Flagellar Function Research Articles

Microcosm

Escherichia coli is omnipresent in the mammalian gastrointestinal tract, but its life in broad daylight is also a fascinating story. In his new book, Microcosm: E. coli and the new science of life, science writer Carl Zimmer reminds us that E. coli has been the substratum for an astonishing number of scientific breakthroughs. The tools of molecular biology were forged on E. coli’s anvil, and application of these tools to the study of the bacterium itself has illuminated genetic regulation and exchange, virulence, nanomachines, bacterial social behavior, and evolution. Carl Zimmer is an American science writer at the zenith of his profession. He contributes to highly visible publications, including the New York Times, National Geographic, and Scientific American, and he is a winner of the 2007 National Academies Communication Award, the highest honor for science writing. He has authored numerous books on topics of biology and medicine, though Zimmer is, impressively, not a professional scientist. Zimmer opens the book with a historical introduction, beginning with the identification of E. coli by the great pediatrician and microbiologist Theodor Escherich, spanning the isolation of the E. coli strain K12 by Edward Tatum, and climaxing with the panorama of Nobel laureates who exploited K12 and its progeny. Zimmer indicates the improbable paradox of E. coli’s existence: “There’s a powerful drive throughout the universe, known as entropy, that pushes order toward disorder . . . [E. coli] does indeed reduce its own entropy, but only by consuming energy.” He likewise devotes considerable attention to E. coli’s remarkable nanomachines (particularly the flagellum), though he stops short of surmising how biological machines can initially achieve their astonishing complexity in the face of entropy’s mandate. These discussions are enlightening for the biology watcher. Zimmer also guides the reader through the highlights of modern E. coli research, lucidly reviewing complex and still-emerging stories. His summary of the complicated regulatory scheme controlling E. coli flagellar function is accurate, as is his discussion of alternative sigma factors. His appreciation of microbial biofilms is similarly clear and current, though the relationship between biofilm formation and intestinal colonization is as murky for Zimmer as it is for the professional microbiologist. Zimmer devotes considerable attention to genome plasticity and natural selection in E. coli genesis, indicating how far we’ve come since Darwin. Zimmer declares that selection can be powered by humans individually (e.g., by antibiotic administration), collectively (e.g., by large-scale industrial food production), or inadvertently (e.g., by acting as host to a microbial pathogen). In an era when the theory of natural selection is under siege politically, exploration of its nuances is welcome. The book has the feel of a personal encounter with great scientists. Yes, Zimmer complements his reading of the primary scientific literature with other written sources but apparently a relatively small number of personal interviews with the giants whose stories he relishes (judging from his notes). The acknowledgment section, in contrast, suggests that Zimmer’s theses have been vetted by a brigade of today’s E. coli community. Regardless of the source, Zimmer’s synthesis provides the texture of humanity for biological research. Though a pleasant read, Zimmer’s work leaves us without the bird’s eye insights characteristic of the best science writers, such as Lewis Thomas, Jared Diamond, and the incomparable Hans Zinsser. Despite, and perhaps because of, the daily immersion of scientists in their experiments, we often lose the grand vision of life’s universal principles. Recognition of these by the gifted science writer invigorates science and enriches the lay public. The value of Zimmer’s contribution will be greatest to the lay science enthusiast who realizes that no matter how sophisticated our scientific tools, biological systems generate an inexhaustible source of wonder. The E. coli biologist may be distracted by several errors and omissions in the book. For example, each chapter is decorated with a figure of a bacterium bearing polar flagella (E. coli is, of course, peritrichous), and antibacterial peptides are positively, not negatively, charged, as Zimmer claims. Prominently superficial is a discussion of E. coli’s true nature: commensal inhabitant of the mammalian gastrointestinal tract. Zimmer admits that E. coli K12 is an aberration, perverted by acts intentional and accidental. But commensal E. coli is the paragon that nature intended, though it is relatively neglected by Zimmer. Perhaps this opportunity provides the fodder for a sequel. Microcosm reminds us that scientific discovery makes compelling reading in the hands of a master storyteller. Mr. Zimmer has woven a fascinating tapestry, intercalating the energy of world-changing scientific discovery with the fascinating complexity of a well-understood living organism. His work will be welcomed by the scientist and the science enthusiast.

Functional studies of an evolutionarily conserved, cytochrome b5 domain protein reveal a specific role in axonemal organisation and the general phenomenon of post‐division axonemal growth in trypanosomes

Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Several recent proteomic studies of cilia and flagella have been published, including a proteome of the flagellum of the protozoan parasite Trypanosoma brucei. Comparing proteomes reveals many novel proteins that appear to be widely conserved in evolution. Amongst these, we found a previously uncharacterised protein which localised to the axoneme in T. brucei, and therefore named it Trypanosome Axonemal protein (TAX)-2. Ablation of the protein using RNA interference in the procyclic form of the parasite has no effect on growth but causes a reduction in motility. Using transmission electron microscopy, various structural defects were seen in some axonemes, most frequently with microtubule doublets missing from the 9+2 arrangement. RNAi knockdown of TAX-2 expression in the bloodstream form of the parasite caused defects in growth and cytokinesis, a further example of the effects caused by loss of flagellar function in bloodstream form T. brucei. In procyclic cells we used a new set of vectors to ablate protein expression in cells expressing a GFP:TAX-2 fusion protein, which enabled us to easily quantify protein reduction and visualise axonemes made before and after RNAi induction. This establishes a useful generic technique but also revealed a specific observation that the new flagellum on the daughter trypanosome continues growth after cytokinesis. Our results provide evidence for TAX-2 function within the axoneme, where we suggest that it is involved in processes linking the outer doublet microtubules and the central pair.

Functional analysis of a large non-conserved region of FlgK (HAP1) from Rhodobacter sphaeroides

The single subpolar flagellum of Rhodobacter sphaeroides shows an enlarged hook-filament junction. One of the two proteins that compose this section of the filament is HAP1(Rs) (FlgK(Rs)) it contains a central non-conserved region of 860 amino acids that makes this protein about three times larger than its homologue in Salmonella enterica serovar Typhimurium. We investigated the role of this central portion of the unusually large HAP1 protein of R. sphaeroides by monitoring the effects of serial deletions in flgK (Rs) , the gene encoding HAP1(Rs), on swimming and swarming. Two deletion mutants did not assemble functional flagella, two were paralyzed and five exhibited reduced free-swimming speeds. Some mutants produced unusual swarming patterns on soft agar without or with Ficoll 400. A segment of approximately 200-aa of the central region of HAP1(Rs) that aligns with the variable region of the flagellin sequence from other gamma- and beta-proteobacteria was also found. Therefore, it is possible that the origin of this large central domain of HAP1(Rs) could be associated with an event of horizontal transfer and subsequent duplications and/or insertions.

Global gene expression profiles for life stages of the deadly amphibian pathogen Batrachochytrium dendrobatidis

Amphibians around the world are being threatened by an emerging pathogen, the chytrid fungus Batrachochytrium dendrobatidis (Bd). Despite intensive ecological study in the decade since Bd was discovered, little is known about the mechanism by which Bd kills frogs. Here, we compare patterns of global gene expression in controlled laboratory conditions for the two phases of the life cycle of Bd: the free-living zoospore and the substrate-embedded sporangia. We find zoospores to be transcriptionally less complex than sporangia. Several transcripts more abundant in zoospores provide clues about how this motile life stage interacts with its environment. Genes with higher levels of expression in sporangia provide new hypotheses about the molecular pathways involved in metabolic activity, flagellar function, and pathogenicity in Bd. We highlight expression patterns for a group of fungalysin metallopeptidase genes, a gene family thought to be involved in pathogenicity in another group of fungal pathogens that similarly cause cutaneous infection of vertebrates. Finally we discuss the challenges inherent in developing a molecular toolkit for chytrids, a basal fungal lineage separated by vast phylogenetic distance from other well characterized fungi.

Swimming with protists: perception, motility and flagellum assembly

In unicellular and multicellular eukaryotes, fast cell motility and rapid movement of material over cell surfaces are often mediated by ciliary or flagellar beating. The conserved defining structure in most motile cilia and flagella is the '9+2' microtubule axoneme. Our general understanding of flagellum assembly and the regulation of flagellar motility has been led by results from seminal studies of flagellate protozoa and algae. Here we review recent work relating to various aspects of protist physiology and cell biology. In particular, we discuss energy metabolism in eukaryotic flagella, modifications to the canonical assembly pathway and flagellum function in parasite virulence.

Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei

In trypanosomes, the flagellum is rooted in the flagellar pocket, a surface micro-domain that is the sole site for endocytosis and exocytosis. By analysis of anterograde or retrograde intraflagellar transport in IFT88RNAi or IFT140RNAi mutant cells, we show that elongation of the new flagellum is not required for flagellar pocket formation but is essential for its organisation, orientation and function. Transmission electron microscopy revealed that the flagellar pocket exhibited a modified shape (smaller, distorted and/or deeper) in cells with abnormally short or no flagella. Scanning electron microscopy analysis of intact and detergent-extracted cells demonstrated that the orientation of the flagellar pocket collar was more variable in trypanosomes with short flagella. The structural protein BILBO1 was present but its localisation and abundance was altered. The membrane flagellar pocket protein CRAM leaked out of the pocket and reached the short flagella. CRAM also accumulated in intracellular compartments, indicating defects in routing of resident flagellar pocket proteins. Perturbations of vesicular trafficking were obvious; vesicles were observed in the lumen of the flagellar pocket or in the short flagella, and fluid-phase endocytosis was drastically diminished in non-flagellated cells. We propose a model to explain the role of flagellum elongation in correct flagellar pocket organisation and function.

Actin‐depolymerizing factor, ADF/cofilin, is essentially required in assembly of Leishmania flagellum

ADF/cofilins are ubiquitous actin dynamics-regulating proteins that have been mainly implicated in actin-based cell motility. Trypanosomatids, e.g. Leishmania and Trypanosoma, which mediate their motility through flagellum, also contain a putative ADF/cofilin homologue, but its role in flagellar motility remains largely unexplored. We have investigated the role of this protein in assembly and motility of the Leishmania flagellum after knocking out the ADF/cofilin gene by targeted gene replacement. The resultant mutants were completely immotile, short and stumpy, and had reduced flagellar length and severely impaired beat. In addition, the assembly of the paraflagellar rod was lost, vesicle-like structures were seen throughout the length of the flagellum and the state and distribution of actin were altered. However, episomal complementation of the gene restored normal morphology and flagellar function. These results for the first time indicate that the actin dynamics-regulating protein ADF/cofilin plays a critical role in assembly and motility of the eukaryotic flagellum.

Control of RpoS in global gene expression of Escherichia coli in minimal media

RpoS, an alternative sigma factor, is critical for stress response in Escherichia coli. The RpoS regulon expression has been well characterized in rich media that support fast growth and high growth yields. In contrast, though RpoS levels are high in minimal media, how RpoS functions under such conditions has not been clearly resolved. In this study, we compared the global transcriptional profiles of wild type and an rpoS mutant of E. coli grown in glucose minimal media using microarray analyses. The expression of over 200 genes was altered by loss of RpoS in exponential and stationary phases, with only 48 genes common to both conditions. The nature of the RpoS-controlled regulon in minimal media was substantially different from that expressed in rich media. Specifically, the expression of many genes encoding regulatory factors (e.g., hfq, csrA, and rpoE) and genes in metabolic pathways (e.g., lysA, lysC, and hisD) were regulated by RpoS in minimal media. In early exponential phase, protein levels of RpoS in minimal media were much higher than that in Luria-Bertani media, which may at least partly account for the observed difference in the expression of RpoS-controlled genes. Expression of genes required for flagellar function and chemotaxis was elevated in the rpoS mutant. Western blot analyses show that the flagella sigma factor FliA was expressed much higher in rpoS mutants than in WT in all phase of growth. Consistent with this, the motility of rpoS mutants was enhanced relative to WT. In conclusion, RpoS and its controlled regulators form a complex regulatory network that mediates the expression of a large regulon in minimal media.

Amino Acid Sequence of Bacterial Microbe-Associated Molecular Pattern flg22 Is Required for Virulence

Flagellin proteins derived from Pseudomonas syringae pv. tabaci 6605 and flg22Pa (QRLSTGSRINSAKDDAAGLQIA), one of the microbe-associated molecular patterns (MAMP) in bacterial flagellin, induce cell death and growth inhibition in Arabidopsis thaliana. To examine the importance of aspartic acid (D) at position 43 from the N-terminus of a flagellin in its elicitor activity, D43 was replaced with valine (V) and alanine (A) in P. syringae pv. tabaci flagellin and flg22Pta. The abilities of flagellins from P. syringae pv. tabaci D43V and D43A to induce cell death and growth inhibition were reduced, whereas the abilities of flg22PtaD43V and flg22PtaD43A were abolished. These results indicate that D43 is important for elicitor activity in P. syringae pv. tabaci. When tobacco plants were inoculated with each bacterium by the spray method, both P. syringae pv. tabaci D43V and D43A mutants had remarkably reduced ability to cause disease symptoms. Both mutants had reduced or no swimming and swarming motilities and adhesion ability. In P. syringae pv. tabaci D43V, little flagellin protein was detected and few flagella were observed by electron microscopy. These results indicate that mutant flagella are unstable and that flagellar motility is impaired. Thus, the amino acid residue required for MAMP activity is important for the intrinsic flagellar function.

Structural Insights into Membrane Targeting by the Flagellar Calcium-binding Protein (FCaBP), a Myristoylated and Palmitoylated Calcium Sensor in Trypanosoma cruzi

The flagellar calcium-binding protein (FCaBP) of the protozoan Trypanosoma cruzi is targeted to the flagellar membrane where it regulates flagellar function and assembly. As a first step toward understanding the Ca(2+)-induced conformational changes important for membrane-targeting, we report here the x-ray crystal structure of FCaBP in the Ca(2+)-free state determined at 2.2A resolution. The first 17 residues from the N terminus appear unstructured and solvent-exposed. Residues implicated in membrane targeting (Lys-19, Lys-22, and Lys-25) are flanked by an exposed N-terminal helix (residues 26-37), forming a patch of positive charge on the protein surface that may interact electrostatically with flagellar membrane targets. The four EF-hands in FCaBP each adopt a "closed conformation" similar to that seen in Ca(2+)-free calmodulin. The overall fold of FCaBP is closest to that of grancalcin and other members of the penta EF-hand superfamily. Unlike the dimeric penta EF-hand proteins, FCaBP lacks a fifth EF-hand and is monomeric. The unstructured N-terminal region of FCaBP suggests that its covalently attached myristoyl group at the N terminus may be solvent-exposed, in contrast to the highly sequestered myristoyl group seen in recoverin and GCAP1. NMR analysis demonstrates that the myristoyl group attached to FCaBP is indeed solvent-exposed in both the Ca(2+)-free and Ca(2+)-bound states, and myristoylation has no effect on protein structure and folding stability. We propose that exposed acyl groups at the N terminus may anchor FCaBP to the flagellar membrane and that Ca(2+)-induced conformational changes may control its binding to membrane-bound protein targets.

Campylobacter-Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells RequiresCampylobacter-Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-κB

Campylobacter jejuni and Campylobacter coli colonize and infect the intestinal epithelium and cause acute inflammatory diarrhea. The intestinal epithelium serves as a physical barrier to, and a sensor of, bacterial infection by secreting proinflammatory cytokines. This study examined the mechanisms for Campylobacter-induced secretion of the proinflammatory chemokine interleukin-8 (IL-8) by using polarized T84 human colonic epithelial cells as a model. C. jejuni increased the secretion of both IL-8 and tumor necrosis factor alpha (TNF-alpha) in polarized epithelial cells. However, the increase in IL-8 secretion was independent of Campylobacter-stimulated TNF-alpha secretion. Polarized T84 cells secreted IL-8 predominantly to the basolateral medium independently of the inoculation direction. While there was a significant correlation between the levels of IL-8 secretion and Campylobacter invasion, all 11 strains tested increased IL-8 secretion by polarized T84 cells despite their differences in adherence, invasion, and transcytosis efficiencies. Cell-free supernatants of Campylobacter-T84-cell culture increased IL-8 secretion to levels similar to those induced by live bacterial inoculation. The ability of the supernatant to induce IL-8 secretion was reduced by flagellum and cytolethal distending toxin (CDT) gene mutants, treatment of the supernatant with protease K or heat, or treatment of T84 cells with the Toll-like receptor (TLR) inhibitor MyD88 inhibitory peptide or chloroquine. NF-kappaB inhibitors or cdtB mutation plus MyD88 inhibitor, but not flaA cdtB double mutations, abolished the ability of the supernatant to induce IL-8 secretion. Taken together, our results demonstrate that Campylobacter-induced IL-8 secretion requires functional flagella and CDT and depends on the activation of NF-kappaB through TLR signaling and CDT in human intestinal epithelial cells.

Light affects motility and infectivity of Agrobacterium tumefaciens

Response to changes in light conditions involves a variety of receptors that can modulate gene expression, enzyme activity and/or motility. For the study of light-regulated effects of Agrobacterium tumefaciens, we used a global analysis approach - proteomics - and compared the protein patterns of dark- and light-grown bacteria. These analyses revealed a significant reduction of FlaA and FlaB - proteins of the flagellum - when the cells were grown in light. The light effect was confirmed by SDS-PAGE with isolated flagella. Quantitative PCR experiments showed a 10-fold increase of the transcription level of flaA, flaB and flaC within 20 min after the transfer from light to darkness. Electron microscopy revealed that these molecular events result in a light-induced reduction of the number of flagella per cell. These changes have major physiological consequences regarding motility, which is considerably reduced with exposure to light. The inhibitory effect of light on the motility is not unique to A. tumefaciens and was also seen in other species of the Rhizobiaceae. Previous studies suggested that the flagella function is significant for bacteria-plant interactions and bacterial virulence. In our studies, light reduced the attachment of A. tumefaciens to tomato roots and the virulence of the bacteria in a cucumber infection assay.

Genomic differences between Campylobacter jejuni isolates identify surface membrane and flagellar function gene products potentially important for colonizing the chicken intestine

Campylobacter spp. are one of the leading bacterial etiologic agents of acute human gastroenteritis among industrialized countries. Poultry are implicated as a major source of the organism for human illness; however, the factors involved with colonization of poultry gastrointestinal systems remain unclear. Genomics and proteomics analyses were used to identify differences between poor- versus robust-colonizing Campylobacter jejuni isolates, 11168(GS) and A74/C, respectively. Sequence analyses of subtracted DNA resulted in A74/C-specifc genes similar to a dimethyl sulfoxide reductase, a serine protease, polysaccharide modification proteins, and restriction modification proteins. DNA microarray analyses were performed for comparison of A74/C to the complete genome sequences published for two C. jejuni. A total of 114 genes (7.1%) were determined absent from A74/C relative to those genomes. Additionally, proteomics was completed on both soluble and membrane protein extracts from 11168(GS) and A74/C. Variation in protein expression and physical characteristics such as pI was detected between the two isolates that included the major outer membrane protein, flagella, and aconitate hydratase. Several proteins including cysteine synthase and a Ni/Fe hydrogenase were determined to be differentially present between the two isolates. Finally, DNA hybridization analyses of 19 C. jejuni isolates recovered from chickens and humans worldwide over the past 20 years were performed to determine the distribution of a subset of differentially identified gene sequences.

Genome Analysis of Chlamydomonas reinhardtii Reveals The Existence of Multiple, Compartmentalized Iron–Sulfur Protein Assembly Machineries of Different Evolutionary Origins

The unicellular green alga Chlamydomonas reinhardtii is used extensively as a model to study eukaryotic photosynthesis, flagellar functions, and more recently the production of hydrogen as biofuel. Two of these processes, photosynthesis and hydrogen production, are highly dependent on iron-sulfur (Fe-S) enzymes. To understand how Fe-S proteins are assembled in Chlamydomonas, we have analyzed its recently sequenced genome for orthologs of genes involved in Fe-S cluster assembly. We found a total of 32 open reading frames, most single copies, that are thought to constitute a mitochondrial assembly pathway, mitochondrial export machinery, a cytosolic assembly pathway, and components for Fe-S cluster assembly in the chloroplast. The chloroplast proteins are also expected to play a role in the assembly of the H-cluster in [FeFe]-hydrogenases, together with the recently identified HydEF and HydG proteins. Comparison with the higher plant model Arabidopsis indicated a strong degree of conservation of Fe-S cofactor assembly pathways in the green lineage, the pathways being derived from different origins during the evolution of the photosynthetic eukaryote. As a haploid, unicellular organism with available forward and reverse genetic tools, Chlamydomonas provides an excellent model system to study Fe-S cluster assembly and its regulation in photosynthetic eukaryotes.

Parkin-co-regulated gene ( PACRG) product interacts with tubulin and microtubules

Parkin-co-regulated gene ( PACRG) is a gene that shares a bidirectional promoter with Parkinson’s disease-related Parkin/Park2 gene. Recently, the PACRG gene product was implicated in the function of flagella. However, its exact function remains unknown. Here, I assessed the interaction between PACRG and tubulin. Co-sedimentation experiments revealed that PACRG directly binds to microtubules and α/β-tubulin heterodimers with high affinity. Microscopic studies showed that PACRG bundles microtubules and forms branched aggregates with unpolymerized tubulin dimers. The amino acid sequence of the microtubule-binding region of PACRG is highly conserved among various organisms, suggesting that tubulin binding is a basic property of PACRG. Structured summary MINT- 6439731, MINT- 6439753, MINT- 6439768: PACRG (genbank_protein_gi: 169219363), tubulin alpha (uniprotkb: P02550), tubulin beta (uniprotkb: Q2XVP4) bind (MI: 0407) by cosedimentation (MI: 0027) MINT- 6439796: PACRG (genbank_protein_gi: 169219363), tubulin alpha (uniprotkb: P02550), tubulin beta (uniprotkb: Q2XVP4) bind (MI: 0407) by pull-down (MI: 0096)

Depletion of plasma membrane PtdIns(4,5)P2 reveals essential roles for phosphoinositides in flagellar biogenesis

Axonemes are microtubule-based organelles of crucial importance in the structure and function of eukaryotic cilia and flagella. Despite great progress in understanding how axonemes are assembled, the signals that initiate axoneme outgrowth remain unknown. Here, we identified phosphatidylinositol phosphates (phosphoinositides) as key regulators of early stages of axoneme outgrowth in Drosophila melanogaster spermatogenesis. In a study of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] function in developing Drosophila male germ cells, we depleted PtdIns(4,5)P2 by expression of a potent phosphoinositide phosphatase. Phosphatase expression dramatically inhibited sperm tail formation and perturbed microtubule organization in a manner reversible by co-expression of a PtdIns 4-phosphate 5-kinase. Depletion of PtdIns(4,5)P2 caused increased levels of basal body gamma-tubulin and altered the distribution of proteins known to be required for axoneme assembly. Examination of PtdIns(4,5)P2-depleted spermatids by transmission electron microscopy revealed defects in basal body docking to the nuclear envelope, and in axoneme architecture and integrity of the developing flagellar axoneme and axial sheath. Our results provide the first evidence that phosphoinositides act at several steps during flagellar biogenesis, coordinately regulating microtubule and membrane organization. They further suggest that phosphoinositides play evolutionarily conserved roles in flagella and cilia, across phyla and in structurally diverse cell types.

Motility allows S. Typhimurium to benefit from the mucosal defence

The mammalian intestine is colonized by a dense bacterial community, called microbiota. The microbiota shields from intestinal infection (colonization resistance). Recently, we have shown that enteropathogenic Salmonella spp. can exploit inflammation to compete with the intestinal microbiota. The mechanisms explaining the enhanced pathogen growth in the inflamed intestine are elusive. Here, we analysed the function of bacterial flagella in the inflamed intestine using a mouse model for acute Salmonella Typhimurium enterocolitis. Mutations affecting flagellar assembly (Fla(-)) and chemotaxis (Che(-)) impaired the pathogen's fitness in the inflamed intestine, but not in the normal gut. This was attributable to a localized source of high-energy nutrients (e.g. galactose-containing glyco-conjugates, mucin) released as an element of the mucosal defence. Motility allows Salmonella Typhimurium to benefit from these nutrients and utilize them for enhanced growth. Thus, nutrient availability contributes to enhanced pathogen growth in the inflamed intestine. Strategies interfering with bacterial motility or nutrient availability might offer starting points for therapeutic approaches.

440. A-kinase anchoring protein 4 in the marsupial

A-Kinase Anchor Protein 4 (AKAP4) is an X-linked member of the AKAP family of scaffold proteins that anchor cAMP-dependent protein kinases and play an essential role in fibrous sheath assembly during spermatogenesis and flagellar function in spermatozoa. Marsupial spermatozoa differ in structural organisation from those of eutherian mammals but data on the molecular control of their structure and function are limited. We therefore cloned and characterised the AKAP4 gene in a marsupial, the tammar wallaby (Macropus eugenii). The gene structure, sequence and predicted protein of AKAP4 were highly conserved with that of eutherian orthologueues and it mapped to the marsupial X-chromosome. There was no AKAP4 expression detected in the developing young and in the adult, expression was limited to the testis with a major transcript of 2.9kb identified by northern blotting. AKAP4 mRNA was detected by in situ hybridisation in the cytoplasm of round and elongated spermatids in the adult testis while its protein was found in the sperm tail from principal piece of the flagellum. This is consistent with its expression in other mammals. Thus this gene appears to have a conserved role in spermatogenesis for at least the last 166 million years of mammalian evolution.

FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis

We report characterization of a novel testis- and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.

Rapid spatiotemporal patterning of cytosolic Ca2+ underlies flagellar excision in Chlamydomonas reinhardtii

Ca(2+)-dependent signalling processes are implicated in many aspects of flagella function in the green alga, Chlamydomonas. In this study, we examine the spatiotemporal dynamics of cytosolic Ca2+ ([Ca2+](cyt)) in single Chlamydomonas cells during the process of flagellar excision, using biolistically loaded calcium-responsive dyes. Acid-induced deflagellation occurred in parallel with a single transient elevation in whole-cell [Ca2+](cyt), which was absent in the acid deflagellation-deficient adf1 mutant. Deflagellation could also be induced by elevated external Ca2+ ([Ca2+](ext)), which promoted very rapid spiking of [Ca2+](cyt) across the whole cell and in the flagella. We also detected very rapid apically localised Ca2+ signalling events with an approximate duration of 500 msec. Ninety-seven per cent of deflagellation events coincided with a rapid elevation in [Ca2+](cyt) in the apical region of the cell, either in the form of a whole cell or an apically localised increase, indicating that [Ca2+](cyt) elevations in the apical region play an underlying role in deflagellation. Our data indicate that elevated [Ca2+](ext) acts to disrupt Ca2+ homeostasis which induces deflagellation by both Adf1-dependent and Adf1-independent mechanisms. Elevated [Ca2+](ext) also results in further [Ca2+](cyt) elevations after the main period of whole cell spiking which are very strongly associated with deflagellation, exhibit a high degree of apical localisation and are largely absent in the adf1 mutant. We propose that these later elevations may act as specific signals for deflagellation.