Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum

Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP.     

Inactivation of Ca2+-induced ciliary reversal by high-salt extraction in the cilia of Paramecium

Intracellular Ca²⁺ induces ciliary reversal and backward swimming in Paramecium. However, it is not known how the Ca²⁺ signal controls the motor machinery to induce ciliary reversal. We found that demembranated cilia on the ciliated cortical sheets from Paramecium caudatum lost the ability to undergo ciliary reversal after brief extraction with a solution containing 0.5 M KCl. KNO₃, which is similar to KCl with respect to chaotropic effect; it had the same effect as that of KCl on ciliary response. Cyclic AMP antagonizes Ca²⁺-induced ciliary reversal. Limited trypsin digestion prevents endogenous A-kinase and cAMP-dependent phosphorylation of an outer arm dynein light chain and induces ciliary reversal. However, the trypsin digestion prior to the high-salt extraction did not affect the inhibition of Ca²⁺-induced ciliary reversal caused by the high-salt extraction. Furthermore, during the course of the high-salt extraction, some axonemal proteins were extracted from ciliary axonemes, suggesting that they may be responsible for Ca²⁺-induced ciliary reversal.     

In vitro phosphorylation of Paramecium axonemes and permeabilized cells

This study seeks to identify phosphoproteins in axonemes from Paramecium tetraurelia whose phosphorylation responses to adenosine 3', 5'-cyclic monophosphate (cAMP) and Ca2+ parallel responses induced by these agents in ciliary behavior in this cell. In purified axonemes, over 15 bands ranging from Mr greater than 300 kDa to 19 kDa on SDS-PAGE incorporate 32P from adenosine 5'-gamma-[32P]triphosphate (gamma-32P-ATP) at pCa 7 in the absence of cAMP. A major band whose label turns over rapidly was identified at Mr 43 kDa. In the presence of 5 microM cAMP, more than eight bands, but not the Mr 43 kDa band, were labeled additionally or enhanced their labeling. These phosphoproteins and their kinases are structural components of the axoneme. Overall, some of the same major bands are labeled in the presence of cAMP in Triton X-100-permeabilized paramecia that retain their behavioral responses and in axonemes mechanically isolated from these cells. In particular, two major bands have been identified whose phosphorylation is greatly enhanced by cAMP at low concentrations: 1) a 29 kDa polypeptide whose cAMP-dependent phosphorylation is diminished at pCa 4 compared with pCa 7 and 2) a 65 kDa polypeptide whose phosphorylation is pCa insensitive. These polypeptides meet minimal criteria for signal-sensitive regulators of motility parameters in the Paramecium axoneme.     

Control of the ciliary beat by cyclic nucleotides in intact cortical sheets from Paramecium

The locomotor behavior of Paramecium depends on the ciliary beat direction and beat frequency. Changes in the ciliary beat are controlled by a signal transduction mechanism that follows changes in the membrane potential. These events take place in cilia covered with a ciliary membrane. To determine the effects of second messengers in the cilia, cortical sheets were used with intact ciliary membrane as a half-closed system in which each cilium is covered with a ciliary membrane with an opening to the cell body. Cyclic nucleotides and their derivatives applied from an opening to the cell body affected the ciliary beat. cAMP and 8-Br-cAMP increased the beat frequency and the efficiency of propulsion and acted antagonistically to the action of Ca(2+). cGMP and 8-Br-cGMP increased the efficiency of propulsion accompanying clear metachronal waves but decreased the beat frequency. These results indicate that the cyclic nucleotides affect target proteins in the ciliary axonemes surrounded by the ciliary membrane without a membrane potential and increase the efficiency of propulsion of the ciliary beat. In vitro phosphorylation of isolated ciliary axonemes in the presence of cyclic nucleotides and their derivatives revealed that the action of cAMP was correlated with the phosphorylation of 29-kDa and 65-kDa proteins and that the action of cGMP was correlated with the phosphorylation of a 42-kDa protein.     

Stimulation of in vitro motility of Chlamydomonas axonemes by inhibition of cAMP-dependent phosphorylation

When demembranated axonemes of Chlamydomonas were reactivated with Mg-ATP, the proportion of motile axonemes was significantly increased by the presence of either phosphodiesterase (PDE) or protein inhibitor of cAMP-dependent kinase (PKI). The effect of PDE was cancelled by the addition of cAMP. These findings strongly suggest that the axoneme samples have endogenous cAMP, which can reduce the proportion of motile axonemes via phosphorylation. This inhibitory effect of cAMP on Chlamydomonas axonemes is opposite to its stimulatory effect on the axonemal motility in other organisms so far reported. PKI or PDE activated the motility either in the absence of Ca2+, when the axonemes beat with an asymmetric waveform, or in 10(-5) M Ca2+, when the axonemes beat with a symmetric waveform. This cAMP-dependent regulation of motility was observed with the axonemes from which detergent-soluble material had been removed, indicating that the proteins responsible for the regulation still remained in the axonemes. Preliminary in vitro phosphorylation studies have implicated two polypeptides as candidates for the target protein of cAMP-dependent protein kinase: one with a molecular weight of 270 kD and the other with a much larger molecular weight.     

Regulation of axonemal Mg2+-ATPase from Paramecium cilia: effects of Ca2+ and cyclic nucleotides

Ciliary activity is regulated by Ca2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca2+ and cyclic nucleotides to alter ciliary Mg2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca2+, but this Ca2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca2+-sensitive component of ciliary Mg2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insensitive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca2+, but it did sediment in the region from 19 S to 14 S. Mg2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleotide-stimulated manner.     

A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.     

Phosphorylation of endogenous proteins of cilia from Paramecium tetraurelia in vitro

Several endogenous substrate proteins of cilia from axenically grown Paramecium tetraurelia were phosphorylated in vitro by inherent protein kinases (PKs). Labeling was stimulated by cAMP and to a lesser extent by cGMP. ATP breakdown was most rapid in cilia and subciliary fractions. Using multiple substrate additions during incubations it was shown that phosphorylation was almost completed within 30 s. Very little dephosphorylation by phosphoprotein phosphatases occurred during 5 min of incubation. Proteins of molecular weight of 103 000 and 46 000 were shown to be particularly associated with axonemal structures of the cilia. No distinct differences in phosphorylation patterns were apparent in ciliary membrane vesicles of low and high buoyant density, which exhibit differential enzyme patterns. cAMP receptor proteins were identified by use of the photoaffinity label 8-azido-[32P]cAMP. Receptor proteins with apparent molecular weights of 43 000, 39 000, 37 000, 31 000 and 30 000 were probably related to the regulatory subunits of cAMP-dependent protein kinases as evidenced by inhibition of incorporation of the photoaffinity label by low concentrations of cAMP. Tagging of a protein of 85 000 molecular weight was specifically inhibited by cGMP, thus in all likelihood it corresponded to a cGMP-dependent protein kinase. Corresponding autophosphorylated protein bands were observed with gamma-[32P]ATP. A functional role for protein phosphorylation in cilia of Paramecium remains to be established.     

The control of ciliary beat frequency

Ciliary movement is powered by axonemal dynein. This article considers how a signal transduction cascade initiated at the cell membrane may activate outer dynein arms to change the velocity of microtubule sliding and the swimming speed of ciliated cells. For Paramecium, a critical event in the cascade is the cAMP-dependent phosphorylation of a 29 kDa polypeptide that is associated with the outer dynein arm.     

Augmented ciliary reorientation response and cAMP-dependent protein phosphorylation induced by glycerol in triton-extracted Paramecium

In the presence of 30% glycerol, the cilia of a permeabilized cell model from Paramecium exhibit dynamic orientation changes while displaying only a restricted cyclic beating with a very small amplitude. The direction of cilia under these conditions corresponds to the direction of the effective power stroke of cilia beating in the absence of glycerol, i.e., pointing posteriorly in the absence of Ca2+ and anteriorly at > 10(-6) M Ca2+. Ciliary reorientation toward the posterior in response to the removal of Ca2+ is particularly conspicuous; all the cilia become predominantly pointing to the posterior end all through their beating phases. Previous studies suggested that the effect of glycerol is caused through modification of cAMP-dependent protein phosphorylation. To determine whether glycerol in fact affects ciliary reorientation through changes in protein phosphorylation, here we examined protein phosphorylation in the axonemes. Glycerol stimulated cAMP-induced phosphorylation of 29-kDa and 65-kDa proteins. The stimulation of phosphorylation was found to be partly due to the inhibition of endogenous phosphodiesterase (PDE), and partly due to the inhibition of the dephosphorylation of the 29-kDa and 65-kDa phosphoproteins within the axoneme. Thus glycerol appears to cause predominant posterior orientation of cilia by stimulating cAMP-dependent phosphorylation on those proteins. In addition, glycerol appears to inhibit ciliary beating through inhibition of dynein ATPase.     

Outer dynein arm light chain 1 is essential for controlling the ciliary response to cyclic AMP in Paramecium tetraurelia

The individual role of the outer dynein arm light chains in the molecular mechanisms of ciliary movements in response to second messengers, such as Ca(2+) and cyclic nucleotides, is unclear. We examined the role of the gene termed the outer dynein arm light chain 1 (LC1) gene of Paramecium tetraurelia (ODAL1), a homologue of the outer dynein arm LC1 gene of Chlamydomonas reinhardtii, in ciliary movements by RNA interference (RNAi) using a feeding method. The ODAL1-silenced (ODAL1-RNAi) cells swam slowly, and their swimming velocity did not increase in response to membrane-hyperpolarizing stimuli. Ciliary movements on the cortical sheets of ODAL1-RNAi cells revealed that the ciliary beat frequency was significantly lower than that of control cells in the presence of ≥ 1 mM Mg(2+)-ATP. In addition, the ciliary orientation of ODAL1-RNAi cells did not change in response to cyclic AMP (cAMP). A 29-kDa protein phosphorylated in a cAMP-dependent manner in the control cells disappeared in the axoneme of ODAL1-RNAi cells. These results indicate that ODAL1 is essential for controlling the ciliary response by cAMP-dependent phosphorylation.     

Cyclic adenosine 3',5' monophosphate, calcium and protein phosphorylation in flagellar motility

cAMP and calcium are two important regulators of sperm flagellar motility. cAMP stimulates sperm motility by activating cAMP-dependent protein kinase and catalyzing the phosphorylation of sperm proteins. The stimulation of sperm motility by cAMP appears to be at two different levels. Evidence has been presented to suggest that cAMP-dependent phosphorylations may be required in order for motility to be initiated. In addition, cAMP-dependent phosphorylation appears to modulate specific parameters of motility resulting in higher beat frequency or greater wave amplitude. Calcium, on the other hand, when elevated intracellularly to 10(-6) M or higher, inhibits flagellar motility. The calcium-binding protein, calmodulin, appears to mediate a large number of effects of calcium on motility. Evidence suggests that calcium-calmodulin may be involved at the level of the membrane to pump calcium out of the flagellum. In addition, calcium-calmodulin may be involved in the control of axonemal function by regulating dynein ATPase and myosin light chain kinase activities. The identification of cAMP-dependent protein kinase, calmodulin and myosin light chain kinase in the sperm head suggests that cAMP and calcium-dependent phosphorylations are also involved in the control of the fertilization process, i.e., the acrosome reaction, in a manner similar to that known for the control of stimulus/secretion coupling. Finally, the effects of cAMP on flagellar motility are mediated by protein phosphorylation while the effects of calcium on motility are also in part, mediated by effects on protein phosphorylation.     

Regulation of 22S dynein by a 29-kD light chain

Previously, a 29-kD axonemal polypeptide (p29) that copurifies with 22S dynein has been shown to be phosphorylated in a cAMP- and Ca(2+)- sensitive manner, consistent with a role for this molecule in the signal transduction cascade leading to fast forward swimming in Paramecium tetraurelia (Hamasaki, T., K. Barkalow, J. Richmond, and P. Satir. 1991. Proc. Natl. Acad. Sci. USA. 88:7912-7922). This study demonstrates the nature of the relationship between p29 and 22S dynein. Chaotropic agents can be used to separate p29 fractions from 22S dynein. When extracted p29 is exchanged into physiological buffers, it regains the ability to recombine with 22S dynein with an apparent dissociation constant of 25 nM; no recombination is seen with 14S dynein or with unrelated control proteins. p29 from Paramecium will also recombine with Tetrahymena 22 but not 14S dynein. After chymotryptic digestion of 22S dynein, p29 preferentially binds to a single-headed fragment, homologous to the alpha H chain of Tetrahymena 22S dynein. 22S dynein treated in vitro by Paramecium protein kinase A in the presence of cAMP and ATP to phosphorylate p29 translocates bovine brain microtubules significantly (1.53x; p < 0.001) faster than before phosphorylation. Similarly, 22S dynein reconstituted in vitro with thiophosphorylated p29 translocates microtubules significantly (1.31x; p < 0.001) faster than controls reconstituted with nonthiophosphorylated p29. p29 is the only moiety thiophosphorylated in the reconstituted dynein. We conclude that p29 functions as a 22S dynein regulatory light chain in that it alone is sufficient to control the rate of microtubule translocation by changes in its phosphorylation state.     

Target molecules of calmodulin on microtubules of Tetrahymena cilia

In the course of an attempt to isolate the calmodulin-binding proteins (CaMBPs) from cilia of Tetrahymena, it was found that some CaMBPs tend to interact with axonemal microtubules. The present study demonstrates this interaction by cosedimentation experiments using in vitro polymerized Tetrahymena axonemal microtubules and Tetrahymena CaMBPs purified from axonemes by calmodulin affinity column chromatography. Analysis by the [125I]calmodulin overlay method showed that at least three CaMBPs (Mr69, 45, and 37 kDa) cosediment with microtubules. Furthermore, without any addition of exogenous CaMBPs, microtubules purified after three cycles of temperature-dependent polymerization and depolymerization included the above CaMBPs and additional CaMBPs (Mr30, 26, and 22 kDa) which could not cosediment with microtubules. From the results, we have classified these microtubule-associated CaMBPs into two groups: (i) CaMBPs which interact with microtubules only during polymerization (30, 26, and 22 kDa), and (ii) CaMBPs which interact not only with microtubules during polymerization, but also with polymerized microtubules (69, 45, and 37 kDa). These results suggest that the microtubule-associated CaMBPs, especially those of the latter group, are located on the surface of ciliary microtubules, and may become the target molecules of calmodulin at Ca2+-triggered ciliary reversal.     

Molecular chaperones in cilia and flagella: implications for protein turnover

The mechanisms of protein incorporation and turnover in 9+2 ciliary axonemes are not known. Previous reports of an HSP70-related protein, first in Chlamydomonas flagella and then in sea urchin embryonic cilia, suggested a potential role in protein transport or incorporation. The present study further explores this and other chaperones in axonemes from a representative range of organisms. Two-dimensional gel electrophoresis proved identity between the sea urchin ciliary 78 kDa HSP and a constitutive cytoplasmic HSP70 cognate (pI = 5.71). When isolated flagella from mature sea urchin sperm were analyzed, the same total amount and distribution of 78 kDa protein as in cilia were found. Antigens of similar size were detected in ctenophore comb plate, molluscan gill, and rabbit tracheal cilia. Absent from sea urchin sperm flagella, TCP-1alpha was detected in sea urchin embryonic and rabbit tracheal cilia; the latter also contained HSP90, detected by two distinct antibodies. Tracheal cilia were shown to undergo axonemal protein turnover while tracheal cells mainly synthesized ciliary proteins. TCP-1alpha progressively appeared in regenerating embryonic cilia only as their growth slowed, suggesting a regulatory role in incorporation or turnover. These results demonstrate that chaperones are widely distributed ciliary and flagellar components, potentially related to axonemal protein dynamics.     

Characterization of an A-kinase anchoring protein in human ciliary axonemes

Although protein kinase A (PKA) activation is known to increase ciliary beat frequency in humans the molecular mechanisms involved are unknown. We demonstrate that PKA is associated with ciliary axonemes where it specifically phosphorylates a 23-kDa protein. Because PKA is often localized to subcellular compartments in proximity to its substrate(s) via interactions with A-kinase-anchoring proteins (AKAPs), we investigated whether an AKAP was also associated with ciliary axonemes. This study has identified a novel 28 kDa AKAP (AKAP28)that is highly enriched in airway axonemes. The mRNA for AKAP28 is up-regulated as primary airway cells differentiate and is specifically expressed in tissues containing cilia and/or flagella. Additionally, both Western blot and immunostaining data show that AKAP28 is enriched in airway cilia. These data demonstrate that we have identified the first human axonemal AKAP, a protein that likely plays a role in the signaling necessary for efficient modulation of ciliary beat frequency.     

Cyclic adenosine monophosphate-dependent phosphorylation of mammalian mitochondrial proteins: enzyme and substrate characterization and functional role.

A study is presented on cyclic adenosine monophosphate- (cAMP-) dependent phosphorylation of mammalian mitochondrial proteins. Immunodetection with specific antibodies reveals the presence of the catalytic and the regulatory subunits of cAMP-dependent protein kinase (PKA) in the inner membrane and matrix of bovine heart mitochondria. The mitochondrial cAMP-dependent protein kinase phosphorylates mitochondrial proteins of 29, 18, and 6.5 kDa. With added histone as substrate, PKA exhibits affinities for ATP and cAMP and pH optimum comparable to those of the cytosolic PKA. Among the mitochondrial proteins phosphorylated by PKA, one is the nuclear-encoded (NDUFS4 gene) 18 kDa subunit of complex I, which has phosphorylation consensus sites in the C terminus and in the presequence. cAMP promotes phosphorylation of the 18 kDa subunit of complex I in myoblasts in culture and in their isolated mitoplast fraction. In both cases cAMP-dependent phosphorylation of the 18 kDa subunit of complex I is accompanied by enhancement of the activity of the complex. These results, and the finding of mutations in the NDUFS4 gene in patients with complex I deficiency, provide evidence showing that cAMP-dependent phosphorylation of the 18 kDa subunit of complex I plays a major role in the control of the mitochondrial respiratory activity.     

Regulation of protein phosphorylation and motility of sperm by cyclic adenosine monophosphate and calcium

Motility and protein phosphorylation have been measured under identical experimental conditions in ejaculated dog sperm lysed with low concentrations of Triton X-100 and reactivated with [gamma-32P]ATP. Cyclic AMP stimulates motility and protein phosphorylation while calcium inhibits motility and the overall incorporation of phosphate into endogenous proteins. Analysis of 32P-labeled sperm proteins on 1- and 2-dimensional polyacrylamide gels demonstrates that an enhanced phosphorylation of a defined number of specific proteins is associated with cAMP-stimulated motility. A major axonemal proteins, namely tubulin, has been tentatively identified as a phosphoprotein subject to regulation by cAMP. The phosphorylation of tubulin is almost completely dependent upon cAMP and is not affected by microM calcium. On the other hand, the cAMP-dependent stimulated phosphorylation of the other sperm proteins still occurs, but in most instances at a reduced rate in the presence of calcium. Two high molecular weight (Mr) phosphoproteins (350,000 and 260,000 daltons) whose phosphorylation states are modified by cAMP and calcium also were identified. It is suggested that 1 or both these proteins may be high Mr subunits of dynein. The phosphorylation of 1 of these proteins is stimulated by cAMP, but not affected by calcium; the other is stimulated by cAMP and inhibited by calcium. Three major cAMP-independent phosphoproteins of Mr 98,000, 43,000 and 26,000 have been identified. The phosphorylation of the 98,000 Mr protein is markedly reduced by micromolar calcium and not restored by cAMP. Using anticalmodulin drugs to inhibit motility, we suggest that the inhibitory effects of calcium on flagellar motility may be mediated in part by calmodulin. We conclude that the regulation of flagellar motility in cAMP and calcium includes mechanisms involving the control of the phosphorylation state of sperm proteins, some of which may be axonemal components.     

Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and beta-tubulin and affect the ciliary matrix in Tetrahymena

Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.     

Rapid tubulin synthesis during ciliated cell differentiation

Using pulse-chase conditions in culture we have investigated the incorporation of 3H-leucine into tubulin of isolated oviducts from 5 day-old mice. Label appears in soluble, particulate and axonemal fractions minutes after incubation. In the latter two fractions, but not in the soluble fraction, this label is rapidly diluted under chase conditions. The data do not fit a simple model of sequential transfer of radioactively labeled, newly synthesized tubulin from a soluble fraction through centriole precursors to assembled ciliary axonemes.