Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii.

Abstract

Mutations at two loci, which cause an altered mobility of the flagella, affected the central pair microtubule complex of Chlamydomonas reinhardtii flagella. The mutations at both loci primarily affected the C1 microtubule of the complex . Three alleles at the PF16 locus affected the stability of the C1 microtubule in isolated axonemes . This phenotype has allowed us to determine that at least ten polypeptides of the central pair complex are unique to the C1 microtubule . The motility defect was correlated with the failure to assemble three of these ten polypeptides in vivo. The structural gene product of the PF16 locus was a polypeptide with molecular weight 57,000 as shown by analysis of five intragenic revertants and by analysis of axonemes from dikaryon rescue experiments. Three alleles at the PF6 locus affected the assembly of one of the two projections of the C1 microtubule and this projection was formed by at least three polypeptide components, which are a subset of polypeptides missing in isolated pfl6 axonemes . No structural gene product has been identified for the PF6 locus . The gene product is probably not one of the identified projection constituents as shown by analysis of dikaryon rescue experiments . Chemical extraction of isolated wild-type axonemes suggests that at least seven polypeptide components are unique to the C2 microtubule .The central pair microtubules offlagella more closely resemble the cytoplasmc microtubules than the outer microtubules of the flagella by virtue of their structure and their lability . They are singlet microtubules with 13 protofilaments unlike the nearby outer doublet microtubules that are composed of acomposite set ofmicrotubules with 13 and 11 protofilaments (1). The outer doublet microtubules, in contrast to the central pair microtubules, arise as a continuation ofthe microtubules of the basal bodies. The central pair complex originates distal to the transition zone of the basal body and terminates in specialized structures associated with the flagellar membrane at its tip (2) . In Chlamydomonas reinhardtii the central pair microtubules are sensitive to depolymerization by low ionic strength dialysis and by exposure to detergents (3, 4), whereas the outer doublet microtubules retain their integrity under these conditions .The two microtubules of the central pair complex also differ from one another (5-7). In squid sperm, Linck and coworkers (8) have shown that the morphology of the Cl microtubule differs from that of the C2 microtubule . In Chlamydomonas the C 1 microtubule has two projections that are 18 nm long as measured by thin-section electron micros-THE JOURNAL OF CELL BIOLOGY " VOLUME 98 JANUARY 1984 229-236 0 The Rockefeller University Press -0021-9525/84/01/0229/08 $1 .00 copy. The C2 microtubule has two shorter projections of -8 nm each. Furthermore, the C2 microtubule is more labile than the C 1 microtubule (4) .We have analyzed mutations at two unlinked loci in Chlamydomonas that affect the C1 microtubule specifically with the aim of unders...

The radial spokes and central apparatus: Mechano‐chemical transducers that regulate flagellar motility

“…Chemical signaling also includes axonemal enzymes that are not directly sensitive to second messengers including casein kinase 1 (CK1), PP1, and PP2A, all of which appear to be involved in the control of dynein-driven motility [Walczak and Nelson, 1993; Central apparatus-associated polypeptides PF6 238-kDa alanine/proline rich protein; pf6 flagella lack the 1a projection on the C1 microtubule and display only twitching motion [Dutcher et al, 1984;Rupp et al, 2001] PF16 57-kDa armadillo repeat containing protein that localizes to the c1 microtubule; pf16 flagella lack the C1 microtubule and are paralyzed. PF16 may correspond to CP14 [Dutcher et al, 1984;Smith and Lefebvre, 1996] PF20 63-kDa WD-repeat containing protein that localizes to the inter-microtubule bridge connecting C1 and C2; pf20 flagella lack the entire central apparatus and are paralyzed [Adams et al, 1981;Smith and Lefebvre, 1997a] PP1c Localized primarily, but not exclusively, to the C1 microtubule [Yang et al, 2001] KLP1 83-kDa kinesin-like protein that localizes to the C2 microtubule [Bernstein et al, 1994] KRP 110-kDa kinesin related protein associated with the central apparatus, most likely the C1 microtubule [Fox et al, 1994;Johnson et al, 1994;Mitchell and Sale, 1999] AKAP240 240-kDa A-kinase anchoring protein; most likely associated with C2 [Gaillard et al, 2001] Calmodulin Most likely associated with C1, sediments as a complex at 10S [Yang et al, 2001;Dymek et al, 2002] Radial spoke-associated polypeptides RSP2 PF24 gene product, GAF, and calmodulin binding domain; pf24 mutants lack radial spoke heads and have paralyzed flagella Piperno et al, 1981;Yang et al, 2002] RSP3 PF14 gene product, 97-kDa A-kinase anchoring protein most likely localized to the base of the spokes; pf14 mutants lack radial spokes and have paralyzed flagella Piperno et al, 1981;Diener et al, 1993;Gaillard et al, 2001] RSP4 PF1 gene product, 49.8-kDa proline rich polypeptide similar to RSP6, pf1 mutants lack spoke heads and have paralyzed flagella Piperno et al, 1981;Curry et al, 1992] RSP6 PF26 gene product, 48.8-kDa proline rich polypeptide similar to RSP4, pf26 mutants lack spoke heads and have paralyzed flagella Piperno et al, 1981;Curry et al, 1992] LC8 RSP2...…”

Three types of cilia including a novel 9+4 axoneme on the notochordal plate of the rabbit embryo

“…This important structure consists of two single microtubules tethered together to make them act as a doublet. Each of the central pair microtubules is equipped with a unique set of accessory proteins distinguishing the two tubules from each other and creating intrinsic asymmetry in the CP: the C1 tubule shows two long and two short projections, the C2 tubule is equipped with 3 short projections (Adams et al, 1981;Dutcher et al, 1984;Mitchell, 2003). In some species, the CP rotates during bending of the cilium, thereby modulating outer doublet dynein activity via the radial spokes, which may account for the variety of waveforms and beating frequencies observed in different cilia and flagella (Omoto et al, 1999;Wargo and Smith, 2003).…”

TheChlamydomonas PF6Locus Encodes a Large Alanine/Proline-Rich Polypeptide That Is Required for Assembly of a Central Pair Projection and Regulates Flagellar Motility

“…Characterization of the bypass mutations has identified defects in genes encoding components of the outer dynein arms (Huang et al, 1982;Porter et al, 1994;Rupp et al, 1996), the inner dynein arms Myster et al, 1997), and the dynein regulatory complex (Huang et al, 1982;Piperno et al, 1992). These and other studies suggest that the central pair microtubules regulate dynein arm activity through a signal transduction cascade that involves the radial spokes and the dynein regulatory complex.Recent work in Chlamydomonas has demonstrated high levels of both structural and biochemical complexity within the central pair microtubules (Dutcher et al, 1984;Mitchell and Sale, 1999). In Chlamydomonas, the C1 microtubule is associated with two long (18 nm) projections (1a and 1b) that repeat at 16-nm intervals and two smaller projections (1c and 1d) that repeat at 32-nm intervals.…”

“…Mutations in at least four loci (PF15, PF18, PF19, and PF20) disrupt the assembly of the entire central pair apparatus and its constituent 25 polypeptides (Adams et al, 1981;Dutcher et al, 1984). Mutations at three other loci (PF16, PF6, and CPC1) result in only partial disruption of central pair structures (Dutcher et al, 1984;Mitchell and Sale, 1999). At least 10 polypeptides are unique to the C1 microtubule, and seven are unique to the C2 microtubule (Dutcher et al, 1984).…”

“…Mutations at three other loci (PF16, PF6, and CPC1) result in only partial disruption of central pair structures (Dutcher et al, 1984;Mitchell and Sale, 1999). At least 10 polypeptides are unique to the C1 microtubule, and seven are unique to the C2 microtubule (Dutcher et al, 1984).To identify central pair components involved in regulating flagellar motility, we used insertional mutagenesis procedures in Chlamydomonas reinhardtii (Tam and Lefebvre, 1993) to recover a new collection of "tagged" motility mutants (Myster et al, 1997). Structural analysis of mutant axonemes identified one strain, 5B9, lacking the 1a projection on the C1 microtubule (Figure 1), a defect that resembles that previously described for pf6-1 (Dutcher et al, 1984;Mitchell and Sale, 1999).…”