processes such as sperm chemotaxis and mucociliary clearance in metazoans and foraging behavior in smaller aquatic eukaryotes. Radial spokes tightly associate with the nine outer doublet microtubules and project toward a central pair apparatus (CP) with which they form transient contacts. During flagellar bending, doublet microtubules slide and spokes must move along the CP surface. Although the CP is nearly circular in cross section, its underlying structure and biochemistry are highly asymmetric (Adams et al., 1981; Mitchell, 2003a). In addition, rows of radial spokes on each doublet align with different surfaces of the CP that each form unique sites of potential interaction, and these interaction sites change as the CP rotates. We recently determined (Mitchell, 2003b) that the Chlamydomonas flagellar CP, like that of Paramecium tetraurelia cilia (Omoto and Kung, 1980), twists in actively beating flagella so that the CP surface facing each row of spokes in bent regions is different from the CP surface facing the same row of spokes in straight regions between bends. When swimming cells were fixed for electron microscopy, the plane through the two CP microtubules was always parallel to the bend plane in curved segments and CP microtubule C1 was nearest the outer edge of each curve. Similar CP orientations in principal and reverse bends were related by 180° twists in interbend regions.
The constant relationship between CP orientation and bend position in Chlamydomonas flagella suggests that bend propagation may drive CP rotation. If the CP is inherently twisted, forced propagation of one CP orientation along with propagation of each bend would result in CP rotation. However, it is equally possible that CP rotation is the driving force that induces bend propagation and that CP twist is caused by the torque of a rotation force. If CP rotation and twist are active processes, then they must occur either through torque generated between CP projections and radial spokes or at sites of CP attachment to flagellar distal tip structures. A rotation force at the tip is unlikely to drive bend propagation because bends form at the proximal end in Chlamydomonas flagella and because the CP continues to rotate after its partial extrusion from flagellar tips (Kamiya, 1982). We set out to determine if CP rotation and twist are causally linked to bend propagation and if radial spoke interactions with CP projections are required for this process. We conclude that bend propagation drives CP rotation, rather than the reverse, and use this conclusion as a basis to constrain models of radial spokecentral pair regulation of flagellar dynein activity.
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Results |
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As illustrated in Fig. 3, formation of CP twists does not depend on interactions between radial spoke heads and CP projections. Straight segments of pf1 and pf17 flagella often contained twisted CP, and in one image (Fig. 3, A and A') two CP twists occur within one visible segment. Where adherent flagella were curved, the CP remained parallel to the section plane (Fig. 3, C and C'), similar to the orientation in curved segments of adherent wild-type flagella (Fig. 2 D). Overall, lack of spoke heads had no apparent effect on CP orientation in quiescent flagella, which suggests that spoke head interactions with the CP are not important determinants of CP shape or orientation under these quiescent conditions.
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Discussion |
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Previous studies of wild-type cells that were fixed while swimming revealed a constant CP orientation in bends, such that both CP microtubules are parallel to the bend plane, with C1 nearest the outer edge of the bend (Mitchell, 2003b). Here we show that this bend-specific orientation is independent of bend propagation because it occurs in curved, quiescent flagella, and is also independent of spoke head interactions because it occurs during bend propagation in spoke head mutants. Similar CP conformations in flagella that retain or lack radial spoke heads show that spokeCP interactions are not important determinants of CP orientation in these flagella. Our data do not rule out an effect of spokecentral pair interactions on CP orientation, but suggest that any such effect provides subtle modulation of a primarily spoke-independent orientation mechanism.
A mechanism consistent with these results would be that an inherently helical CP conforms to axonemal curvature generated by dynein-dependent doublet sliding. When the axoneme curves, the CP always curves with C1 along its outer edge because this shape constitutes a minimum energy CP conformation. Straight flagellar segments between bends force the CP to straighten, which can only be accommodated by a CP twist. During active bend propagation, successive CP curves and twists must also propagate, and it is this propagation of a twisted conformation that results in CP rotation. As each bend elongates, inherent curvature of the CP forces an orientation that keeps specific CP projections directed toward radial spokes along doublets with active dyneins. This relationship is maintained for every bend, in both the principal and reverse bend directions, and propagates with each bend along the axoneme. Regulatory signals, transmitted from CP projections through spokes to modulate dyneins, could simultaneously alter the activity of dyneins in every bend.
Of what advantage is a rotating CP, and why has rotation been abandoned in some cell types? We contend that bend-dependent CP orientation automatically adjusts the CP regulatory machinery to changes in principal bend direction, and therefore is retained in organelles that must change beat direction. For example, in P. tetraurelia, major changes in effective stroke direction are modulated by changes in calcium ion concentration through a beat-independent mechanism (Naitoh and Kaneko, 1972). Simultaneously, beat frequency, as well as more subtle changes in effective stroke direction, are modulated by changes in cAMP and cGMP (Bonini and Nelson, 1988), which act in part through changes in the phosphorylation of dynein subunits (Hamasaki et al., 1991; Noguchi et al., 2000). The signal transduction pathway from cyclic nucleotide to outer row dynein phosphorylation has been well studied in P. tetraurelia (Barkalow et al., 1994; Satir et al., 1995), and a similar pathway involving inner row dyneins has been identified in Chlamydomonas. Phosphorylation of Chlamydomonas inner row I1 dynein occurs through a CPradial spokemodulated cAMP-dependent protein kinase cascade (Howard et al., 1994; Habermacher and Sale, 1997), alters doublet sliding velocities (Smith and Sale, 1992), and has been linked to phototaxis (King and Dutcher, 1997). Thus, although CP orientation is itself determined initially by bend formation, the resulting CP orientation provides a platform for CPradial spoke modulation of the bend shape (waveform) and propagation velocity (beat frequency), and this regulation of waveform and beat frequency can be independent of effective stroke direction. Not surprisingly, Chlamydomonas flagella continue to beat with essentially planar waveforms and to respond in a limited way to shifts in calcium concentration even in the absence of radial spokecentral pair interactions (Wakabayashi et al., 1997), confirming that CP orientation is a response to, not a determinant of, bend plane. Our model predicts that cell types in which CP orientation has become fixed should beat with a fixed bend plane that does not require subtle changes in effective stroke direction (Mitchell, 2004). In the case of ctenophore macrocilia, in which the CP does not rotate (Tamm and Tamm, 1981), hundreds of cilia must beat in unison within each macrocilium, and their geometry precludes effective strokes that vary from a fixed bend plane. These cilia can reverse effective stroke direction in response to calcium signals, but maintain the same axonemal bend plane during forward and reverse beating episodes (Tamm and Tamm, 1981). Likewise, typical metazoan spermatozoa, which have a fixed CP orientation and planar bends, modulate asymmetry, rather than bend plane, to effect tactic responses (Brokaw, 1979; Cook et al., 1994). The apparently fixed CP orientation of lamellibranch gill cilia (Gibbons, 1961; Warner and Satir, 1974) may also correspond with a single effective stroke direction, as the primary response of these organelles to calcium-mediated signals is quiescence, rather than reorientation (Walter and Satir, 1978). This model also explains evolutionary loss of the CP in organelles that beat with helical, rather than planar, bends (Gibbons et al., 1983; Nonaka et al., 1998) because these organelles need not modulate bend direction or waveform symmetry.
Confirmation of this model requires more information on CP orientation from organisms that can modulate effective stroke orientation, as well as better information about the ability of Chlamydomonas to alter beat orientation during tactic responses. To understand the mechanisms of CP-based waveform and beat frequency regulation, we also need better information on central pair proteins and their interactions with radial spoke heads. Recent advances in analyzing proteins of the Chlamydomonas radial spoke (Yang et al., 2001, 2004) and central pair (Smith and Lefebvre, 1996, 1997; Mitchell and Sale, 1999; Rupp et al., 2001; Zhang and Mitchell, 2004), together with continued characterization of mutations affecting CP function (Wargo and Smith, 2003; Wargo et al., 2004; Zhang and Mitchell, 2004), provide promise that advances should not be far off.
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Materials and methods |
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Extruded central pair complexes were produced spontaneously from reactivated axonemes as follows. Flagella isolated from wild-type cells by standard procedures (Witman, 1986) were resuspended in HMDEK (30 mM Hepes, 5 mM MgSO4, 1 mM DTT, 0.5 mM EGTA, 25 mM potassium acetate, and 1 mM PMSF, pH 7.4) and mixed with an equal volume of HMDEK containing 1% NP-40 and 2 mM ATP to initiate reactivation. Reactivated axonemes were set at RT until beating stopped (3060 min). This preparation was triturated gently to dissociate CP complexes from the distal ends of axonemes and spun in a microfuge at 6,000 rpm for 4 min. The supernatant solution was retained as an enriched CP fraction. Extruded CP were imaged under darkfield illumination (Aksiokop; Carl Zeiss MicroImaging, Inc.; 40x oil immersion, irised 0.51.0 NA objective), or were applied to carbon formvar-coated grids and negatively stained with 1% uranyl acetate for EM. Darkfield images were recorded at RT on film (TMax P3200; Kodak), and negatives were scanned at 2,400 dpi on a flatbed scanner. Positive (inverted) images were adjusted for contrast and gamma in Photoshop 6.0.
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Acknowledgments |
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Submitted: 23 June 2004
Accepted: 22 July 2004
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References |
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