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PubMed1989

Isolation and Ultrastructure of the Flagella of Methanococcus thermolithotrophicus and Methanospirillum hungatei.

The flagella of the archaebacteria Methanococcus thermolithotrophicus and Methanospirillum hungatei enter the cells in regions with ultrastructure resembling that of the polar organelles found in a variety of eubacteria. Flagella of both organisms consist of a filament, a hook, and a basal body with two rings similar to those of gram-positive eubacteria. The integrity of the flagella of M. thermolithotrophicus is lost in the absence of high salt concentrations, and those of both organisms are unstable at high pH. The flagellar filaments of M. hungatei are composed of two flagellins of 24 and 26 kilodaltons.

Journal Article↗

PubMed1984

Flagella help Salmonella typhimurium survive within murine macrophages.

In this study, we evaluated how flagella enhance the pathogenicity of Salmonella typhimurium in strain C57BL/6J mice. When mice were infected orally with flagellated or nonflagellated S. typhimurium, equivalent numbers of bacteria colonized the gastrointestinal tracts of the animals, but the number of flagellated organisms increased faster once colonization began in the spleens and livers. To evaluate this differential rate of Salmonella growth, the rate of blood clearance, and the kinetics of net multiplication of salmonellae in splenic tissue after intravenous challenge, the two groups of mice were compared. We found that clearance of bacteria from the blood was the same for flagellated or nonflagellated strains. However, the number of flagellated bacteria in the spleen increased logarithmically until the death of the animals, whereas the number of nonflagellated salmonellae increased only slightly. In contrast, both flagellated and nonflagellated strains grew exponentially in the spleens of mice pretreated with silica, a macrophage toxic agent. In an in vitro macrophage assay, flagellated salmonellae survived longer than nonflagellated organisms. These results indicate that flagella either protect S. typhimurium from the intracellular killing mechanisms of murine macrophages or that flagella enhance the ability of S. typhimurium to multiply within murine macrophages.

Animals↗

PubMed1991

Proteus mirabilis flagella and MR/P fimbriae: isolation, purification, N-terminal analysis, and serum antibody response following experimental urinary tract infection.

Urinary tract infection with Proteus mirabilis may lead to serious complications, including cystitis, acute pyelonephritis, fever, bacteremia, and death. In addition to the production of hemolysin and the enzyme urease, fimbriae and flagellum-mediated motility have been postulated as virulence factors for this species. We purified mannose-resistant/proteuslike (MR/P) fimbriae and flagella from strains CFT322 and HU2450, respectively. Electron microscopy revealed highly concentrated preparations of fimbriae and flagella. Fimbrial and flagellar structural subunits were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 18.5 and 41 kDa, respectively. N-terminal sequencing revealed that 10 of the first 20 amino acids of the major MR/P subunit matched the sequence of the P. mirabilis uroepithelial cell adhesin N terminus and 11 of 20 amino acids matched the predicted amino acid sequence of the Escherichia coli P fimbriae structural subunit, PapA. In addition, 90 and 80% homologies were found between the first 20 amino acids of P. mirabilis flagellin and those of Salmonella typhimurium phase-1 flagellin and the E. coli hag gene product, respectively. An enzyme-linked immunosorbent assay using purified antigens showed a strong reaction between the MR/P fimbriae or flagella and sera of CBA mice challenged transurethrally with P. mirabilis. A possible role for MR/P fimbriae in the pathogenesis of urinary tract infection is supported by (i) a strong immune response to the antigen in experimentally infected animals, (ii) amino acid sequence similarity to other enteric surface structure, and (iii) our previously reported observation that MR/P fimbriae are expressed preferentially as the sole fimbrial type in human pyelonephritis isolates.

Amino Acid Sequence↗

PubMed2002

Isolation and expression of the human hPF20 gene orthologous to Chlamydomonas PF20: evaluation as a candidate for axonemal defects of respiratory cilia and sperm flagella.

Primary ciliary dyskinesia (PCD) is a heterogeneous congenital disorder characterized by bronchiectasis and chronic sinusitis, sometimes associated with situs inversus (i.e., Kartagener's syndrome) and male infertility. At the cell level, the disease phenotype includes various axonemal abnormalities of respiratory cilia and sperm flagella. We have previously isolated DNAI1, the first gene involved in these diseases in patients lacking outer dynein arms. In this study, designed to find additional genes for other axonemal defects, we report the isolation of a novel human gene, hPF20, which is orthologous to Chlamydomonas pf20. The hPF20 gene is expressed as two major transcripts: one is expressed in testis only, whereas the second is weakly expressed in many other tissues. As flagella of Chlamydomonas strains carrying pf20 mutations lack the axonemal central complexes, we tested the involvement of the hPF20 gene in the disease phenotype of five patients in whom cilia or flagella display abnormal central complexes. Five intragenic polymorphisms were identified and used to exclude hPF20 in two consanguineous patients, while no mutation was found in the remaining patients. However, given the genetic heterogeneity of PCD, we consider that this gene remains a good candidate to be investigated in patients with abnormal central complexes.

Adult↗

PubMed2000

Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella.

The small GTPase Rho; functions as a molecular switch that regulates various cellular processes such as cell adhesion, motility, gene expression and cytokinesis. We previously isolated several putative Rho; targets including rhophilin which bound selectively to the GTP-bound form of Rho;. Rhophilin is expressed highly in testis and is localized specifically in sperm flagella. The presence of a PDZ domain at the carboxy terminus of rhophilin suggested that rhophilin works as an adaptor molecule. To test this hypothesis, we employed a yeast two hybrid system using the rhophilin PDZ domain as a bait, and screened a mouse testis cDNA library. We isolated several positive clones containing the same insert. The open reading frame of the cDNA encoded a novel protein of 212 amino acids designated as ropporin from a Japanese word 'oppo' (the tail). The amino-terminal 40 amino acid sequence of ropporin showed high homology to that of the regulatory subunit of type II cAMP-dependent protein kinase, which is involved in dimerization and binding to A-kinase anchoring proteins. Consistently, a yeast two hybrid assay and gel filtration of recombinant ropporin indicated that ropporin dimerizes through this domain. Deletion analysis indicated that the carboxy-terminal four amino acids are essential for binding of ropporin to rhophilin, and ropporin and RhoV14 coprecipitated in the presence of rhophilin in vitro. Northern blot analysis showed that ropporin is exclusively expressed in testis, and induced at the late stage of spermatogenesis. This induction paralleled that of rhophilin. Immunocytochemistry using anti-ropporin antibody showed that ropporin is localized in the principal piece and the end piece of sperm flagella. Electronmicroscopy revealed that ropporin is mostly localized in the inner surface of the fibrous sheath while rhophilin is present in the outer surface of the outer dense fiber. These results suggest that rhophilin and ropporin may form a complex in sperm flagella.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Effect of beat frequency on the velocity of microtubule sliding in reactivated sea urchin sperm flagella under imposed head vibration.

The heads of demembranated spermatozoa of the sea urchin Tripneustes gratilla, reactivated at different concentrations of ATP, were held by suction in the tip of a micropipette and vibrated laterally with respect to the head axis. This imposed vibration resulted in a stable rhythmic beating of the reactivated flagella that was synchronized to the frequency of the micropipette. The reactivated flagella, which in the absence of imposed vibration had an average beat frequency of 39 Hz at 2 mmol l-1 ATP, showed stable beating synchronized to the pipette vibration over a range of 20-70 Hz. Vibration frequencies above 70 Hz caused irregular, asymmetrical beating, while those below 20 Hz induced instability of the beat plane. At ATP concentrations of 10-100 mumol l-1, the range of vibration frequency capable of maintaining stable beating was diminished; an increase in ATP concentration above 2 mmol l-1 had no effect on the range of stable beating. In flagella reactivated at ATP concentrations above 100 mumol l-1, the apparent time-averaged sliding velocity of axonemal microtubules decreased when the imposed frequency was below the undriven flagellar beat frequency, but at higher imposed frequencies it remained constant, with the higher frequency being accompanied by a decrease in bend angle. This maximal sliding velocity at 2 mmol l-1 ATP was close to the sliding velocity in the distal region of live spermatozoa, possibly indicating that it represents an inherent limit in the velocity of active sliding.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1979

[Ultrastructure of the double and multiple flagella of the sperm of the drake].

The structure of double and multiple flagella of the spermatozoa of a drake was investigated. In spermatozoa with this defect one or two implantation pits were observed in the head of the spermatozoon with one or two proximal centrioles situated in the extended base of the nucleus. The structure of the proximal centrioles was frequently disturbed. Distal centrioles were placed separately without any marked figural or structural disturbance. The mitochondrial sheath was formed around both or more flagella, and between the flagella only one line of mitochondria was usually found. The ring showed defects in shape and changes in the deposition. An amorphous sheath was formed around each flagellum separately without any marked defects. In single cases insignificant changes were recorded in the arrangement of fibrils of the axial bundle.

Animals↗

PubMed1982

Microtubule sliding in reactivated flagella.

Recent experimental studies of microtubule sliding in demembranated sea urchin sperm flagella are described. A local iontophoretic application of ATP to a Triton-extracted flagellum elicits a local bending response whose form is in exact conformity with the predictions of the sliding microtubule model. Cinematographic analysis of the microtubule sliding initiated by treating fragments of demembranated flagella with trypsin in the presence of ATP reveals that the speed of sliding is almost constant. This implies that the speed does not depend on the number of dynein arms participating in the generation of sliding force. The distribution of apparent sliding velocities indicates that there is no difference in sliding velocity among the doublets. The sliding velocity depends on MgATP concentration in a manner consistent with Michaelis-Menten kinetics. The sliding velocity of doublets in trypsin-treated axonemes is close to the maximum velocity of relative sliding taking place between adjacent doublets in beating flagella reactivated at the same MgATP concentration.

Adenosine Triphosphate↗

PubMed2026

A Novel Homozygous Mutation in ARL2BP Causes Multiple Morphological Abnormalities of the Flagella and Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) and multiple morphological abnormalities of the sperm flagella (MMAF) frequently co-occur in male infertility. However, the genetic basis of this syndromic presentation remains unclear. Using whole-exome sequencing, we identified a novel homozygous ARL2BP splice-site mutation (c.294-2A>G) in a 23-year-old infertile male from a consanguineous family who presented with syndromic PCD and MMAF. This variant causes aberrant pre-mRNA splicing and triggers nonsense-mediated mRNA decay, resulting in the complete absence of ARL2BP protein expression. Transmission electron microscopy revealed extensive disorganization of flagellar axonemes with consistent central pair (CP) microtubule depletion and disorganization of peripheral doublets. Immunofluorescence confirmed a severe deficiency of the CP protein SPAG6 in the sperm flagella. Notably, the patient presented without retinal symptoms. Given that ARL2BP-related retinitis pigmentosa generally emerges during the third decade, long-term ophthalmological follow-up is essential to detect delayed-onset retinal degeneration. In conclusion, these findings confirm ARL2BP as a causative gene for both PCD and MMAF, expanding the genotypic and phenotypic spectrum of ciliopathies.

Humans↗

PubMed1997

Beat frequency difference between the two flagella of Chlamydomonas depends on the attachment site of outer dynein arms on the outer-doublet microtubules.

The two flagella of Chlamydomonas, although similar to each other at first glance, differ in functional properties. A clear difference exists in the beat frequency: the trans-flagellum (the one farthest from the eyespot) beats with 30-40% higher frequency than the cis-flagellum (the one nearest to the eyespot) in demembranated and reactivated cell models. This difference is considered to be influenced by outer arm dynein, because the two flagella beat at almost the same frequency in cell models of oda mutants lacking the outer dynein arm. When a sample of outer arm dynein extracted and purified from the wild-type axoneme was mixed with the cell models of an oda mutant, oda1, an almost normal number of outer dynein arms became attached to the axonemes, and the wild-type level of beat frequency was recovered on reactivation with ATP addition. The frequency imbalance, however, was not restored. Unexpectedly, when a similar experiment was performed with the cell model of another oda mutant, oda6, the addition of outer arm dynein restored the cis-trans frequency imbalance in addition to the normal number of outer arms and the higher level of reactivated motility. Among other oda mutants, oda3 yielded results similar to those with oda1, whereas oda2, oda4, and oda5 yielded results similar to those with oda6. Because the only structural difference between the two groups of oda mutants is that the oda1 and oda3 axonemes lack the outer arm attachment site on the outer doublet A-tubule while the axonemes of the other mutants retain it, these findings suggest that the attachment site for the outer dynein arm is important in determining the flagellar beat frequency. This suggests that the basal portion of the outer arm dynein is important in regulating the flagellar activity and therefore the behavior of the cell.

Animals↗

PubMed2001

Measurement of the force and torque produced in the calcium response of reactivated rat sperm flagella.

Rat sperm that are demembranated with Triton X-100 and reactivated with Mg-ATP show a strong mechanical response to the presence of free calcium ion. At pCa < 4, the midpiece region of the flagellum develops a strong and sustained curvature that gives the cell the overall appearance of a fishhook [Lindemann and Goltz, 1988: Cell Motil. Cytoskeleton 10:420-431]. In the present study, the force and torque that maintain the calcium-induced hook have been examined quantitatively. In addition, full-length and shortened flagella were manipulated to evaluate the plasticity of the hooks and determined the critical length necessary for maintaining the curvature. The hooks were found to be highly resilient, returning to their original configuration (>95%) after being straightened and released. The results from manipulating the shortened flagella suggest that the force holding the hook in the curved configuration is generated in the basal 60 microm of the flagellum. The force required to straighten the calcium-induced hooks was measured with force-calibrated glass microprobes, and the bending torque was calculated from the measured force. The force and torque required to straighten the flagellum were found to be proportional to the change in curvature of the hooked region of the flagellum, suggesting an elastic-like behavior. The average torque to open the hooks to a straight position was 2.6 (+/-1.4) x 10(-7) dyne x cm (2.6 x 10(-14) N x m) and the apparent stiffness was 4.3 (+/-1.3) x 10(-10) dyne x cm(2) (4.3 x 10(-19) N x m(2)). The stiffness of the hook was determined to be approximately one quarter the rigor stiffness of a rat sperm flagellum measured under comparable conditions.

Animals↗

PubMed2005

Computer simulation of flagellar movement IX. Oscillation and symmetry breaking in a model for short flagella and nodal cilia.

A computer model of flagella in which oscillation results from regulation of active sliding force by sliding velocity can simulate the movements of very short flagella and cilia. Of particular interest are the movements of the short (2-3 microm) nodal cilia of the mammalian embryo, which determine the development of the asymmetry of the internal organs. These cilia must generate a counterclockwise (viewed from base to tip) circling motion. A three-dimensional computer model, with active force generated by a simple mathematical formulation and regulated by sliding velocity, can generate this circling motion if a time delay process is included in the control specification. Without the introduction of a symmetry-breaking mechanism, the computer models start randomly in either direction, and maintain either clockwise or counterclockwise circling. Symmetry can be broken by at least two mechanisms: (1) control of dynein activity on one outer doublet by sliding velocity can be influenced by the sliding velocity experienced on an adjacent outer doublet, or (2) a constant twist of the axoneme caused by an off-axis component of dynein force. This second mechanism appears more reasonable, but its effectiveness is highly dependent upon specifications for the elastic resistances of the model. These symmetry-breaking mechanisms need to be present only at the beginning of circling. With these models, once a circling direction is established, it remains stable even if the symmetry-breaking mechanism is removed.

Animals↗

PubMed1983

Bending patterns of chlamydomonas flagella I. Wild-type bending patterns.

Using a uniflagellate mutant of Chlamydomonas and flash photomicrography at 300 Hz, we have obtained detailed information on the forward and reverse beating modes of Chlamydomonas flagella and on the relationship between rotation of the uniflagellate cell and the bending cycle of the forward mode. Flagella ranging in length from 5 to 15.5 micron were photographed. There is a decrease in wavelength and an increase in curvature in the principal bends when the length of the flagellum is less than the normal length of 12-13 micron, but these changes are not sufficient to maintain similarity of the bending pattern. In the reverse mode, the flagellum propagates symmetrical, planar, undulatory waves with a shear amplitude which is the same as in the forward mode; there is a 19% increase in beat frequency and a similar decrease in wave length. The reorientation of the flagellar beat direction towards the axis of the cell in the reverse mode is caused both by the decrease in asymmetry of beat and by activation of sliding in the principal bends at an earlier time in the beat cycle, relative to the time of activation of sliding in reverse bends. There are additional rare modes of beating which may be related to intermediate stages in the transition between forward and reverse beating modes.

Chlamydomonas↗

PubMed1996

A moving image of flagella: news and views on the mechanisms involved in axonemal beating.

Beating of cilia and flagellae allows movement of the fluid surrounding isolated cells (for example: protists) or epithelia (bronchial tissue) but is also responsible for the movement of unicellular organisms in this medium (such as spermatozoa or protists). This paper aims to describe: (1) the biochemical and structural elements of the '9 + 2' structure called the axoneme; (2) the mechanisms of wave generation and propagation along the axoneme of cilia and flagellae are then described, stating that in most models of wave propagation, a clear distinction is made between the dynein-dependent microtubule sliding which represents the oscillatory motor and the bending mechanism which regulates wave propagation. In current models, the bending propagation is supported by a bind/relax cyclic mechanism which propagates in register, but frame-shifted, with the powering action of the dynein motor along the axoneme. While a large amount of knowledge was accumulated about the motor, little is known about the resisting elements regulating the bending. (3) The present study also puts forward ideas as to how these organelles have been highly conserved throughout eucaryotic evolution, and concludes with suggestions for further fields of investigation into this unique mechanical device used for cell movement.

Animals↗

PubMed1996

Defined mutants of Proteus mirabilis lacking flagella cause ascending urinary tract infection in mice.

A clinical isolate of Proteus mirabilis (Pr 990) and an isogenic non-flagellate allelic replacement mutant (Pr M9) were tested for their ability to cause infection in the ascending mouse model of urinary tract infection. Wild-type Pr 990 differentiates into swarmer cells in brain-heart infusion broth. Pr M9 neither has flagella nor does it apparently differentiate into swarmer cells after subculturing. The infectivity of both strains from an initial culture and the sixth subculture was assessed in the ascending urinary tract infection mouse model. Infection was ascertained by determining colony forming units from kidney and bladder homogenates from individual mice 7 days after inoculation. In all cases the nonflagellate mutant Pr M9 was at least as infective as Pr 990. Using bacteria from the first culture, Pr M9 infected 61.5% and Pr 990 infected 45.5% of mice tested. The levels of viable counts were similar between the Pr M9 and the Pr 990 infections. Using bacteria from the sixth subculture, Pr M9 infected 75% and Pr 990 infected 76.5% of mice tested. Again viable counts were similar. Pr 990 increased in infectivity from the first to the sixth subculture, whereas Pr M9 did not, but this may be a reflection of the high initial rate of infectivity with first culture Pr M9. These results suggest that neither flagella nor swarmer cells are required for P. mirabilis infectivity in ascending urinary tract infections in mice.

Alleles↗

PubMed1996

Small G proteins of two green algae are localized to exocytic compartments and to flagella.

The Ypt/Rab proteins are small GTPases, which belong to the Ras superfamily and have been shown to be involved in endo- and exocytosis in mammalian cells and yeast. Using affinity-purified antibodies specific for four Ypt proteins, namely Ypt1p, Ypt4p, Ypt5p and Ypt6p, of the multicellular green alga Volvox carteri (YptVp) and its close unicellular relative Chlamydomonas reinhardtii (YptCp), we examined the abundance of the corresponding antigens during the asexual life cycle of Volvox, and their intracellular localization. The YptV proteins were found in all stages throughout the asexual life cycle and are tightly associated with intracellular membranes. Indirect immunofluorescence revealed that YptV4p, YptV5p and YptV6p are present in perinuclear regions of the cell, indicating an association with the Golgi region. Golgi localization of YptV4p and YptV6p in Volvox was confirmed by immunogold electron microscopy. In contrast, we found Ypt1p associated with the contractile vacuole in both V. carteri and C. reinhardtii. Furthermore, the YptV proteins were also detected along the entire length of the flagella of somatic Volvox cells. This flagellar location was substantiated by western blot analysis of extracts prepared from isolated flagella of both algae. While localization to exocytic compartments is in agreement with the established Ypt/Rab function in intracellular vesicle transport of eukaryotic cells, presence in the algal flagellum is the first hint of a possible role for small G proteins also in motility organelles.

Blotting, Western↗

PubMed1976

Regulation of polar surface structures in Caulobacter crescentus: pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors.

A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages. The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile. Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25 degrees C. However, at a higher temperature (35 degrees C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35 degrees C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects. It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.

Bacteria↗

PubMed1975

Production of potent salmonella H antisera by immunization with flagellae, isolated by immunosorption.

For serotyping Salmonella H sera should exhibit a high H titer and preferably a low O titer which does not interfere with the H slide agglutination at the working dilution of the serum. By conventional immunization with motile bacteria OH sera are always produced. We isolated flagellae from highly motile Salmonella cultures, which were detached from the bacterial cells by a waring blendor, with an immunosorbent, i.e. cyanobromide-activated Sepharose to which the respective anti H antibody was coupled. The desorption of the flagella from the immunosorbent was done by lowering the pH to 2.3 with glycine-HC1 buffer. 4.2 mg of flagellar protein were recovered from 20 agar swarm plates. 10 mug of the antigen incorporated into complete Freund adjuvant was found to immunize a rabbit successfully. The H titers varied from 10,000-160,000 and the majority of the O titers did not exceed 160. Only one immunosorbent was necessary to isolate a group of related H antigens (i.e., lv, lw, lz13, lz28). After regeneration the immunosorbent can be reused many times.

Adsorption↗