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

Abstract

Efficient motility of the eukaryotic flagellum requires precise temporal and spatial control of its constituent dynein motors. The central pair and its associated structures have been implicated as important members of a signal transduction cascade that ultimately regulates dynein arm activity. To identify central pair components involved in this process, we characterized a Chlamydomonas motility mutant (pf6-2) obtained by insertional mutagenesis. pf6-2 flagella twitch ineffectively and lack the 1a projection on the C1 microtubule of the central pair. Transformation with constructs containing a full-length, wild-type copy of the PF6 gene rescues the functional, structural, and biochemical defects associated with the pf6 mutation. Sequence analysis indicates that the PF6 gene encodes a large polypeptide that contains numerous alanine-rich, proline-rich, and basic domains and has limited homology to an expressed sequence tag derived from a human testis cDNA library. Biochemical analysis of an epitope-tagged PF6 construct demonstrates that the PF6 polypeptide is an axonemal component that cosediments at 12.6S with several other polypeptides. The PF6 protein appears to be an essential component required for assembly of some of these polypeptides into the C1-1a projection. INTRODUCTIONCilia and flagella are highly conserved structures found on diverse cell types, ranging from single-cell protozoa to multicellular tissues in humans, where they function to propel cells through a fluid environment or to transport fluid across a cell surface. Motile cilia are also found in the embryonic node (Sulik et al., 1994; Bellomo et al., 1996), where their motility appears to be critical for establishing the morphogenetic gradient that determines the left-right body axis in mammals (Nonaka et al., 1998). Defects in the assembly or activity of cilia and flagella result in a variety of abnormalities, including defects in left-right axis asymmetry, infertility, and respiratory disease (Afzelius et al., 1975; Afzelius, 1995;Supp et al., 1999). Most motile cilia and flagella contain an axoneme that consists of nine outer doublet microtubules surrounding two central singlet microtubules. The outer doublets contain binding sites for the inner and outer dynein arms, the molecular motors that power axoneme motility (reviewed by Porter, 1996;King, 2000). The dynein arms on one outer doublet interact transiently with the adjacent doublet to generate the force for interdoublet microtubule sliding. Other structures within the axoneme constrain and coordinate the activity of the multiple dynein motors and thereby convert microtubule sliding into flagellar bending.Both structural and genetic evidence indicate that the central pair microtubules and radial spokes play an important role in coordinating dynein activity. The two central pair microtubules are structurally asymmetric, and in several organisms, this apparatus has been shown to undergo clockwise rotation at a rate of approximately one turn per beat (reviewed by Omoto et al., 1999). Thes...

Regulation of flagellar dynein activity by a central pair kinesin

“…5 Inset), and are associated with an almost total lack of motility (10,29). Flagella of pf6 -1 cells occasionally beat with frequencies as high as 5 Hz.…”

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

“…For example, several subunits of dynein motors are EF-hand proteins that may bind calcium directly, altering dynein activity [Piperno et al, 1992;King and Patel-King, 1995;Yanagisawa and Kamiya, 2001;Casey et al, 2003;Guerra et al, 2003]. 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 e...…”

Haploinsufficiency for the murine orthologue of Chlamydomonas PF20 disrupts spermatogenesis

“…However, studies of Chlamydomonas rheinhardtii revealed important roles for the central apparatus. Mutant Chlamydomonas strains lacking functional PF16, PF6, and PF20 have paralyzed flagella, and the C1 microtubule, the projections connecting the C1 microtubule and the entire central pair, respectively, are missing in isolated axonemes (11)(12)(13).To understand the function of the central apparatus in mammals, we cloned the human and mouse orthologues of Chlamydomonas, PF16 (SPAG6) (14, 15) and PF20 (16). SPAG6-deficient mice are hydrocephalic, and males surviving to maturity are infertile as a result of a marked sperm motility defect associated with disorganization of flagellar structures, including loss of the central pair microtubules and disorganization of the outer dense fibers and fibrous sheath (6).…”

“…However, studies of Chlamydomonas rheinhardtii revealed important roles for the central apparatus. Mutant Chlamydomonas strains lacking functional PF16, PF6, and PF20 have paralyzed flagella, and the C1 microtubule, the projections connecting the C1 microtubule and the entire central pair, respectively, are missing in isolated axonemes (11)(12)(13).…”