A NIMA-related kinase,CNK4, regulates ciliary stability and length

NIMA-related kinases (Nrks or Neks) have emerged as key regulators of ciliogenesis. In human, mutations in Nek1 and Nek8 cause cilia-related disorders. The ciliary functions of Nrks are mostly revealed by genetic studies; however, the underlying mechanisms are not well understood. Here we show that a Chlamydomonas Nrk, CNK4, regulates ciliary stability and length. CNK4 is localized to the basal body region and the flagella. The cnk4-null mutant exhibited long flagella, with formation of flagellar bulges. The flagella gradually became curled at the bulge formation site, leading to flagellar loss. Electron microscopy shows that the curled flagella involved curling and degeneration of axonemal microtubules. cnk4 mutation resulted in flagellar increases of IFT trains, as well as its accumulation at the flagellar bulges. IFT speeds were not affected, however, IFT trains frequently stalled, leading to reduced IFT frequencies. These data are consistent with a model in which CNK4 regulates microtubule dynamics and IFT to control flagellar stability and length.     

The GTPase Activity of FlhF Is Dispensable for Flagellar Localization,but Not Motility,in Pseudomonas aeruginosa

The opportunistic human pathogen Pseudomonas aeruginosa uses two surface organelles, flagella and pili, for motility and adhesion in biotic and abiotic environments. Polar flagellar placement and number are influenced by FlhF, which is a signal recognition particle (SRP)-type GTPase. The FlhF proteins of Bacillus subtilis and Campylobacter jejuni were recently shown to have GTPase activity. However, the phenotypes associated with flhF deletion and/or mutation differ between these organisms and P. aeruginosa, making it difficult to generalize a role for FlhF in pseudomonads. In this study, we confirmed that FlhF of P. aeruginosa binds and hydrolyzes GTP. We mutated FlhF residues that we predicted would alter nucleotide binding and hydrolysis and determined the effects of these mutations on FlhF enzymatic activity, protein dimerization, and bacterial motility. Both hydrolytically active and inactive FlhF point mutants restored polar flagellar assembly, as seen for wild-type FlhF. However, differential effects on flagellar function were observed in single-cell assays of swimming motility and flagellar rotation. These findings indicate that FlhF function is influenced by its nucleotide binding and hydrolytic activities and demonstrate that FlhF affects P. aeruginosa flagellar function as well as assembly.     

Control of flagellar motility in Euglena and Chlamydomonas. Microinjection of EDTA, EGTA, Mn(2)+, and Zn(2)+.

When a Euglena, in a medium containing ATP, is microinjected with 7 × 10^?14 l of 0.02 M EDTA, which binds Ca²⁺ and Mg²⁺, flagellar motility stops. Flagellar arrest in Chlamydomonas occurs with the injection of 2 × 10^?14 l of 0.02 M EDTA. The injection of similar amounts (7 × 10^?14 l in Euglena and 3 × 10^?14 l in Chlamydomonas) of 0.02 M EGTA, which preferentially binds Ca²⁺, did not significantly alter flagellar motility. This suggests that a decrease in the internal Ca²⁺ concentration in Euglena or Chlamydomonas did not stimulate flagellar beating. Further, flagellar motility decreased when internal Mg²⁺ was chelated. The microinjection of Zn²⁺ into these cells caused a decrease in flagellar frequency analogous to the decrease in frequency caused by the injection of Ca²⁺ and EDTA. The microinjection of 7 × 10^?14 l of 0.2 M Mn²⁺ caused an approx. 1.5-fold increase in Euglena flagellar motility. Chlamydomonas flagella, which cease to beat upon impalement in an Mg²⁺-free medium, resume a flagellar frequency of 18 Hz when injected with 3 × 10^?14 l of 0.2 M Mn²⁺. In the experiments reported here, Mn²⁺ acts as an analog of Mg²⁺.     

Identification and Characterization of a Novel Flagellum-Dependent Salmonella-Infecting Bacteriophage,iEPS5

A novel flagellatropic phage of Salmonella enterica serovar Typhimurium, called iEPS5, was isolated and characterized. iEPS5 has an icosahedral head and a long noncontractile tail with a tail fiber. Genome sequencing revealed a double-stranded DNA of 59,254 bp having 73 open reading frames (ORFs). To identify the receptor for iEPS5, Tn5 transposon insertion mutants of S. Typhimurium SL1344 that were resistant to the phage were isolated. All of the phage-resistant mutants were found to have mutations in genes involved in flagellar formation, suggesting that the flagellum is the adsorption target of this phage. Analysis of phage infection using the ΔmotA mutant, which is flagellated but nonmotile, demonstrated the requirement of flagellar rotation for iEPS5 infection. Further analysis of phage infection using the ΔcheY mutant revealed that iEPS5 could infect host bacteria only when the flagellum is rotating counterclockwise (CCW). These results suggested that the CCW-rotating flagellar filament is essential for phage adsorption and required for successful infection by iEPS5. In contrast to the well-studied flagellatropic phage Chi, iEPS5 cannot infect the ΔfliK mutant that makes a polyhook without a flagellar filament, suggesting that these two flagellatropic phages utilize different infection mechanisms. Here, we present evidence that iEPS5 injects its DNA into the flagellar filament for infection by assessing DNA transfer from SYBR gold-labeled iEPS5 to the host bacteria.     

Regulation of Flagellar Biogenesis by a Calcium Dependent Protein Kinase in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a bi-flagellated green alga, is a model organism for studies of flagella or cilia related activities including cilia-based signaling, flagellar motility and flagellar biogenesis. Calcium has been shown to be a key regulator of these cellular processes whereas the signaling pathways linking calcium to these cellular functions are less understood. Calcium-dependent protein kinases (CDPKs), which are present in plants but not in animals, are also present in ciliated microorganisms which led us to examine their possible functions and mechanisms in flagellar related activities. By in silico analysis of Chlamydomonas genome we have identified 14 CDPKs and studied one of the flagellar localized CDPKs – CrCDPK3. CrCDPK3 was a protein of 485 amino acids and predicted to have a protein kinase domain at the N-terminus and four EF-hand motifs at the C-terminus. In flagella, CrCDPK3 was exclusively localized in the membrane matrix fraction and formed an unknown 20 S protein complex. Knockdown of CrCDPK3 expression by using artificial microRNA did not affect flagellar motility as well as flagellar adhesion and mating. Though flagellar shortening induced by treatment with sucrose or sodium pyrophosphate was not affected in RNAi strains, CrCDPK3 increased in the flagella, and pre-formed protein complex was disrupted. During flagellar regeneration, CrCDPK3 also increased in the flagella. When extracellular calcium was lowered to certain range by the addition of EGTA after deflagellation, flagellar regeneration was severely affected in RNAi cells compared with wild type cells. In addition, during flagellar elongation induced by LiCl, RNAi cells exhibited early onset of bulbed flagella. This work expands new functions of CDPKs in flagellar activities by showing involvement of CrCDPK3 in flagellar biogenesis in Chlamydomonas .     

ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE IN CHLAMYDOMONAS REINHARDTII : Influence on Flagellar Function and Regeneration

Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.     

PflI, a protein involved in flagellar positioning in Caulobacter crescentus

The bacterial flagellum is important for motility and adaptation to environmental niches. The sequence of events required for the synthesis of the flagellar apparatus has been extensively studied, yet the events that dictate where the flagellum is placed at the onset of flagellar biosynthesis remain largely unknown. We addressed this question for alphaproteobacteria by using the polarly flagellated alphaproteobacterium Caulobacter crescentus as an experimental model system. To identify candidates for a role in flagellar placement, we searched all available alphaproteobacterial genomes for genes of unknown function that cluster with early flagellar genes and that are present in polarly flagellated alphaproteobacteria while being absent in alphaproteobacteria with other flagellation patterns. From this in silico screen, we identified pflI. Loss of PflI function in C. crescentus results in an abnormally high frequency of cells with a randomly placed flagellum, while other aspects of cell polarization remain normal. In a wild-type background, a fusion of green fluorescent protein (GFP) and PflI localizes to the pole where the flagellum develops. This polar localization is independent of the flagellar protein FliF, whose oligomerization into the MS ring is thought to define the site of flagellar synthesis, suggesting that PflI acts before or independently of this event. Overproduction of PflI-GFP often leads to ectopic localization at the wrong, stalked pole. This is accompanied by a high frequency of flagellum formation at this ectopic site, suggesting that the location of PflI is a sufficient marker for a site for flagellar assembly.     

Programmed Flagellar Ejection in Caulobacter crescentus Leaves PL-subcomplexes

The bacterial flagellum consists of a long extracellular filament that is rotated by a motor embedded in the cell envelope. While flagellar assembly has been extensively studied,¹ the disassembly process remains less well understood. In addition to the programmed flagellar ejection that occurs during the life cycle of Caulobacter crescentus, we and others have recently shown that many bacterial species lose their flagella under starvation conditions, leaving relic structures in the outer membrane.^2–7 However, it remains unknown whether the programmed flagellar ejection of C. crescentus leaves similar relics or not. Here, we imaged the various stages of the C. crescentus life cycle using electron cryo-tomography (cryo-ET) and found that flagellar relic subcomplexes, akin to those produced in the starvation-induced process, remain as a result of flagellar ejection during cell development. This similarity suggests that the programmed flagellar ejection of C. crescentus might share a common evolutionary path with the more general, and likely more ancient,³ starvation-related flagellar loss.     

Roles of CytR,an anti-activator of cyclic-AMP receptor protein (CRP) on flagellar expression and virulence in uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infection (UTI), a common bacterial infectious disease. This bacterium invades the urinary tract cells, where it aggregates, and subsequently forms multicellular colonies termed intracellular bacterial communities (IBCs). The motility of the bacteria plays a key role in the mechanism of virulence in the host bladder. Here, we show that CytR is a modulator of bacterial internalization and aggregation within the bladder epithelial cells sustained by CRP in UPEC. Mutational analyses and gel-shift assays indicated that CytR represses the expression of flhD, thereby encoding a master regulator for flagellar expression that is responsible for bacterial motility when CRP is present, whereas CRP is an activator of flhD expression. Thus, elevated flagellar expression was involved in promoted virulence in the cytR mutant. These combined observations suggest another regulatory layer of flagellar expression and the role of CytR in UPEC virulence.     

A flagellar-specific ATPase (FliI) is necessary for flagellar export in Helicobacter pylori

Although flagellar motility is essential for the colonisation of the stomach by Helicobacter pylori, little is known about the regulation of flagellar biosynthesis in this organism. We have identified a gene in H. pylori, designated fliI, whose deduced amino acid sequence revealed extensive homology with the FliI/LcrB/InvC family of proteins which energise the export of flagellar and other virulence factors in several bacterial species. An isogenic mutant of fliI was non-motile and synthesised reduced amounts of flagellin and hook protein subunits. The majority (>99%) of mutant cells were completely aflagellate. These results suggest that FliI is a novel ATPase involved in flagellar export in H. pylori.     

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.     

Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.     

EXTRACELLULAR MICROTUBULES : The Origin, Structure, and Attachment of Flagellar Hairs in Fucus and Ascophyllum Antherozoids

Mastigonemes (Flimmer) from the sperm of Ascophyllum and Fucus were found to consist of a tripartite structure—a ca. 2000-A tapered basal region, a closed microtubular shaft, and a group of terminal filaments. Each of these regions appears to be constructed of globular subunits with a center-to-center distance of about 45 A. The mastigoneme microtubule is of smaller diameter (170–190 A) than cytoplasmic microtubules in these or other plant cells. During the initial stages of flagellar ontogeny, structures similar to mastigonemes (presumptive mastigonemes) are found within membrane-limited sacs in the cytoplasm or within the perinuclear space. Mastigonemes at this time are generally not found on the flagellar surface. Later, when the anterior flagellum acquires mastigonemes, the presumptive mastigonemes are absent from the cytoplasm. The regularity of attachment of mastigonemes to the flagellar surface suggests that specific attachment sites are constructed on the plasma membrane during flagellar ontogeny. No evidence for penetration of the mastigoneme through the plasma membrane was obtained. The origin and structure of mastigonemes are discussed in relation to reports of the origin and structure of other microtubular systems.     

Transient Photoresponses of a Phototactic Microorganism, Haematococcus Pluvialis, Revealed by Light Scattering

A new method, using incoherent light scattering, has been developed to measure the flagellar beating frequency of swimming microorganisms. By means of this method, transient changes of flagellar beating frequency in response to white light flashes have been revealed in samples of a phototactic microorganism, Haematococcus pluvialis. An increase of flagellar beating frequency occurs when the flash dose (flash intensity × flash duration) is sufficient. Reciprocity between light intensity and flash duration holds for durations not exceeding 60-80 ms. For lower doses a bimodal distribution of flagellar beating frequency is revealed. No effect is observed for very low flashes or for red stimuli, whereas green light is effective. A detailed analysis of experimental results has allowed us to determine the characteristic time of the effect and follow its evolution. The correlation of this effect with visually observed behavior is discussed and a possible underlying mechanism is suggested.