Protein phosphorylation is a key event of flagellar disassembly revealed by analysis of flagellar phosphoproteins during flagellar shortening in Chlamydomonas

Cilia are disassembled prior to cell division, which is proposed to regulate proper cell cycle progression. The signaling pathways that regulate cilia disassembly are not well-understood. Recent biochemical and genetic data demonstrate that protein phosphorylation plays important roles in cilia disassembly. Here, we analyzed the phosphoproteins in the membrane/matrix fraction of flagella undergoing shortening as well as flagella from steady state cells of Chlamydomonas. The phosphopeptides were enriched by a combination of IMAC and titanium dioxide chromatography with a strategy of sequential elution from IMAC (SIMAC) and analyzed by tandem mass spectrometry. A total of 224 phosphoproteins derived from 1296 spectral counts of phosphopeptides were identified. Among the identified phosphoproteins are flagellar motility proteins such as outer dynein arm, intraflagellar transport proteins as well as signaling molecules including protein kinases, phosphatases, G proteins, and ion channels. Eighty-nine of these phosphoproteins were only detected in shortening flagella, whereas 29 were solely in flagella of steady growing cells, indicating dramatic changes of protein phosphorylation during flagellar shortening. Our data indicates that protein phosphorylation is a key event in flagellar disassembly, and paves the way for further study of flagellar assembly and disassembly controlled by protein phosphorylation.     

Flagellar waveform and rotational orientation in a Chlamydomonas mutant lacking normal striated fibers

The Chlamydomonas mutant vfl-3 lacks normal striated fibers and microtubular rootlets. Although the flagella beat vigorously, the cells rarely display effective forward swimming. High speed cinephotomicrography reveals that flagellar waveform, frequency, and beat synchrony are similar to those of wild-type cells, indicating that neither striated fibers nor microtubular rootlets are required for initiation or synchronization of flagellar motion. However, in contrast to wild type, the effective strokes of the flagella of vfl-3 may occur in virtually any direction. Although the direction of beat varies between cells, it was not observed to vary for a given flagellum during periods of filming lasting up to several thousand beat cycles, indicating that the flagella are not free to rotate in the mature cell. Structural polarity markers in the proximal portion of each flagellum show that the flagella of the mutant have an altered rotational orientation consistent with their altered direction of beat. This implies that the variable direction of beat is not due to a defect in the intrinsic polarity of the axoneme, and that in wild-type cells the striated fibers and/or associated structures are important in establishing or maintaining the correct rotational orientation of the basal bodies to ensure that the inherent functional polarity of the flagellum results in effective cellular movement. As in wild type, the flagella of vfl-3 coordinately switch to a symmetrical, flagellar-type waveform during the shock response (induced by a sudden increase in illumination), indicating that the striated fibers are not directly involved in this process.     

A protein methylation pathway in Chlamydomonas flagella is active during flagellar resorption

During intraflagellar transport (IFT), the regulation of motor proteins, the loading and unloading of cargo and the turnover of flagellar proteins all occur at the flagellar tip. To begin an analysis of the protein composition of the flagellar tip, we used difference gel electrophoresis to compare long versus short (i.e., regenerating) flagella. The concentration of tip proteins should be higher relative to that of tubulin (which is constant per unit length of the flagellum) in short compared with long flagella. One protein we have identified is the cobalamin-independent form of methionine synthase (MetE). Antibodies to MetE label flagella in a punctate pattern reminiscent of IFT particle staining, and immunoblot analysis reveals that the amount of MetE in flagella is low in full-length flagella, increased in regenerating flagella, and highest in resorbing flagella. Four methylated proteins have been identified in resorbing flagella, using antibodies specific for asymmetrically dimethylated arginine residues. These proteins are found almost exclusively in the axonemal fraction, and the methylated forms of these proteins are essentially absent in full-length and regenerating flagella. Because most cells resorb cilia/flagella before cell division, these data indicate a link between flagellar protein methylation and progression through the cell cycle.     

Analysis of flagellar size control using a mutant of Chlamydomonas reinhardtii with a variable number of flagella

A mutant of Chlamydomonas reinhardtii with a variable number of flagella per cell has been used to investigate flagellar size control. The mutant and wild-type do not differ in cell size nor in flagellar length, yet the size of the intracellular pool of flagellar precursor protein can differ dramatically among individual mutant cells, with, for example, triflagellate cells having three times the pool of monoflagellate cells. Because cells of the same size, but with very different pool sizes, have flagella of identical length, it appears that the concentration of the unassembled flagellar precursor protein pool does not regulate flagellar length. The relation between cell size, pool size, and flagellar length has also been investigated for wild-type cells of different sizes and ploidies. Again, flagellar length appears to be maintained independent of pool size or concentration.     

Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii

Previously, we reported that flagellar excision in Chlamydomonas reinhardtii is mediated by an active process whereby microtubules are severed at select sites within the flagellar-basal body transition zone (Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751- 1760). At the time of flagellar excision, stellate fibers of the transition zone contract and displace the microtubule doublets of the axoneme inward. The resulting shear force and torsional load generated during inward displacement leads to microtubule severing immediately distal to the central cylinder of the transition zone. In this study, we have used a detergent-extracted cell model of Chlamydomonas that allows direct experimental access to the molecular machinery responsible for microtubule severing without the impediment of the plasma membrane. We present four independent lines of experimental evidence for the essential involvement of centrin-based stellate fibers of the transition zone in the process of flagellar excision: (a) Detergent-extracted cell models excise their flagella in response to elevated, yet physiological, levels of free calcium. (b) Extraction of cell models with buffers containing the divalent cation chelator EDTA leads to the disassembly of centrin-based fibers and to the disruption of transition zone stellate fiber structure. This treatment results in a complete loss of flagellar excision competence. (c) Three separate anti-centrin monoclonal antibody preparations, which localize to the stellate fibers of the transition zone, specifically inhibit contraction of the stellate fibers and block calcium-induced flagellar excision, while control antibodies have no inhibitory effect. Finally, (d) cells of the centrin mutant vfl-2 (Taillon, B., S. Adler, J. Suhan, and J. Jarvik. 1992. J. Cell Biol. 119:1613-1624) fail to actively excise their flagella following pH shock in living cells or calcium treatment of detergent-extracted cell models. Taken together, these observations demonstrate that centrin-based fiber contraction plays a fundamental role in microtubule severing at the time of flagellar excision in Chlamydomonas.     

Polarity of flagellar assembly in Chlamydomonas.

During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.     

Experimental dissection of flagellar surface motility in chlamydomonas

Experiments have explored the possible relationships between the flagellar surface motility of chlamydomonas, visualized as translocation of polystyrene beads by paralyzed (pf) mutants (Bloodgood, 1977, J. Cell Biol. 15:983-989), and the capacity of gametic flagella to participate in the mating reaction. While vegetative and gametic flagella bind beads with equal efficiencies and are capable of transporting them along entire flagellar lengths, beads on vegetative flagella are primarily associated with the proximal half of the flagella whereas those of gametic flagella exhibit no such preference. This difference may relate to the "tipping" response of gametes during sexual flagellar agglutination (Goodenough and Jurivich, 1978, J. Cell Biol. 79:680-693). Colchicine, vinblastine, chymotrypsin, cytochalasins B and D, and anti-β-tubulin antiserum are all able to inhibit the binding of beads to the flagellar suface. Trysin digestion and an antiserum directed against whole chlamydomonas flagella have no effect on the ability of flagella to bind beads, but the beads remain immobile. These results suggest that at least two flagellar activities participate in surface motility: (a) bead binding, which may involve a tubulin-like component at the flagellar surface; and (b) bead translocation, which may depend on a second component (e.g. an ATPase) of the flagellar surface. Surface motility is shown to be distinct from gametic adhesiveness per se, but it may participate in concentrating dispersed agglutinins, in driving them toward the flagellar tips, and/or in generating a signal-to-fuse from the flagellar tips to the cell body. Directly supporting these concepts is the observation that bound beads remain immobilized at the flagellar tips during the "tip-locking" stage of pf x pf matings, and the observation that bound ligands such as antibody fail to be tipped by trypsinized flagella.     

Polar flagellar biosynthesis and a regulator of flagellar number influence spatial parameters of cell division in Campylobacter jejuni

Spatial and numerical regulation of flagellar biosynthesis results in different flagellation patterns specific for each bacterial species. Campylobacter jejuni produces amphitrichous (bipolar) flagella to result in a single flagellum at both poles. These flagella confer swimming motility and a distinctive darting motility necessary for infection of humans to cause diarrheal disease and animals to promote commensalism. In addition to flagellation, symmetrical cell division is spatially regulated so that the divisome forms near the cellular midpoint. We have identified an unprecedented system for spatially regulating cell division in C. jejuni composed by FlhG, a regulator of flagellar number in polar flagellates, and components of amphitrichous flagella. Similar to its role in other polarly-flagellated bacteria, we found that FlhG regulates flagellar biosynthesis to limit poles of C. jejuni to one flagellum. Furthermore, we discovered that FlhG negatively influences the ability of FtsZ to initiate cell division. Through analysis of specific flagellar mutants, we discovered that components of the motor and switch complex of amphitrichous flagella are required with FlhG to specifically inhibit division at poles. Without FlhG or specific motor and switch complex proteins, cell division occurs more often at polar regions to form minicells. Our findings suggest a new understanding for the biological requirement of the amphitrichous flagellation pattern in bacteria that extend beyond motility, virulence, and colonization. We propose that amphitrichous bacteria such as Campylobacter species advantageously exploit placement of flagella at both poles to spatially regulate an FlhG-dependent mechanism to inhibit polar cell division, thereby encouraging symmetrical cell division to generate the greatest number of viable offspring. Furthermore, we found that other polarly-flagellated bacteria produce FlhG proteins that influence cell division, suggesting that FlhG and polar flagella may function together in a broad range of bacteria to spatially regulate division.     

Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas Flagella

A dramatic stimulation of synthesis of flagellar proteins occurs in Chlamydomonas following flagellar removal or experimentally induced resorption of the flagella into the cell. In this report we show that this stimulation involves an increase in the levels of mRNAs for tubulin and many other flagellar proteins. Total RNA and poly(A) RNA were isolated from cells after deflagellation or flagellar resorption, and were then translated in the reticulocyte lysate system. Two-dimensional gel analysis of the translation products demonstrates that the RNA-directed in vitro synthesis of α and β tubulins, and a number of other flagellar proteins, increases after deflagellation or flagellar resorption. Surprisingly, the α-tubulin synthesized in vitro does not co-migrate on two-dimensional gels with mature flagellar α-tubulin. Moreover, in vivo labeling experiments show that the major α-tubulin synthesized in the cell after deflagellation co-migrates with the major α-tubulin made in vitro, not with the major α-tubulin present in the flagella. These results suggest that flagellar α-tubulin is synthesized as a precursor, and undergoes post-translational modification before assembly into the flagella. In addition, we report that the synthesis of tubulin and other flagellar proteins can be specifically inhibited, as well as stimulated. Treatment of cells with IBMX, which induces flagellar resorption, causes a marked decrease in the levels of translatable mRNAs for tubulin and other flagellar proteins, without affecting levels of mRNAs for nonflagellar proteins.     

The influence of the herbicide trifluralin on flagellar regeneration in Chlamydomonas

The mode of action of trifluralin is known to include disruption of cell division in root meristems by causing an absence of spindle microtubules. It has also been shown that trifluralin binds to tubulin isolated and purified from Chlamydomonas flagella. In this paper the kinetics of in vivo flagellar regeneration was used as a model to determine the influence of trifluralin on tubulin assembly. Chlamydomonas cells were grown in synchronous culture using a 12 h light-dark cycle. At 3 h into the light cycle the cells were subjected to shear force to induce flagellar abortion. Flagellar regeneration, in the presence of varying concentrations of trifluralin, was observed by Nomarski interference microscopy. After 1.5 h, trifluralin concentrations below 0.1 μM had not affected the regeneration rate, while concentrations above 5 μM prevented the onset of regeneration. As the concentration between 0.1 and 5 μM was increased, the final length of all flagella decreased. Using combinations of cycloheximide and trifluralin it was determined that trifluralin did not influence tubulin synthesis, and removing trifluralin only restored 50% of the regeneration capacity present at the beginning of treatment. By comparing groups of cells where the tubulin pool was depleted or present, it was found that trifluralin prevented assembly rather than causing a breakdown of previously assembled flagella. The research reported here supports the theory that the mechanism of action of trifluralin is an interaction of trifluralin and tubulin in a way that prevents tubulin assembly into spindle microtubules.     

Analysis of force generation during flagellar assembly through optical trapping of free-swimming Chlamydomonas reinhardtii

Many studies have used velocity measurements, waveform analyses, and theoretical flagella models to investigate the establishment, maintenance, and function of flagella of the biflagellate green algae Chlamydomonas reinhardtii. We report the first direct measurement of Chlamydomonas flagellar swimming force. Using an optical trap ("optical tweezers") we detect a 75% decrease in swimming force between wild type (CC124) cells and mutants lacking outer flagellar dynein arms (oda1). This difference is consistent with previous estimates and validates the force measurement approach. To examine mechanisms underlying flagella organization and function, we deflagellated cells and examined force generation during flagellar regeneration. As expected, fully regenerated flagella are functionally equivalent to flagella of untreated wild type cells. However, analysis of swimming force vs. flagella length and the increase in force over regeneration time reveals intriguing patterns where increases in force do not always correspond with increases in length. These investigations of flagellar force, therefore, contribute to the understanding of Chlamydomonas motility, describe phenomena surrounding flagella regeneration, and demonstrate the advantages of the optical trapping technique in studies of cell motility.     

Ciliary contractile model applied to sperm flagellar motion

The contractile model developed previously to describe ciliary motions was applied to the case of invertebrate sperm flagella. The model could describe waveforms of these flagella as a function of the flagellar frequency and as a function of the external viscosity. Rapid spontaneous transients from rest to motion were also correctly described by the model. The calculations showed that the time course of the internal, active, moments during a cycle is only weakly displayed in the time course of the transverse motion of the flagella. This is apparently due to nonlinearities which suppress higher harmonics in the frequency spectrum of the active moments. The local curvature of the flagella was found to follow the time course of the internal, active moments directly. It is concluded that to derive the time course of the active contractile moments from the transverse motion, data on the flagellar motion with a position accuracy of 0·1 μm and with a time resolution of 1 msec are required. For curvature data an accuracy of 50 cm^?1 and a time resolution of 2·5 msec would suffice.     

Flagellar elongation and shortening in Chlamydomonas. IV. Effects of flagellar detachment, regeneration, and resorption on the induction of flagellar protein synthesis

Synthesis of new proteins is required to regenerate full length Chlamydomonas flagella after deflagellation. Using gametes, which have a low basal level of protein synthesis, it has been possible to label and detect the synthesis of many flagellar proteins in whole cells. The deflagellation-induced synthesis of the tubulins, dyneins, the flagellar membrane protein, and at least 20 other proteins which co- migrate with proteins in isolated axonemes, can be detected in gamete cytoplasm, and the times of initiation and termination of synthesis for each of the proteins can be studied. The nature of the signal that stimulates the cell to initiate flagellar protein synthesis is unknown. Flagellar regeneration and accompanying pool depletion are not necessary for either the onset or termination of flagellar protein synthesis, because colchicine, which blocks flagellar regeneration, does not change the pattern of proteins synthesized in the cytoplasm after deflagellation or the timing of their synthesis. Moreover, flagellar protein synthesis is stimulated after cells are chemically induced to resorb their flagella, indicating that the act of deflagellation itself is not necessary to stimulate synthesis. Methods were defined for inducing the cells to resorb their flagella by removing Ca++ from the medium and raising the concentration of K+ or Na+. The resorption was reversible and the flagellar components that were resorbed could be re-utilized to assemble flagella in the absence of protein synthesis. This new technique is used in this report to study the control of synthesis and assembly of flagella.     

Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas

Ciliary membranes have a large repertoire of receptors and ion channels that act to transduce information from the environment to the cell. Chlamydomonas offers a tractable system for dissecting the transport and function of ciliary and flagellar membrane proteins. Isolation of ergosterol and sphingolipid-enriched Chlamydomonas flagellar membrane domains identified potential signaling molecules by mass spectroscopy. These include a membrane protein and a matrix flavodoxin protein that are encoded by the AGG2 and AGG3 genes, respectively. Agg2p localizes to the proximal flagellar membrane near the basal bodies. Agg3p is distributed throughout the flagellar matrix, with an increased concentration in the proximal regions where Agg2p is located. Chlamydomonas cells sense light by using a microbial-type rhodopsin , transduce a signal from the cell body to the flagella, and alter the waveform of the flagella to turn a cell toward the light. Protein depletion by RNA interference reveals that both AGG gene products play roles in the orientation of cells to a directional light source. The depleted strains mimic the phenotype of the previously identified agg1 mutant, which swims away from light. We propose that the localization of Agg2p and Agg3p to the proximal region of the flagella may be important for interpreting light signals.     

Uncoupling of Chlamydomonas flagellar gene expression and outgrowth from flagellar excision by manipulation of Ca2+

Chlamydomonas cells respond to certain environmental stimuli by shedding their flagella. Flagellar loss induces a rapid, transient increase in expression of a specific set of genes encoding flagellar proteins, and assembly of a new flagellar pair. While flagellar gene expression and initiation of flagellar outgrowth are normally tightly coupled to flagellar excision, our results demonstrate that these processes can be uncoupled by manipulating Ca2+ levels or calmodulin activity. In our experiments, wild-type cells were stimulated to excise their flagella using mechanical shearing, and at times after deflagellation, flagellar lengths were measured and flagellar mRNA abundance changes were determined by S1 nuclease protection analysis. When extracellular Ca2+ was lowered by addition of EGTA to cultures before excision, flagellar mRNA abundance changes and flagellar outgrowth were temporally uncoupled from flagellar excision. When extracellular Ca2+ was lowered immediately after excision or when calmodulin activity was inhibited with W-7, flagellar outgrowth was uncoupled from flagellar excision and flagellar mRNA abundance changes. Whenever events in the process of flagellar regeneration were temporally uncoupled, the magnitude of the flagellar mRNA abundance change was reduced. These results suggest that flagellar gene expression may be regulated by multiple signals generated from these events, and implicate Ca2+ as a factor in the mechanisms controlling flagellar regeneration.     

Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a "high-load environment," we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters.     

Atypical flagellar structure associated with endocrine cells of rat pituitary

Atypical flagellar structures containing eight subfibers arrayed around a single, central element (8 + 1) are associated with pituitary secretory cells, largely gonadotropes. Less frequently, flagella with a 7 + 2 pattern are seen. Both types appear commonly in pituitary tissues obtained from both male and female rats above 1 year of age and appear to penetrate the cells. Except for the pattern of the array, the structures are similar in dimension to commonly observed flagellar structures (9 + 2) when viewed in sections cut either in perpendicular or sagittal to the major axis. The (8 + 1) flagella are observed both singly and in pairs. Steroidal milieu (ovariectomy with or without steroid replacement) does not seem to influence their appearance. Flagella with the common 9 + 2 arrangement are not observed in the rat pituitary.     

Formation of flagella during interphase in secondary spermatocytes from Xenopus laevis in vitro

In cell culture, single motile flagella, 1 micron in length, were observed to grow from secondary spermatocytes of Xenopus laevis within 2-3 hours after telophase I, at 22 degrees C. About 90% of the secondary spermatocytes formed flagella as observed by phase-contrast microscopy. The flagella grew up to 2-6 microns in length during interphase II, which lasted about 18 hours. The presence of the "9 + 2" microtubular structure of the flagellar axonemes of secondary spermatocytes was confirmed by electron microscopy. When chromosomal condensation began (prophase II), the flagella were resorbed into the cells and, after the second meiotic division, a flagellum was formed again by each of the round spermatids. Thus, there appears to be a close relationship between the meiotic division cycle and the formation of flagella. The possible contribution of Sertoli cells to the formation of flagella in secondary spermatocytes was examined by reducing the number of Sertoli cells to less than ten per culture. Under these conditions, flagella formed in secondary spermatocytes with very high efficiency. It is very likely that secondary spermatocytes form flagella in vivo, since the secondary spermatocytes were observed to have flagella immediately after dissociation of the testes.     

A complete set of flagellar genes acquired by horizontal transfer coexists with the endogenous flagellar system in Rhodobacter sphaeroides

Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure. Rhodobacter sphaeroides has two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system of R. sphaeroides can be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system of R. sphaeroides produces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a gamma-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other alpha-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster.