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PubMed1971

Purification and thermal stability of intact Bacillus subtilis flagella.

Flagella were prepared and purified in a relatively intact form from bacterial lysates. Immunochemical tests showed that over 95% of the protein in the final preparation consisted of flagellar antigen. These flagella are more stable to thermal denaturation than flagella filaments obtained by shearing. Their thermal properties more closely resemble those of flagella in the native state on bacteria. The presence of the hook structure is responsible for this extra stability.

Antigens↗

PubMed1996

A novel type of mechanoreception by the flagella of Chlamydomonas

A novel type of mechanosensory mechanism is found in Chlamydomonas reinhardtii. When a cell is captured with a suction pipette and a negative pressure is applied, the cell produces repetitive Ca2+ impulses at a frequency of 0.5-1.0 Hz. The impulse frequency increases with the applied pressure. The impulses are produced when the flagella are sucked into the pipette but not when the cell body is sucked in leaving the flagella outside the pipette. Cells with short flagella produce impulses of small amplitude. Thus, the site where the cell senses mechanical stimuli and generates the impulse current must be localized at the flagella. The amplitude, shape and ion selectivity of the pressure-induced impulses are distinct from the all-or-none flagellar current that is evoked by photostimulation. The impulses are possibly produced by a combination of currents passing through mechanosensitive channels and Ca2+ channels. This response probably functions to modulate flagellar beating and thereby to regulate the behaviour of the cell.

Journal Article↗

PubMed1989

Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella.

A calcium and calmodulin-regulated cyclic nucleotide phosphodiesterase has been shown to be an integral component of both rat and bovine sperm flagella. The calcium-activated enzyme was inhibited by both trifluoperazine (ID50 = 10 microM) and [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid (EGTA), and the basal activity measured in the presence of EGTA was stimulated by limited proteolysis to that observed in the presence of calcium/calmodulin. 125I-Calmodulin binding to purified rat sperm flagella has been characterized and the flagellar-associated calmodulin-binding proteins identified by a combination of gel and nitrocellulose overlay procedures and by chemical cross-linking experiments using dimethyl suberimidate. 125I-Calmodulin bound to demembranated rat sperm flagella in a time- and concentration-dependent manner. At equilibrium, 30-40% of the bound 125I-calmodulin remains associated with the flagella after treatment with EGTA or trifluoperazine. The majority of the bound 125I-calmodulin, both the Ca2+-dependent and -independent, was displaced by excess calmodulin. A 67-kDa calmodulin-binding protein was identified by both the gel and nitrocellulose overlay procedures. In both cases, binding was dependent on Ca2+ and was totally inhibited by trifluoperazine, EGTA, and excess calmodulin. On nitrocellulose overlays, the concentration of calmodulin required to decrease binding of 125I-calmodulin by 50% was between 10(-10) and 10(-11) M. Limited proteolysis resulted in the total loss of all Ca2+-dependent binding to the 67-kDa polypeptide. Chemical cross-linking experiments identified a major calcium-dependent 125I-calmodulin:polypeptide complex in the 84-90-kDa molecular mass range and a minor complex of approximately 200 kDa. Immunoblot analysis showed that the major 67-kDa calmodulin-binding protein did not cross-react with polyclonal antibodies raised against either the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase or phosphoprotein phosphatase (calcineurin) from bovine brain.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1997

Induction of temporary beating in paralyzed flagella of Chlamydomonas mutants by application of external force.

To help understand the mechanism by which the sliding movement of outer-doublet microtubules in cilia and flagella is converted into bending waves, we examined the effect of mechanical force imposed on the flagella of Chlamydomonas mutants lacking the central pair or multiple dyneins. These mutants were almost completely nonmotile under normal conditions. A bend was produced in a flagellum either by holding a cell with a micropipette and quickly moving it with a piezoelectric actuator; or by pushing a flagellum with a microneedle. After removal of the external force, mutants lacking the central pair (pf18 and pf19) displayed beating at irregular intervals of > 1 second for one to several cycles. Similarly, a double mutant (ida2ida4) lacking four species of inner-arm dynein displayed beating at intervals of > 0.1 second for up to 80 cycles. However, paralyzed flagella of double mutants that lack the outer dynein arm in addition to the central pair or the inner dynein arm did not show cyclical movements upon application of external force. These results indicate that the central pair and the inner dynein arm are important for both stable bend formation at the base and efficient bend propagation along the flagellar length. They also suggest that the outer dynein arm, and not the inner dynein arm, enables the flagellar axoneme to propagate bends independently of the central pair. We propose that the axoneme is equipped with two independent motor systems for oscillatory movements: an outer-arm system controlled by the axonemal mechanical state independently of the central pair/radial spoke system, and an inner-arm system controlled by both the axonemal mechanical state and the central pair/radial spokes.

Animals↗

PubMed1997

Reactivation at low ATP distinguishes among classes of paralyzed flagella mutants.

Paralyzed flagella (pf) mutants of Chlamydomonas have been distinguished by the inability of the intact cells to move. Demembranated flagella from these mutants are also immotile when reactivated under standard conditions, with millimolar ATP concentrations. Three of these pf mutants were previously found to be motile when reactivated under 3 alternate reactivation conditions: low ATP concentration (< or =50 microM); 0.1 mM ATP combined with >0.5 mM ADP; or 0.1 mM ATP combined with non-reactivating ATP analogs anthraniloyl ATP or methylanthraniloyl ATP. We have now surveyed all pf mutants in the Chlamydomonas Culture Collection and discovered that a great majority of these mutants can move under these alternate nucleotide conditions. Only pf22 and pf23, mutants missing multiple subsets of dynein arms, did not reactivate under those conditions. This suggests that the paralysis observed in most pf mutants is the result of inhibition by physiological ATP. Except for pf12, which has an abnormally symmetric bending pattern, all other pf mutants exhibit asymmetric bending patterns similar to wild-type. Previously, motility that was restored by the presence of suppressor mutations was found to lack the normal asymmetry of wild-type flagella or the suppressor by itself. The waveform of pf mutants at alternate reactivation conditions in the absence of suppressor shows that pf mutants with radial-spoke or central-pair defects are capable of asymmetric bending similar to wild-type. A complete radial-spoke/central-pair complex is not essential for the production of asymmetric bending patterns. Furthermore, this suggests that the symmetric waveform observed previously in suppressed pf mutants is due to the interaction between the pf and suppressor mutations.

Adenosine Triphosphate↗

PubMed1999

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.

Animals↗

PubMed1996

SksC, a fertilization-related protein kinase in Chlamydomonas, is expressed throughout the cell cycle and gametogenesis, and a phosphorylated form is present in both flagella and cell bodies.

Fertilization in the biflagellated eukaryote, Chlamydomonas, is initiated by flagellar adhesion between gametes of opposite mating types. An early event in the signal transduction pathway induced by these cell-cell interactions is the rapid inactivation of a flagellar protein kinase that phosphorylates a 48 kDa flagellar protein. Molecular cloning and characterization indicated that the 48 kDa substrate, termed SksC, itself is a novel protein kinase. Here, we have determined that its transcript levels were unchanged during prolonged flagellar adhesion. Moreover, resynthesis of new flagellar proteins following deflagellation was not accompanied by increases in transcript levels of SksC, suggesting that expression of this soluble protein kinase might not be restricted to flagella. Immunoblot analysis indicated that expression of SksC was ubiquitous: this soluble protein was found in both flagella and cell bodies and was expressed throughout the cell cycle and gametogenesis. Immunoprecipitation experiments indicated that SksC was phosphorylated in both flagella and cell bodies. Thus, in addition to its potential role in fertilization, this novel protein kinase may play a role in other signaling events in Chlamydomonas.

Animals↗

PubMed1986

The association of distinct acid phosphatases with the flagella pocket and surface membrane fractions obtained from bloodstream forms of Trypanosoma rhodesiense.

Cell fractionation of bloodstream Trypanosoma rhodesiense, using isopycnic sucrose gradient centrifugation, reveals acid phosphatase activities against a range of substrates to be associated, to varying degrees, with subcellular particle populations identified as derived from flagella pocket membrane and surface membrane. Using these same substrates (alpha and beta glycerophosphate, p-nitrophenyl phosphate and glucose-6-phosphate) at least two distinct acid phosphatase activities can be distinguished. One is thermolabile (approximately 80% inactivated after 30 min. at 60 degrees C), sensitive to tartrate (50% inhibited at 1.8 mM Na tartrate) with a pH optimum approximately 4.5 and appears to exhibit little substrate preference. The other acid phosphatase is relatively heat stable (approximately 30% inactivated), insensitive to tartrate (greater than 5.0% inhibited using 1.8 mM Na tartrate) exhibits a somewhat higher pH optimum (approximately 6.0) and is more substrate specific (6X more active toward glucose-6-PO4 than beta-glycerophosphate). Further cell fractionation experiments reveal 85% of the tartrate sensitive acid phosphatase to be associated with flagella pocket membrane and to account for 80% of the organisms hydrolytic activity toward beta-glycerophosphate. The tartrate resistant acid phosphatase however, has a much less exclusive localization being almost equally distributed between surface membrane (40%) and flagella pocket membrane (60%).

Acid Phosphatase↗

PubMed1988

Triton-labile antigens in flagella isolated from Giardia lamblia.

Sheared flagella from Giardia lamblia were freed from cytoskeleton fragments and other cell contaminants by centrifuging in a density gradient. The purified organelles contain many polypeptides, including a set of low-molecular-weight antigens [apparent molecular weights (MWs) = 31, 32, 34, 35 and 37 kD] in the same size range as the approximately 30 kD structural giardins of the cytoskeleton. However, on sodium dodecyl sulphate-polyacrylamide gels, the mobilities of individual flagellar polypeptides do not correspond exactly to the cytoskeleton bands, and, unlike the cytoskeleton proteins, the flagellar components are easily extracted by Triton demembranation. The pattern of flagellar isoforms after isoelectric focussing (IEF) and electrophoresis in two dimensions is also clearly different from that of the cytoskeleton proteins. The fact that at least some approximately 30 kD flagellar antigens are localised by immunofluorescence specifically in the two ventral flagella suggests that these proteins may be components of the paraflagellar structures found beneath the membrane of these organelles. In electron micrographs of the isolated flagella, the paraflagellar rods are seen to bridge the membrane to three adjacent doublet microtubules of the axoneme.

Animals↗

PubMed1995

Enzyme immunoassay for detection of antibodies against isolated flagella of Legionella pneumophila.

Flagella of Legionella pneumophila serogroup 1, strain Philadelphia (ATCC 33152), were isolated and used as antigen to detect antibodies by enzyme immunoassay (EIA) in sera of patients with significant titers against Legionella as determined by the indirect immunofluorescence assay (IFA). Healthy blood donors served as a control group. Whereas the sera of blood donors were negative in both assays, sera of patients showed two patterns of reactivity. Sera with elevated titers against Legionella pneumophila serogroup 1 or Legionella gormanii in IFA usually demonstrated high titers in EIA. However, high IFA titers against Legionella bozemanii were associated with low titers in EIA. The data show that flagella protein of Legionella pneumophila serogroup 1 strain Philadelphia is of limited value for the detection of antibodies against Legionella other than Legionella pneumophila serogroup 1, although in vitro studies revealed genus-specific epitopes. This might be due to unpredictable variabilities in the expression of flagella in vivo.

Antibodies, Bacterial↗

PubMed2000

Transient invagination of flagella by Tritrichomonas foetus.

We describe an experimental system for the study of rapid and reversible formation of pseudocysts, which are spherical forms that lack a true cyst wall, by Tritrichomonas foetus, a trichomonad parasite of the bovine genitourinary tract. It highlights the dynamics of the plasma membrane and cytoskeleton of this parasite, which is perpetually devoid of any sort of protective cell wall, and can reflect a responsive survival mechanism. We have found that cooling of axenic cultures of T. foetus from their normal 37 degrees C to below about 16 degrees C can trigger pseudocyst formation. The three anterior flagella and the single recurrent flagellum can be fully internalized within 1 3 min at 37 degrees C, with the axonemes and flagellar membranes remaining intact within the cell body. Electron microscopy confirms that the internalized flagella are surrounded by a separate membrane. At 37 degrees C the internalized flagella can resume beating movements and become externalized as quickly as within 10 min. We have begun to elucidate the mechanisms of this unusual phenomenon, characterizing its temperature dependence and exploring the effects of agents that interfere with various aspects of the cytoskeleton, phagocytosis, endocytosis, and exocytosis.

Animals↗

PubMed1987

Intrinsic difference in beat frequency between the two flagella of Chlamydomonas reinhardtii.

The flagellar beat frequency of the biflagellated green alga Chlamydomonas reinhardtii was measured by fast Fourier transform analysis of the light intensity fluctuation in microscope images of swimming cells. Live cells had a mean beat frequency of 48-53 Hz at 20 degrees C. However, detergent-extracted "cell models," when reactivated in the presence of 1 mM ATP, appeared to have two different beat frequencies of about 30 and 45 Hz. Measurements in cell models in which only one of the two flagella was beating indicated that the lower and higher frequencies most likely represented the beat frequency of the flagellum nearer to the eyespot (the cis-flagellum) and that of the flagellum farther from it (the trans-flagellum), respectively. In live cells also, the trans-flagellum beat at a frequency about 30% higher than that of the cis-flagellum when the cells were rendered uniflagellated by mechanical treatment, whereas both flagella beat at the frequency of the cis-flagellum under normal conditions. These observations suggest that the two flagella of Chlamydomonas have different intrinsic beat frequencies but that they are somehow synchronized when beating together on a live swimming cell.

Animals↗

PubMed2005

Chlamydomonas shortens its flagella by activating axonemal disassembly, stimulating IFT particle trafficking, and blocking anterograde cargo loading.

Almost all eukaryotic cells form cilia/flagella, maintain them at their genetically specified lengths, and shorten them. Here, we define the cellular mechanisms that bring about shortening of flagella prior to meiotic cell division and in response to environmental cues in the biflagellated green alga Chlamydomonas. We show that the flagellar shortening pathway is distinct from the one that enforces transient shortening essential for length control. During flagellar shortening, disassembly of the axoneme is stimulated over the basal rate, and the rate of entry into flagella of intraflagellar transport (IFT) particles is increased. Moreover, the particles entering the disassembling flagella lack cargo. Thus, flagellar shortening depends on the interplay between dynamic properties of the axoneme and the IFT machinery; a cell triggered to shorten its flagellum activates disassembly of the axoneme and stimulates entry into the flagellum of IFT particles possessing empty cargo binding sites available to retrieve the disassembled components.

Animals↗

PubMed2004

Flagella-mediated bacterial motility accelerates but is not required for Salmonella serotype Enteritidis invasion of differentiated Caco-2 cells.

The relative contributions of the flagellum and the flagella-associated bacterial motility in the invasion of Caco-2 cells by Salmonella serotype Enteritidis were investigated using an fliC mutant defective in flagellin production and a motA mutant that carries flagella but is non-motile. Infection assays demonstrated that, at 1 h of infection, both the fliC and the motA mutants were severely impaired in bacterial invasion compared to the parental strain. Infection assays at 3 h infection demonstrated virtually equal invasion levels for both non-motile mutants and the parental strain. Together these data suggest that flagella-mediated bacterial motility accelerates the invasion of Salmonella but is not required for the invasion event per se.

Bacterial Adhesion↗

PubMed2004

A short flagella mutant of Dunaliella sallina (Chlorophyta, Cholorophyceae).

Dunaliella salina (Chlorophyta, Chlorophyceae) is a unicellular wall-less biflagellate alga. In this paper we describe a spontaneous mutant of D. salina, isolated from wild type cultures, which is characterized by very short flagella. The ultrastructure showed the basic 9 + 2 organization of wild-type flagella. Immunofluorescence localization of tubulin in this mutant confirmed the normal construction of the axoneme. Although, the mutant does not swim, still it is able to move and perform photobehavior. As shown by track reconstruction, and rotation movements, observed by means of reflection microscopy, this mutant can move, probably gliding by means of its stumpy flagella. A possible model to explain the mutant motion pattern is discussed.

Chlorophyta↗

PubMed1985

Digitized precision measurements of the movements of sea urchin sperm flagella.

High speed cinemicrographs were made of sea urchin sperm at temperatures varying from 22 to 6 degrees C. Apparatus, combining a television camera and a video digitizer, was constructed to scan individual flagellar images and to digitize the flagellar waveforms. With appropriate smoothing and averaging procedures, the rough data were condensed by a microcomputer into the coordinates of 20 points along a flagellum, spaced 2 microns apart. The curvature of the flagellum at these points was also computed. The coordinates of the flagellar positions were obtained to an accuracy of approximately +/- 0.1 micron, flagellar curvature to an accuracy of approximately +/- 50 cm-1. At all temperatures the amplitude of the flagella was found to vary with time in a purely sinusoidal fashion to within +/- 2%. The local curvature of the flagella had basically a purely sinusoidal time course to within +/- 50 cm-1, but a varying amount of asymmetry was present in the distal and the proximal ends of the flagella. This asymmetry in the curvature was related to the radius of the circular path of the sperm. The flagellar waveforms can probably be summarized in simple algebraic functions.

Animals↗

PubMed1985

Algebraic expressions for the waveforms of sea urchin sperm flagella.

The waveforms of live sea urchin sperm flagella were digitized with video-digitizing apparatus. The flagellar waveforms were expressed by the coordinates of 20 points spaced 2 micron apart along the flagella. The waveforms were condensed into simple algebraic expression with four parameters. Each of these parameters showed a systematic variation with the flagellar frequency. These average trends of the parameters made it possible to define an average, idealized, waveshape as a function of the flagella frequency, in the range 8-80 Hz.

Animals↗

PubMed1994

Characterization of antisera raised against Treponema denticola (ATCC 33521) whole cell, outer sheath, protoplasmic cylinder, and axial flagella.

In this study we produced polyclonal antisera directed to whole cell, outer sheath, protoplasmic cylinder and axial flagella sonicates of Treponema denticola (ATCC 33521) reference strain. Furthermore, the reactivity of the antisera was determined, using the enzyme-linked immunosorbent assay and immunoblotting techniques. As control antigen, other gram-negative bacteria (Salmonella minnesota, Escherichia coli) and related pathogenic spirochetes (Borrelia burgdorferi and Treponema pallidum) were used. It could be shown that the purified antibodies were specific for Treponema denticola and did not cross-react with the control antigens tested. Interestingly, with one exception, the anti-axial flagella antibody reacted with the flagellin of Treponema pallidum but not with Borrelia burgdorferi flagella. It is intended to use these antisera for the characterization of patient isolates in further studies.

Antibodies, Bacterial↗