essential for flagellar motility, as revealed by the paralyzed flagella pheno- ... with C1 defined as the microtubule with longer projections ( Hopkins, 1970 ;.
CHAPTER 8
The Flagellar Central Pair Apparatus
David R. Mitchell Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York, USA
C H A P T E R C O N TE N T S I. Introduction II. Central pair structure III. Genetic dissection of central pair structure and function IV. Biochemical identification of central pair proteins V. Central pair regulation of flagellar motility VI. Future directions References
235 236 239 242 244 247 248
I. INTRODUCTION The central pair of singlet microtubules and their associated structures (the central pair apparatus, central pair complex, or simply the central pair) are essential for flagellar motility, as revealed by the paralyzed flagella phenotype of central pair assembly mutants. Analysis of Chlamydomonas mutations that disrupt different central pair structures has provided a basis for detailed structural characterization of the central pair and aided in biochemical identification of proteins located in specific central pair associated complexes. Because radial spoke heads form intimate contacts with central pair projections, regulation is thought to work through central pair modulation of spokes, which in turn alter the activity of doublet-associated dynein motors. Several proteins with known signaling properties, including a protein phosphatase, an A kinase anchoring protein (AKAP), and calmodulin, have been localized to the central pair and may participate in these
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CHAPTER 8: The Flagellar Central Pair Apparatus
regulatory pathways. In addition, the central pair harbors motor proteins of the kinesin family, and proteins that may play important roles in flagellar nucleotide metabolism. This structure appears to have evolved prior to the divergence of eukaryotes from a common ancestor (Mitchell, 2004), and hence extensive work on the Chlamydomonas central pair has provided a basis for studies of central pair function in systems as distantly related as mammals (Zhang et al., 2004) and trypanosomes (Branche et al., 2006). As genome sequences have become available from all branches of eukaryotic organisms, the conservation of central pair proteins, as well as central pair structure, has been broadly confirmed. Because of its dominant role in studies of central pair biology, much of what we know about the Chlamydomonas central pair has been discussed in two general reviews of this suborganellar structure that cover all work up to that time (Smith and Lefebvre, 1997b; Smith and Yang, 2004).
II. CENTRAL PAIR STRUCTURE Our current understanding of central pair structure derives primarily from work in Chlamydomonas using electron microscopy of freeze-etch replicas, negative stain preparations, and thin sections. Early work identified differences in the length of prominent projections from the two microtubules, with C1 defined as the microtubule with longer projections (Hopkins, 1970; Witman et al., 1978; Dutcher et al., 1984). The two longer projections from C1 are designated 1a and 1b, and those from C2 are 2a and 2b. Additional densities were characterized following image averaging of central pair cross sections (Figure 8.1A) (Mitchell and Sale, 1999), as summarized in Figure 8.1B. The axial periodicity of some C1 projections is 32 nm (four tubulin dimers), whereas others repeat at 16 nm (two tubulin dimers) (Figure 8.1D, summarized diagrammatically in Figure 8.2); in contrast, all C2 projections appear to repeat every 16 nm (Goodenough and Heuser, 1985; Mitchell and Sale, 1999; Mitchell, 2003b). The central pair microtubules are held together along most of their length by bridging structures that also have a 16-nm repeat period. Additional connections between the two microtubules as seen in cross section include a diagonal bridge between C1 projection 1b and the C2 microtubule wall, and an interaction between the tips of projections 1b and 2b. Thin circumferential arcs or sheath elements (sh in Figure 8.2) link the tips of some projections and give the entire central pair a circular cross section. These arcs may be lacking from central pair structure in metazoans. The two microtubules of the central pair both start from a region above the basal body and transition zone (Ringo, 1967; Rosenbaum et al., 1969) (see Figure 10.1A and F in Chapter 10), and end in a membrane-associated cap structure at the flagellar tip (Ringo, 1967; Dentler and Rosenbaum, 1977) (see Figure 10.3C in Chapter 10). Work in other organisms indicates
Central Pair Structure
PF 6(
1a
)
oa 1c
2a 2c
C1a C2
ia
KLP
1d
PF20
1
PF16
C1
C1 CP
DL 2b
(1 b)
(A)
C1d
(D)
(C)
(B)
FIGURE 8.1 (A) TEM image average of the central region of wild-type Chlamydomonas axonemes, with central pair orientation maintained constant. Because the central pair does not maintain a fixed orientation relative to the surrounding doublet microtubules and associated radial spokes, spoke head images blur into a ring around the central pair complex. Adapted from Figure 8.4 of Yokoyama et al., 2004, Proc. Natl. Acad. Sci. 101:17398–17403, by copyright permission of the National Academy of Sciences. (B) Diagram of the central pair in which the two microtubules (C1 and C2) and prominent cross sectional densities (1a–1d and 2a–2c) are labeled. The locations of complexes containing identified central pair proteins (PF6, PF16, PF20, CPC1, and KLP1) are also indicated; in addition, hydin is located in 2b. DL, diagonal link. (C) Electron micrograph of a cross section through a wild-type axoneme, showing the relationship between outer doublets, radial spokes, and the central pair complex. (D) A longitudinal ultrathin section through a pf14 cpc1 axoneme reveals the periodicity of C1a projections (16 nm), C1d projections (32 nm), outer row dyneins (oa; 24 nm), and inner row dyneins (ia, 96 nm). The central pair does not remain centered in the axoneme when spokes are absent, as in this mutant. Proximal end to the left. Scale bar 100 nm.
proximal 1c
1a
sh 2a
1d
2b
2c 2b
1b
1c
bridge 2a 2c
bridge
2b
1c
1d
1a
C2
1b
link
sh
1b
1a 2c
sh
32 nm
1a
2a
sh
1c
1d
16 nm
1d sh 1b link (A)
2b
C1 (B)
C2
C1 (C)
sh C2 proximal
proximal (D)
FIGURE 8.2 Color-coded diagrams summarizing the relationships among structural elements of the central pair. (A) Cross sectional view of the central pair as seen from the proximal toward the distal end. (B) View of the 1b and 2b projections and associated structures with the proximal end toward the top of the page. Note the 32-nm spacing of projection 1d (yellow), and an associated attachment point of the 1b sheath (blue ovals), whereas most other structures visible from this view repeat every 16 nm. The topmost 1b projection and its associated sheath element (sh) have been omitted to provide a clearer view of the diagonal links (green) that connect 1b projections to the C2 microtubule. (C) The view in B has been flipped end-for-end to expose projections 1a, 2a, and 2c. The sheath connects every other 1a projection to a 1c density. (D) The view in C has been rotated by 90° to reveal the unusually dense particle distribution on the surface of C1, and the tilt of projections toward the distal end of the axoneme. Modified from Mitchell, 2003b, Cell Motil. Cytoskeleton 55:188–199, by copyright permission of Wiley-Liss, Inc.
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that gamma-tubulin is important for central pair nucleation (McKean et al., 2003), but no structural or biochemical specializations have been identified at the proximal end of the Chlamydomonas central pair, which can appear coincident with the distal end of the stellate transition zone structure (Ringo, 1967), or begin after a short gap (Mitchell et al., 2005). The transition zone may function as a physical barrier that prevents the central pair from sliding into the basal body cavity, as this defect occurs frequently in transition zone mutants (Jarvik and Suhan, 1991). Addition of new tubulin subunits to the central pair occurs at the distal end (Johnson and Rosenbaum, 1992), consistent with the view that these microtubules are nucleated by gamma-tubulin in the transition zone (Silflow et al., 1999; McKean et al., 2003) and with the observation that mutations affecting central pair microtubule assembly, such as beta-tubulin modifications in Tetrahymena (Thazhath et al., 2004) or Drosophila (Nielsen et al., 2001), can result in complete absence of the central pair, or in a central pair that is only present in proximal regions of the flagellum, but never in a central pair that is only assembled in distal regions. Assembly may be space-limited since a second, parallel central pair apparatus will assemble within the cage of outer doublets when space permits. This occurs when radial spokes are missing and the central pair cross sectional area is reduced by projection assembly mutations, such as in the triple mutant pf14 cpc1 pf6 (Figure 8.3), or when the cage of doublets is enlarged from 9 to 10 in the basal body defective mutant strain bld12 (Nakazawa et al., 2007; Nakazawa et al., personal communication). The central pair extends distally beyond the end of the outer doublet microtubules by as much as 0.5 μm. This distal extension appears free of associated projections and is distinguished by an electron-dense sheet between the two microtubules (Ringo, 1967) (and see Figure 10.3B and C in Chapter 10). Because the central pair complex rotates relative to the outer doublet microtubules (see section V), a shear zone must exist within the cap structure itself
8
9 1
7
2
6
3 5
4
FIGURE 8.3 A cross sectional micrograph of a pf14 pf6 cpc1 axoneme in which two central pair complexes have assembled (9 2 2). The absence of radial spokes (pf14) and a reduced cross sectional area of the central pair from loss of projections 1a (pf6) and 1b (cpc1) provides room for two central pair complexes to assemble within the normal nine doublets. Both central pair complexes are viewed from their proximal ends with C1 to the left. Doublet numbers are based on identification of doublet one, which lacks outer row dyneins. Scale bar 100 nm.
Genetic Dissection of Central Pair Structure and Function
or within the adjacent membrane. Although the flagellar tip region also plays an important role as the site of reversal of IFT motility (see Chapter 4), the central pair (and by inference the cap) are not required for this process, as IFT appears unaltered in strains carrying central pair assembly mutations. Central pair tip structures have not been characterized at the biochemical level.
III. GENETIC DISSECTION OF CENTRAL PAIR STRUCTURE AND FUNCTION Mutations at four loci destabilize the entire central pair complex: pf15, pf18, pf19, and pf20 (Table 8.1). These loci were some of the earliest to be mapped in Chlamydomonas (Ebersold et al., 1962) and analyzed for structural defects by electron microscopy (Warr et al., 1966), and were later studied in greater biochemical and structural detail (Witman et al., 1978; Adams et al., 1981). Phenotypically, these mutations all result in primarily nonmotile cells with flagella that do not beat, although a few cells in populations of these mutants display limited motility (Warr et al., 1966). Unlike the paralyzed flagella of radial spoke mutants (see Chapter 7), which are curved (most likely due to the intrinsic helical curvature of the central pair; see below), flagella of central pair mutants are straight. Although the central pair microtubules fail to assemble in these mutants, many central pair associated proteins are still present in the flagellar compartment and may interact to form a higher-order dense core structure (Witman et al., 1978; Adams et al., 1981). These remaining proteins and structures partition into the soluble phase when flagellar membranes are removed with detergents. Two of these four loci have been identified at the molecular level. The PF15 locus encodes a p80 (noncatalytic) subunit of the microtubule-severing protein
Table 8.1 Mutations affecting central pair structure Strain
Structural defect in flagella
Structural defect in axonemes
Gene product
Referencesa
pf6
9 2, 1a missing
9 2, 1a missing
PF6
[1], [2]
pf15
9 dense core
90
Katanin p80
[3], [4], [5], [6]
pf16
92
9 1, C1 missing
PF16
[1], [7]
pf18
9 dense core
90
[3], [5]
pf19
9 dense core
90
[3], [4], [5]
pf20
9 2, 9 1, 9 0
90
PF20
[3], [5], [8]
cpc1
1b missing
1b missing
CPC1
[9], [10]
a [1] Dutcher et al. (1984); [2] Rupp et al. (2001); [3] Starling and Randall (1971); [4] Witman et al. (1978); [5] Adams et al. (1981); [6] Dymek et al. (2004); [7] Smith and Lefebvre (1996); [8] Smith and Lefebvre (1997a); [9] Mitchell and Sale (1999); [10] Zhang and Mitchell (2004).
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CHAPTER 8: The Flagellar Central Pair Apparatus
katanin (Dymek et al., 2004), supporting a role for katanin in regulation of central pair microtubule assembly. The PF15 protein itself, although associated with the axoneme, does not appear to reside within the central pair structure. PF20 encodes a WD-repeat protein that localizes to the bridge between C1 and C2 (Smith and Lefebvre, 1997a). The mammalian orthologue of PF20, SPAG16L, is also localized to the central pair (Zhang et al., 2002), and is essential for normal sperm motility, but not for central pair assembly (Zhang et al., 2006). Gene products of PF18 and PF19 have not been identified. Three additional mutations have been characterized that partially disrupt the C1 microtubule or its associated structures: pf6, pf16, and cpc1. Nonconditional mutations at PF16 destabilize the entire C1 microtubule, which remains assembled in intact pf16 flagella but is selectively lost upon flagellar demembranation (Dutcher et al., 1984; Mitchell and Sale, 1999). Phenotypically, pf16 flagella twitch but fail to propagate bends, and cells are nonmotile. When isolated pf16 axonemes are exposed to reactivation conditions in the presence of a protease, they display an ability to disintegrate by doublet sliding, indicating residual dynein activity. However, the sliding rate is considerably slower than that of wild-type axonemes, and is indistinguishable from the sliding rate of axonemes that completely lack the central pair such as pf15 and pf18 (Smith, 2002a, b). The PF16 locus encodes an armadillo-repeat protein associated with the C1 microtubule (Smith and Lefebvre, 1996, 2000). The orthologue in mammals (SPAG6) is also a central pair protein essential for ciliary and flagellar motility (Sapiro et al., 2002), and yeast two-hybrid studies suggest that, in mammals, PF16 interacts directly with PF20 (Zhang et al., 2002) and is therefore likely localized at the interface between C1 and C2 (Figure 8.1B). A similar location is supported by the structural defects in pf16 axonemes under conditions that partially stabilize the C1 microtubule (Mitchell and Sale, 1999). However, selection of extragenic suppressors of a temperature-sensitive Chlamydomonas pf16 allele identified pf9-2, an inner row I1 dynein assembly mutation, as a bypass suppressor specific to the pf16-BR3ts allele (Porter et al., 1992), a result that is hard to reconcile with a positioning of PF16 in the bridge region. In vitro doublet microtubule sliding rate assays have also shown that I1 assembly mutations relieve the inhibition of doublet sliding caused by pf16 (Smith, 2002b). These results suggest that some regulatory signals generated by the central pair complex and involving PF16-associated or C1-associated proteins may specifically control I1 dynein activity (see Chapter 9 for additional information on I1 dynein regulation). Mutations at PF6 disrupt assembly of the C1a projection, and result in flagella that may beat, but too slowly for effective swimming (Dutcher et al., 1984). The gene product is a large (⬃240 kD) structural protein (Rupp et al., 2001) associated with five other subunits (Table 8.2), including both calmodulin and an 86-kD calmodulin-binding protein that has limited
Genetic Dissection of Central Pair Structure and Function
Table 8.2 Central pair complex proteins Structure/ location
Protein/ mutanta
Massb (kD)
pIb
Accession number
Properties
Referencesc
C1a
PF6 C1a-86 C1a-34 C1a-32 C1a-18 Calmodulin
240 86 34 32 18 18
4.84 5.54 5.63 5.56 5.70 4.30
AAK38270 AAZ31187 AAZ31186 AAZ31185 AAZ31184 AAA33083
Alanine-proline rich; ASH domain PKA RII-like LRR domain Coiled-coil, dimerizes with C1a-32 Resembles C1a-34 MORN domains Ca2-binding protein
[1], [2] [2] [2] [2] [2] [2]
C1b
C1b-350 (FAP42) CPC1 C1b-135 (FAP69) HSP70 Enolase
350
5.37
EDP00757
[3]
265 135
5.45 5.61
AAT40992 EDP06190
Five guanylate kinase domains, one adenylate kinase domain CH, adenylate kinase domains Armadillo repeat protein
78 56
5.25 5.26
P25840 P13683
Chaperone Glycolytic enzyme
[3], [5] [3]
[3], [4] [3]
C1
PF16 PP1c
57 35
6.0 4.8
AAC49169 AAD38850
Armadillo repeat protein Protein phosphatase 1c
[6] [7]
C2b
Hydin
540
5.80
EDP09735
ASH domains
[8]
C2c
KLP1
96
6.6
P46870
Kinesin-like protein
[9], [10]
C1–C2 bridge
PF20
63
7.7
AAB41727
WD-repeat protein
[11]
a Proteins listed in bold face are associated with loci at which Chlamydomonas mutations have been characterized; protein names correspond to locus names (Table 8.1). b
Mass as estimated by SDS-PAGE, pI as calculated from primary sequence.
c
[1] Rupp et al. (2001); [2] Wargo et al. (2005); [3] Mitchell et al. (2005); [4] Zhang and Mitchell (2004); [5] Bloch and Johnson (1995); [6] Smith and Lefebvre (1996); [7] Yang et al. (2000); [8] Lechtreck and Witman (2007); [9] Bernstein et al. (1994); [10] Yokoyama et al. (2004); [11] Smith and Lefebvre (1997a).
homology to protein kinase A (PKA) RII subunits (Wargo et al., 2005). Analysis of dynein regulation by calcium suggests that the PF6-calmodulin complex contributes to calcium-dependent regulation of dynein activation patterns, as seen by the pattern of doublet microtubule sliding in proteasetreated axonemes (Wargo et al., 2004). Immunolocalization studies in mice show that the mouse orthologue of PF6 is also a central pair protein (Zhang et al., 2005). One region of retained sequence similarity between the algal and vertebrate PF6 proteins also appears in hydin, another conserved central pair protein (see below and Table 8.2), and defines a domain of unknown function that has been termed the ASH domain (Ponting, 2006). The CPC1 locus, similar to the PF6 locus, encodes a large protein essential for assembly of a C1 microtubule projection (C1b) (Zhang and Mitchell, 2004). The tip of C1 projection 1b appears to interact with the tip of C2 projection 2b (Figure 8.1), and the absence of 1b correlates with frequent absence of projection 2b in cpc1 axonemes (Mitchell and Sale, 1999). Although a domain homologous to adenylate kinases appears within the
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CHAPTER 8: The Flagellar Central Pair Apparatus
CPC1 sequence, the functional significance of this domain has not been determined and axonemal adenylate kinase activity appears unaltered by this mutation (Zhang and Mitchell, 2004; Mitchell et al., 2005). A calponin homology (CH) domain near the N-terminus of CPC1 resembles CH domains of known microtubule-binding proteins such as EB1 (Bu and Su, 2003) and Spef1/CLAMP (Dougherty et al., 2005; Chan et al., 2005). The CPC1 protein can be extracted as part of a complex that contains another large (350 kD) protein with homology to nucleoside diphosphate kinases, a novel 135-kD structural protein, the heat shock protein HSP70, and enolase (Mitchell et al., 2005). Mutations at cpc1 disrupt assembly of these subunits and result in motility with a reduced beat frequency. This phenotype can be largely attributed to a reduction in intraflagellar ATP concentrations that result from the loss of glycolytic ATP synthesis supported by enolase. Other enzymes necessary (along with enolase) for one of the ATP-generating steps of glycolysis have also been localized to the flagellar compartment, but do not appear to interact with the central pair complex (Mitchell et al., 2005). KPL2, the mammalian orthologue of CPC1 (Ostrowski et al., 2002; Zhang and Mitchell, 2004), occurs in several splice variants, at least one of which is essential for normal central pair assembly and motility in mammalian spermatozoa (Sironen et al., 2006). In addition to the Chlamydomonas mutations listed above, whose characterizations first identified the gene products as central pair components and led to their later identification in mammals, one mammalian mutation became the starting point for identification of a Chlamydomonas central pair protein. The mouse hy3 mutation in the gene encoding Hydin, a large protein expressed in ciliated cells (Davy and Robinson, 2003), results in hydrocephalus. The knockdown of the Trypanosoma brucei (Broadhead et al., 2006) or Chlamydomonas (Lechtreck and Witman, 2007) orthologues results in reduced motility, and the Chlamydomonas protein was further localized to the C2b central pair projection, where it stabilizes the association of kinesin-like protein KLP1 with the C2 microtubule (Lechtreck and Witman, 2007). The presence of a conserved ASH domain in both PF6 and hydin (Table 8.2) together with their locations at rotationally symmetric positions on the C1 and C2 microtubules suggests that these proteins may play similar roles as scaffolds for other central pair components.
IV. BIOCHEMICAL IDENTIFICATION OF CENTRAL PAIR PROTEINS Comparative gel analysis was used over 20 years ago to catalog 23 proteins that are reduced or missing from axonemes of Chlamydomonas central pair assembly mutants (Adams et al., 1981; Dutcher et al., 1984) and to further show that 11 of these are likely associated with C1, based on their
Biochemical Identification of Central Pair Proteins
depletion from pf16 axonemes. Reassessment of those results based on data from the molecular studies of pf6, pf16, and cpc1 suggest that fewer than half of these probable central pair proteins have been characterized at the molecular level (Table 8.2). However, in addition to the proteins identified through analysis of mutants, biochemical methods have been used to identify a few central pair proteins in two categories: kinesin-like proteins and signal transduction proteins. Kinesin-like proteins were first identified in the Chlamydomonas central pair through western blot analysis with pan-kinesin antibodies (Fox et al., 1994; Johnson et al., 1994), and through amplification of Chlamydomonas kinesin-related sequences (Bernstein et al., 1994). Two proteins with kinesin-like properties were localized to the central pair in these studies, but only one has been verified as a bona fide kinesin-related protein at the molecular level. The KLP1 protein was first identified through PCR of kinesin-related sequences, and complete characterizations of the gene and cDNA show that KLP1 has a conserved amino-terminal motor domain, but a carboxy-terminal tail unrelated to other kinesins (Bernstein et al., 1994). Sequence comparisons show KLP1 to be a member of the kinesin9 family (Miki et al., 2005) that appears widely in the genomes of organisms with motile 9 2 organelles (Wickstead and Gull, 2006; Richardson et al., 2006), including mammals, whose kinesin-9 homologues are highly represented in testis and lung cDNA libraries (Miki et al., 2003). An antibody to Chlamydomonas KLP1 recognizes a 96-kD central pair protein and decorates the C2 microtubule. RNAi depletion of KLP1 results in abnormal motility and disrupts structures on the outer margin of the C2 microtubule (Yokoyama et al., 2004) (Figure 8.1). KLP1 shows nucleotide-sensitive binding to reassembled microtubules in vitro, but its association with the central pair is not nucleotide sensitive (Bernstein, 1995; Yokoyama et al., 2004). Although KLP1 has been hypothesized to act as a motor for rotation of the central pair in Chlamydomonas, more recent studies conclude that central pair rotation can occur as a passive response to bend propagation (see section V), and the activity of a central pair-associated motor is not required. In addition, kinesin-9 family members appear in the genomes of organisms such as mammals and trypanosomes whose flagella have fixed (nonrotating) central pairs (Omoto et al., 1999; Branche et al., 2006; Gadelha et al., 2006). Instead, it may be of value to consider KLP1 as a conformational switch whose activation could be linked to a change in central pair curvature, or to a change in the conformation of associated proteins important for signal transduction. The second central pair kinesin is a 110-kD protein that reacts with antibodies directed against two conserved kinesin sequences, HIPYR and LAGSE, and that is missing from axonemes of central pair mutants such as pf18 and pf19 (Fox et al., 1994; Johnson et al., 1994). The molecular identity and central pair location of this protein are not known. Immunolocalization
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with an antibody directed against conserved kinesin neck domain sequences (αHD antibody) identified the C1 microtubule as a site of kinesin-like protein attachment (Johnson et al., 1994). The αHD antibody also detects a 110 -kD band on blots, but this band is retained in pf18 axonemes, and its relationship to the HIPYR and LAGSE band has not been determined. Two biochemical studies suggest that additional signaling events feature central pair proteins. Through blot overlays with the PKA RII subunit, Sale and colleagues identified two AKAP-like proteins in the Chlamydomonas flagellum (Gaillard et al., 2001). One of these is missing from the radial spoke assembly mutant pf14 and was identified by Gaillard et al. as the PF14 gene product (see Chapter 7 for additional details). The other AKAP is missing from central pair mutant axonemes. This 240-kD central pair AKAP is retained in pf16 axonemes, and thus likely associates with C2, but its molecular identity and functional role have not been determined. Biochemical and western blot analysis of protein phosphatases in the Chlamydomonas flagellum showed that most of the flagellar serine/ threonine phosphatase PP1c is linked to the C1 central pair microtubule (Yang et al., 2000). PP1 is known to regulate axonemal dynein activity in Chlamydomonas (see Chapter 9), so it is tempting to assume that the central pair-linked PP1c is involved. However, some PP1c immunoreactive signal was also found associated with outer doublet microtubules, where dyneins are anchored. Therefore, the central pair PP1c may not be involved directly in this dynein regulatory pathway, and downstream targets of the central pair enzyme remain unknown.
V. CENTRAL PAIR REGULATION OF FLAGELLAR MOTILITY The mechanisms through which the central pair influences axonemal motility have apparently gone through one major evolutionary shift, since all metazoans examined to date have a central pair complex that remains fixed in its orientation relative to the surrounding doublet microtubules, whereas protists from several clades have a central pair that is twisted, and which rotates during bend propagation (Omoto et al., 1999). Chlamydomonas has long been recognized as an organism with a central pair complex that could rotate, based on an observed variation in central pair orientation (relative to doublets and basal bodies) in electron micrographs of fixed cells, and on rotation of the extruded tip of the central pair in disintegrating reactivated axonemes (Kamiya, 1982; Kamiya et al., 1982; see videos “Central pair rotating in demembranated Chlamydomonas cell model” and “Central pair rotating in Chlamydomonas axoneme” at http://www.elsevierdirect.com/companions/ 9780123708731). The entire central pair complex in Chlamydomonas has a helical conformation, best observed when the central pair has been released from the rest of the flagellum (Figure 8.4) (Kamiya et al., 1982; Mitchell
Central Pair Regulation of Flagellar Motility
FIGURE 8.4 Dark-field light micrographs of central pair complexes extruded from in vitro-reactivated Chlamydomonas axonemes. The central pair is helical when not constrained by surrounding doublet microtubules. Scale bar 5 μm.
}
B
}C
}D
(A)
C1 C2 (B)
(C)
(D)
FIGURE 8.5 (A) Diagram of a cell during forward swimming. Brackets indicate the principal bend (B), interbend (C), and reverse bend (D) regions illustrated in the diagrams to the right. (B) Central pair orientation in principal bends. The C1 microtubule is distinguished by a row of projections (dark ovals) repeating every 32 nm. (C) The central pair twists in interbend regions. (D) Central pair orientation in the reverse bend region. Arrows point toward distal end of flagellum. Adapted from Mitchell, 2005, Am. Inst. Phys. Conf. Proc. 555:130–136, by copyright permission of the American Institute of Physics.
and Nakatsugawa, 2004). Constraint of this helix within the cage of doublet microtubules results in a fixed relationship between central pair orientation and flagellar bends, such that the C1 microtubule faces the outside of every bend (Figure 8.5); the central pair twists by 180° between alternating bends to maintain the bend-dependent orientation (Mitchell, 2003a). As a result, during bend propagation, the twisted central pair rotates by 360° between successive beats. The relationship between central pair rotation and regulation of bend formation has been partially resolved through experiments using suppressor mutations. As detailed in Chapters 7 and 9, the paralyzed phenotype that results from disruption of either radial spoke or central pair assembly can be partially overcome by bypass suppressors that restore bend propagation, without restoring the disrupted structures. When one of these suppressors, suppf1, was combined with pf1, which blocks radial spoke head assembly, the resulting flagella could beat in the absence of normal interactions between radial spokes and the central pair. In these flagella, the central pair maintained its twisted conformation and its bend-dependent orientation, showing that central pair rotation is independent of spoke/central pair interactions (Mitchell and Nakatsugawa, 2004). Bend propagation must be sufficient to
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drive central pair rotation, as first suggested by Kamiya (1982), without the requirement of a rotational motor linked to the central pair microtubules. To determine the exact contribution of the central pair complex to motility regulation, one can start from the premise that the central pair complex must provide regulation for an aspect of motility that is specifically and consistently missing in central pair–defective mutants. When flagella from such mutants beat in the presence of suppressor mutations, they beat with a reduced frequency and an altered waveform (Brokaw et al., 1982; Brokaw and Luck, 1985), suggesting that both of these parameters may be modified through central pair/radial spoke interactions. During forward swimming, wild-type Chlamydomonas flagella beat with a highly asymmetric waveform in which bends are largely restricted to a single plane (rather than being helical) and principal bends have a much greater curvature than reverse ones. The waveform remains essentially planar in suppressed central pair mutants, indicating that the central pair is not essential for this form of beat asymmetry, but principal bend curvature is reduced and reverse bend curvature increased, suggesting that the central pair may be involved in regulation of the relative contributions of dyneins on different doublets to bend curvature at the low flagellar calcium concentrations typical of forward swimming. This would be consistent with a model in which the central pair regulates the underlying curvature-dependent activation of dynein arms on different doublets and modulates bending patterns. When isolated Chlamydomonas axonemes from central pair assembly mutants are reactivated, they fail to beat except under highly selective buffer or nucleotide conditions. As was the case for suppression by mutations, these conditions generally only permit bend propagation with reduced frequency, reduced asymmetry of principal and reverse bends, and a tendency to a more three-dimensional (helical) waveform (Omoto et al., 1996; Frey et al., 1997; Wakabayashi et al., 1997; Yagi and Kamiya, 2000). Although reactivation in 10 mM MgSO4 produced similar beat frequencies and waveforms in pf18 and wild-type axonemes (Yagi and Kamiya, 2000), the beat frequency of the wild-type axonemes was reduced by these conditions compared with standard conditions (and compared with living cells). The calcium-dependent switch between asymmetric and symmetric (planar) waveforms (see Chapter 13) does not depend on the central pair (Wakabayashi et al., 1997), and thus must not be a function of the calmodulin complex associated with PF6. Studies of doublet sliding in protease-treated axonemes have clearly shown a relationship between central pair/radial spoke interactions and regulation of I1 inner row dynein, as described in detail in Chapter 9. The central pair–specific aspects of that regulation are worth emphasizing in this chapter. Doublet sliding rates at low calcium concentrations are reduced in both central pair and radial spoke mutants compared to wild-type axonemes, and this reduced sliding rate is seen in pf16 axonemes, which lack
Future Directions
only C1, as well as in mutants such as pf15 and pf18 that lack the entire central pair, but not in pf6 axonemes, which are only missing the 1a projection (Smith, 2002b). Doublet sliding rates in pf16 and pf18 axonemes return to wild-type levels when I1 dynein assembly is blocked (ida1), or when the serine/threonine protein kinase CK1 is inhibited. At high calcium concentrations, sliding rates are wild type in central pairless axonemes (e.g., pf15, pf18), but are reduced in C1-defective pf16 axonemes (Smith, 2002a). This result suggests that the pf16 central pair modulates radial spokes so that spoke-dependent calcium activation, seen in the absence of the central pair, is prevented. Because the pattern of doublets that slide in proteasetreated axonemes is related to central pair orientation (Wargo and Smith, 2003) and calcium concentration (Wargo et al., 2004), these results can be interpreted to show that different central pair projections send operationally unique signals through radial spokes, and that modulation of dynein activity patterns is dependent on central pair orientation. The mechanism of signal transduction between the central pair and radial spokes remains unclear. In metazoans, which lack central pair rotation, radial spokes tilt in the plane of the bend in response to bend-induced displacement of doublets relative to the central pair (Warner and Satir, 1974). The spoke tilt shows that spoke heads physically interact with central pair projections during bend formation. As each bend grows, tilt increases and reaches a maximum, after which spoke heads must translocate along the central pair without further tilt. Axonemal geometry requires that for a planar bend, the doublets in the bend plane (numbers 1 and 5–6) show little or no sliding displacement relative to their neighboring doublets, and therefore the dyneins on these doublets should contribute little or nothing to bend formation. Therefore, spokes tilt maximally along doublets that have largely inactive dyneins, which should require little regulation, and minimally along doublets with highly active dyneins, whose regulation would modify beat asymmetry (waveform). How then do spoke/central pair interactions regulate motility? Spoke tilt could function as a mechanism for the central pair/radial spoke system to locally sense both the direction and the extent of curvature. In this way, signals transmitted by spokes to dyneins could be modulated locally by the extent of tilt (as well as globally by physiological signal modulations such as changes in intraflagellar calcium concentration), so that dynein regulation would always be influenced by both the direction and the size of the bend. Whether similar longitudinal spoke tilt occurs in Chlamydomonas, or spoke tilt includes a radial displacement due to central pair rotation, has not been established.
VI. FUTURE DIRECTIONS At this time the central pair field has at least as many questions as answers, but the many advances from work with Chlamydomonas show the
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continuing power of this organism for unraveling the complexities of flagellar mechanics and motility regulation. Especially needed is a more complete proteomic analysis coupled with a more precise three-dimensional structure of the central pair. The completion of a radial spoke proteome (Yang et al., 2006) and the discovery of methods to purify intact radial spokes (Yang et al., 2001) provide hope that some aspects of radial spoke/central pair interactions may yield to in vitro rebinding and biochemical approaches. Selection of additional mutants or depletion of proteins through RNAi technology will also be needed to clarify the in vivo role of specific central pair proteins in regulatory pathways. Finally, further comparative studies will ultimately be essential for understanding the evolution of this complex structure and its central importance in flagellar motility.
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