Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.Cilia and flagella are prominent organelles of many eukaryotic cells. The names “cilia” and “flagella” are often related to historical reasons but they correspond to the same entity: a cylindrical organelle surrounded by a membrane and composed of an axoneme, a set of nine doublet microtubules originating from the basal body. Motile cilia usually contain a central pair of single microtubules and various substructures involved in the generation or the control of flagellar or ciliary beating, such as dynein arms, radial spokes, or central pair projections. This structural organization is remarkably well conserved across evolution, being encountered from protists to mammals (1). The conservation is also found at the molecular level as observed by comparative genomics between species with or without cilia and flagella (2, 3). Nevertheless, proteomic analysis revealed that in addition to the common core, many components unique to each group of eukaryotes are also present (4–8).The cilium represents a separate compartment from the cell body and does not contain any ribosomes or vesicles of any kind. The base of cilia and flagella contains projections that link each microtubule triplet of the basal body to the flagellum membrane (9). This region has been proposed to act as a barrier restricting entry of cytoplasmic proteins and ensuring retention of flagellum matrix elements (10). The transition zone is found in-between this area and the axoneme and contains several complexes of proteins (many of whom are mutated in the case of ciliopathies, genetic diseases affecting cilia function and/or formation) that contribute to the definition of the ciliary compartment (11, 12). Recent data showed that dextrans of low molecular weight are free to diffuse in the ciliary compartment as well as in the nucleus, whereas molecules of higher size (30 kDa or above) could not access these organelles. This led to the finding that a structure equivalent to the nucleopore complex is localized at the basal body area and could control access to the ciliary compartment (13). Finally, a septin barrier appears to be present close to the basis of the cilium and could control the trafficking of specific ciliary membrane proteins (14). The existence of a specific compartment comprising a large number of skeletal, matrix, and membrane proteins raises the issue of its internal organization. Key questions include the distribution of proteins, the mechanisms involved in specific distribution and the turnover during the life of the organelle.We selected to address these basic phenomena in the protist Trypanosoma brucei, well known as the etiological agent of sleeping sickness in Africa, but that is also an amenable model for cilia studies (15). It possesses a single flagellum that contains a typical 9 + 2 axoneme emerging from a depression of the cell surface called the flagellar pocket. This structure can be related to the ciliary pocket found at the base of different types of cilia in mammalian cells (16, 17). The axoneme is flanked by a lattice-like structure called the paraflagellar rod (PFR)¹ that is present as soon as the flagellum emerges from the pocket and runs to its distal end (18). The PFR contains at least 30 different proteins (19) and has been proposed to contribute to cell motility because its ablation results in cell paralysis in T. brucei (20) and in the related parasite Leishmania mexicana (21). The flagellum is attached to the cell body for most of its length, with the PFR lying close to the cell body side where a specific cytoskeletal structure termed the flagellum attachment zone (FAZ) is found (22). It is made of a unique filament composed of trypanosome-specific proteins (23, 24) and of four specialized microtubules flanked by the smooth endoplasmic reticulum (25). The flagellum plays key cellular functions as it drives cell motility (4, 26, 27), controls cell morphogenesis (28) and is responsible for parasite attachment during invasion of the salivary glands in the tsetse fly (29). Moreover, it could perform sensory functions and contribute to detection of the environment during the parasite life cycle (30). Recent data revealed the essential role of flagellum beating during fly invasion (31) but surprisingly reduction of forward motility did not affect infectivity in a mouse model (32).Purification of intact flagella from trypanosomes is a challenging task because of the adhesion to the cell body. Detergent and high-salt treatment have been used to efficiently purify the skeletal fraction of the flagellum that contains the axoneme, the PFR, and the basal body but that also includes the kinetoplast (mitochondrial genome), the FAZ, and the flagellar pocket collar (4, 33, 34). However, membrane and matrix components are totally lost during this procedure. For example, none of the intraflagellar transport (IFT) proteins that normally traffic in the flagellum matrix along peripheral microtubules (35) could be detected in samples purified by this procedure (4). We therefore decided to purify intact flagella by using a mutant strain called FLA1^RNAi where expression of an mRNA encoding a protein essential for flagellum attachment to the cell body (36) can be conditionally knocked-down by RNAi (37). FLA1^RNAi cells exhibit detached flagella from the main cell body, with the exception of the anchoring point at the basal body (37). By mechanical shearing, we found out that flagella could be severed from the cell body while preserving their membrane and their matrix elements. After purification, flagellar fractions were exhaustively characterized at the level of light and electron microscopy and their content was determined by mass spectrometry that confirmed the presence of the majority of known flagellar markers and revealed novel flagellar components. Three previously characterized proteins (the arginine kinase and two 14-3-3 proteins) and 10 hypothetical proteins were investigated in detail. Out of these 13 candidate proteins, 10 turned out to be associated to the flagellum whereas the others could not be detected experimentally. The novel ones were termed FLAM, for Flagellum Members. Remarkably, these proteins showed very specific location patterns within the flagellum including the membrane, the distal tip of the axoneme or the first proximal half of the axoneme, and displayed unexpected variations in their turnover rate. Overall, we revealed the existence of multiple subdomains within the flagellum with very specific dynamics, further demonstrating the highly sophisticated organization of the organelle.     

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

Background

Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.

Methods

Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.

Results

A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.

Conclusion

Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.     

Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum

Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort.The eukaryotic cilium/flagellum is a multifunctional organelle involved in an array of biological processes ranging from cell motility to cell signaling. Many cells in the human body, across a range of tissues and organs, produce either single or multiple, motile or nonmotile cilia where they perform diverse biological processes essential for maintaining human health. This diversity of function is reflected in an equally diverse range of pathologies and syndromes that result from ciliary/flagellar dysfunction via inherited mutations. This diversity is a reflection of the molecular complexity, both in components and in protein interactions of this organelle (1, 2).The canonical eukaryotic flagellum displays a characteristic “9 + 2” microtubular profile, where nine outer doublet microtubules encircle two singlet central pair microtubules, an arrangement found in organisms as diverse as trypanosomes, green algae, and mammals. Although this 9 + 2 microtubule arrangement has been highly conserved through eukaryotic evolution, there are examples where this standard layout has been modified, including the “9 + 0” layout of primary cilia and the “9 + 9 + 2” of many insect sperm flagella. In addition to this highly conserved 9 + 2 microtubule structure, flagella and cilia show a vast range of discrete substructures, such as the inner and outer dynein arms, nexin links, radial spokes, bipartite bridges, beak-like projections, ponticuli, and other microtubule elaborations that are essential for cilium/flagellum function. Cilia and flagella can also exhibit various extra-axonemal elaborations, and although these are often restricted to specific lineages, there is evidence that some functions, such as metabolic specialization, provided by these diverse structures are conserved (3, 4). Examples of such extraaxonemal elaborations include the fibrous or rod-like structures in the flagellum of the parasite Giardia lamblia (5), kinetoplastid protozoa (6, 7), and mammalian sperm flagella, along with extra sheaths of microtubules in insect sperm flagella (8).Several recent studies have set out to determine the protein composition of the flagellum and demonstrated the existence of both an evolutionarily conserved core of flagellum/cilium proteins and a large number of lineage-restricted components (9–13). Although these approaches provide an invaluable catalogue of the protein components of the flagellum, they provide only limited information on the substructural localization of proteins and do not address either the likely protein-protein interactions or the function of these proteins within the flagellum. To address these issues, the protein composition of some axonemal substructures (radial spoke complexes; for example see Ref. 14) has been determined by direct isolation of these structures, and a number of complexes have been resolved by the use of co-immunoprecipitation of indicator proteins (for example see Refs. 15 and 16). In addition the localization and function of a number of flagellar proteins have been investigated by detailed analysis of mutant cell lines (particularly of Chlamydomonas reinhardtii) that exhibit defined structural defects within the assembled axoneme. Early studies employed two-dimensional PAGE to compare the proteomic profile of purified flagella derived from C. reinhardtii mutants and wild type cells (17–22) that showed numerous proteomic differences in the derived profiles. The available technology did not allow identification of the individual proteins within the profiles. Recent proteomic advances offer the opportunity for this identification. For instance the comparative proteomic technique isotope coded affinity tagging has been used to identify components of the outer dynein arm (23). This technique utilizes stable isotope tagging to quantify the relative concentration of proteins between two samples.Trypanosomatids are important protozoan parasites whose flagellum is a critical organelle for their cell biology and pathogenicity. Their experimental tractability also provides opportunities for generic insights to the eukaryotic flagellum. They are responsible for a number of devastating diseases of humans and other mammals, including commercially important livestock, in some of the poorest areas of the world (24–26). All kinetoplastids build a flagellum that contains an extra-axonemal structure termed the paraflagellar rod (PFR).³ In the case of the African trypanosome Trypanosoma brucei brucei, this consists of a complex subdomain organization of a proximal, intermediate, and distal domain as well as links to specific doublets of the axoneme and a structure known as the flagellum attachment zone (FAZ) by which the flagellum is attached to the cell body for much of its length (6, 7). The PFR is required for cell motility (27, 28) and serves as a scaffold for metabolic and signaling enzymes (3, 29, 30). We have previously shown that the presence of this structure is essential for the survival of the mammalian bloodstream form of the parasite both in vitro (in culture) (12) and in vivo (in mice) (31) as part of a wider requirement for motility in this life cycle stage (12, 32, 33).Two major protein components of the PFR (PFR1 and PFR2) have been identified (34–38) along with several minor PFR protein components (3, 29, 30, 39–43). The availability of RNAi techniques in T. brucei allowed the generation of the inducible mutant cell line snl2 (44), in which RNAi-mediated ablation of the PFR2 protein causes the specific loss of both the distal and intermediate PFR subdomains (see Fig. 1A). After RNAi induction cells become paralyzed but remain viable (44). Our laboratory (3) has previously identified two PFR-specific adenylate kinases by comparing two-dimensional SDS-PAGE gels of purified flagella from induced and noninduced snl2 cells. These proteins cannot be incorporated into the PFR after PFR2 ablation.Open in a separate windowFIGURE 1.A, electron microscopy images (prepared as described previously (12)) of T. brucei snl2 noninduced and RNAi-induced flagellar transverse sections shows the loss of a large part of the PFR structure. Bar, 100 nm. B, frequencies (resolution 0.25) of log₂ protein abundance ratios of noninduced to noninduced samples from quadruplex iTRAQ. C, averaged frequencies (resolution 0.25) of log₂ protein abundance ratios of induced to noninduced samples from quadruplex iTRAQ. D, log₂ protein abundance ratios of induced to noninduced samples from all iTRAQ experiments for all proteins that show at least a 2-fold decrease after RNAi induction of snl2. α- and β-tubulin show a less than 2-fold change as expected. The results of individual sample pairs are graphed separately as per key.The ability to ablate PFR2 and hence disable assembly of a major portion of the PFR affords an opportunity to apply advanced proteomic approaches to identify additional PFR proteins. In this present study we have used two complementary proteomic approaches, two-dimensional fluorescence difference gel electrophoresis (DIGE) (45) and isobaric tags for relative and absolute quantitation (iTRAQ; Applied Biosystems), to investigate PFR+ and PFR–flagella to define 30 components of these two PFR subdomains. We have also conducted a bioinformatic analysis of amino acid motifs present in this protein cohort to gain insights into the possible functions of novel proteins and used epitope tagging approaches to confirm the PFR localization of a test set of identified proteins. We then asked whether it was possible to combine comparative proteomics with further analysis of RNAi mutant trypanosomes to provide detailed information on the individual interactions and assembly dependences within the novel PFR components we had identified. By iterating the subtractive proteomic analysis with novel putative PFR proteins, we were able to reveal the existence of distinct PFR protein dependence relationships and provide intriguing new insight into regulatory processes potentially operating within the trypanosome flagellum. Finally, this study establishes the mutant/proteomic combination as a powerful enabling approach for revealing dependences within subcohorts of the flagellar proteome.     

Effect of Oxygen on Cardiac Differentiation in Mouse iPS Cells: Role of Hypoxia Inducible Factor-1 and Wnt/Beta-Catenin Signaling

Background

Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.

Objective

We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.

Methods

Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.

Results

At 14 days of differentiation, 59±2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.

Conclusion

Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.     

Acceptance of Wife Beating and Its Association with Physical Violence towards Women in Nepal: A Cross-Sectional Study Using Couple’s Data

Background

Intimate partner violence (IPV) is a serious global public health issue. Acceptance of wife beating is known to be associated with IPV, but few studies have analysed the acceptance of wife beating from both women and men’s points of view. The objective of this study was to examine whether acceptance of wife beating among couples is associated with lifetime and past one-year physical IPV perpetration towards wives in Nepal.

Methods

A cross-sectional study was conducted from August to September 2011, with 717 randomly selected couples with wives aged 18 to 49 years old from the Kirtipur municipality and Bhaktapur district of Nepal. Wives’ and husbands’ acceptance of wife beating was measured by six scale items, while physical IPV experience among wives was measured by seven physical assault scale items. To assess the association between acceptance of wife beating and physical IPV, multiple logistic regression analysis was used.

Results

Nearly 30% of wives and husbands indicated that beating of wives is acceptable under certain circumstances. Statistically, no significant difference was detected between wives’ and husbands’ level of acceptance of wife beating. However, husbands’ acceptance of wife beating was positively associated with lifetime and past one-year perpetration of physical IPV, whereas wives’ acceptance of wife beating was neither associated with lifetime nor past one-year victimization of physical IPV. The positive association for husbands remained even after controlling for their partner’s factors.

Conclusions

Acceptance of wife beating is an important risk factor, which must be considered to prevent perpetration of physical IPV towards wives in Nepal. Future studies should include men to better understand the structure and dynamics of IPV in Nepal, and prevention programs should also target men to change their attitudes or to identify which couples are at more risk of physical IPV occurring toward wives.     

Intraflagellar Transport (IFT) Protein IFT25 Is a Phosphoprotein Component of IFT Complex B and Physically Interacts with IFT27 in Chlamydomonas

Background

Intraflagellar transport (IFT) is the bidirectional movement of IFT particles between the cell body and the distal tip of a flagellum. Organized into complexes A and B, IFT particles are composed of at least 18 proteins. The function of IFT proteins in flagellar assembly has been extensively investigated. However, much less is known about the molecular mechanism of how IFT is regulated.

Methodology/Principal Findings

We herein report the identification of a novel IFT particle protein, IFT25, in Chlamydomonas. Dephosphorylation assay revealed that IFT25 is a phosphoprotein. Biochemical analysis of temperature sensitive IFT mutants indicated that IFT25 is an IFT complex B subunit. In vitro binding assay confirmed that IFT25 binds to IFT27, a Rab-like small GTPase component of the IFT complex B. Immunofluorescence staining showed that IFT25 has a punctuate flagellar distribution as expected for an IFT protein, but displays a unique distribution pattern at the flagellar base. IFT25 co-localizes with IFT27 at the distal-most portion of basal bodies, probably the transition zones, and concentrates in the basal body region by partially overlapping with other IFT complex B subunits, such as IFT46. Sucrose density gradient centrifugation analysis demonstrated that, in flagella, the majority of IFT27 and IFT25 including both phosphorylated and non-phosphorylated forms are cosedimented with other complex B subunits in the 16S fractions. In contrast, in cell body, only a fraction of IFT25 and IFT27 is integrated into the preassembled complex B, and IFT25 detected in complex B is preferentially phosphorylated.

Conclusion/Significance

IFT25 is a phosphoprotein component of IFT particle complex B. IFT25 directly interacts with IFT27, and these two proteins likely form a subcomplex in vivo. We postulate that the association and disassociation between the subcomplex of IFT25 and IFT27 and complex B might be involved in the regulation of IFT.     

<Emphasis Type="Italic">Chlamydomonas fla</Emphasis> mutants reveal a link between deflagellation and intraflagellar transport

Background

Cilia and flagella are often lost in anticipation of mitosis or in response to stress. There are two ways that a cell can lose its flagella: resorption or deflagellation. Deflagellation involves active severing of the axoneme at the base of the flagellum; this process is defective in Chlamydomonas fa mutants. In contrast, resorption has been thought to occur as a consequence of constitutive disassembly at the tip in the absence of continued assembly, which requires intraflagellar transport (IFT). Chlamydomonas fla mutants are unable to build and maintain flagella due to defects in IFT.

Results

fla10 cells, which are defective in kinesin-II, the anterograde IFT motor, resorb their flagella at the restrictive temperature (33°C), as previously reported. We find that in standard media containing ~300 microM calcium, fla10 cells lose flagella by deflagellation at 33°C. This temperature-induced deflagellation of a fla mutant is not predicted by the IFT-based model for flagellar length control. Other fla mutants behave similarly, losing their flagella by deflagellation instead of resorption, if adequate calcium is available. These data suggest a new model whereby flagellar resorption involves active disassembly at the base of the flagellum via a mechanism with components in common with the severing machinery of deflagellation. As predicted by this model, we discovered that deflagellation stimuli induce resorption if deflagellation is blocked either by mutation in a FA gene or by lack of calcium. Further support for this model comes from our discovery that fla10-fa double mutants resorb their flagella more slowly than fla10 mutants.

Conclusions

Deflagellation of the fla10 mutant at the restrictive temperature is indicative of an active disassembly signal, which can manifest as either resorption or deflagellation. We propose that when IFT is halted by either an inactivating mutation or a cellular signal, active flagellar disassembly is initiated. This active disassembly is distinct from the constitutive disassembly which plays a role in flagellar length control.

     

Drosophila sperm swim backwards in the female reproductive tract and are activated via TRPP2 ion channels

Background

Sperm have but one purpose, to fertilize an egg. In various species including Drosophila melanogaster female sperm storage is a necessary step in the reproductive process. Amo is a homolog of the human transient receptor potential channel TRPP2 (also known as PKD2), which is mutated in autosomal dominant polycystic kidney disease. In flies Amo is required for sperm storage. Drosophila males with Amo mutations produce motile sperm that are transferred to the uterus but they do not reach the female storage organs. Therefore Amo appears to be a mediator of directed sperm motility in the female reproductive tract but the underlying mechanism is unknown.

Methodology/Principal Findings

Amo exhibits a unique expression pattern during spermatogenesis. In spermatocytes, Amo is restricted to the endoplasmic reticulum (ER) whereas in mature sperm, Amo clusters at the distal tip of the sperm tail. Here we show that flagellar localization of Amo is required for sperm storage. This raised the question of how Amo at the rear end of sperm regulates forward movement into the storage organs. In order to address this question, we used in vivo imaging of dual labelled sperm to demonstrate that Drosophila sperm navigate backwards in the female reproductive tract. In addition, we show that sperm exhibit hyperactivation upon transfer to the uterus. Amo mutant sperm remain capable of reverse motility but fail to display hyperactivation and directed movement, suggesting that these functions are required for sperm storage in flies.

Conclusions/Significance

Amo is part of a signalling complex at the leading edge of the sperm tail that modulates flagellar beating and that guides a backwards path into the storage organs. Our data support an evolutionarily conserved role for TRPP2 channels in cilia.     

Effects of calcium on the longituindal flagellum of Oxyrrhis marina

Summary— 2–4 nm filaments represent a new class of cytoskeletal components. They are found in ciliary and flagellar roots and centrosomes of all eucaryotes. They are also the major components of paraflagellar rods (PFR) in Euglena, trypanosomes and dinoflagellates. Oxyrrhis marina, a marine dinoflagellate, possesses a transverse and a longitudinal flagellum. Only the longitudinal flagellum carries the PFR along the proximal two-thirds of its length. This flagellum is not only capable of the classic flagellar beat but is also able to retract and bend, a property mediated by external calcium. To determine if calcium has a direct role in the bending, experimental conditions were established to permeabilization and reactivation. Our conditions to reactivate the axoneme function (wave propagation) appear similar to those observed in the case of the sea urchin sperm. The results show that in vitro, an increase in calcium concentration induces a conformational change of the longitudinal flagellum in the absence of ATP with a half maximum effect at 0.1 μM. In the presence of ATP, this morphology modification causes a total inhibition of the wave propagation which is replaced by non-propulsive contractions of low amplitude. As these properties are not shared by reactivated sea urchin sperm flagella or the transverse flagellum of O marina devoid of PFR, we propose that PFR are responsible for the bending phenomenon. A calcium shock also induces flagellar excision with a half maximum effect at 0.3 μM, and immunofluorescence results suggest that a centrin-like protein is present in O marina and is responsible for this excision.     

Subject positioning in the BOD POD® only marginally affects measurement of body volume and estimation of percent body fat in young adult men

Introduction

The aim of the study was to evaluate whether subject positioning would affect the measurement of raw body volume, thoracic gas volume, corrected body volume and the resulting percent body fat as assessed by air displacement plethysmography (ADP).

Methods

Twenty-five young adult men (20.7±1.1y, BMI = 22.5±1.4 kg/m²) were measured using the BOD POD® system using a measured thoracic gas volume sitting in a ‘forward bent’ position and sitting up in a straight position in random order.

Results

Raw body volume was 58±124 ml (p<0.05) higher in the ‘straight’ position compared to the ‘bent’ position. The mean difference in measured thoracic gas volume (bent-straight = −71±211 ml) was not statistically significant. Corrected body volume and percent body fat in the bent position consequently were on average 86±122 ml (p<0.05) and 0.5±0.7% (p<0.05) lower than in the straight position respectively.

Conclusion

Although the differences reached statistical significance, absolute differences are rather small. Subject positioning should be viewed as a factor that may contribute to between-test variability and hence contribute to (in)precision in detecting small individual changes in body composition, rather than a potential source of systematic bias. It therefore may be advisable to pay attention to standardizing subject positioning when tracking small changes in PF are of interest.The cause of the differences is shown not to be related to changes in the volume of isothermal air in the lungs. It is hypothesized and calculated that the observed direction and magnitude of these differences may arise from the surface area artifact which does not take into account that a subject in the bent position exposes more skin to the air in the device therefore potentially creating a larger underestimation of the actual body volume due to the isothermal effect of air close to the skin.     

Mathematical model of fructan biosynthesis and polymer length distribution in plants

Background and Aims

There are many unresolved issues concerning the biochemistry of fructan biosynthesis. The aim of this paper is to address some of these by means of modelling mathematically the biochemical processes.

Methods

A model has been constructed for the step-by-step synthesis of fructan polymers. This is run until a steady state is achieved for which a polymer distribution is predicted. It is shown how qualitatively different distributions can be obtained.

Key Results

It is demonstrated how a set of experimental results on polymer distribution can by simulated by a simple parameter adjustments.

Conclusions

Mathematical modelling of fructan biosynthesis can provide a useful tool for helping elucidate the details of the biosynthetic processes.     

Evidence for axonemal distortion during the flagellar beat of Chlamydomonas

In order to understand the working mechanism that governs the flagellar beat it is essential to know if the axoneme undergoes distortion during the course of the beat cycle. The rapid fixation method employed by Mitchell was able to preserve the waveform of Chlamydomonas flagella much as it appears during normal flagellar beating [Mitchell, Cell Motil Cytoskeleton 2003;56:120-129]. This conservation of the waveform suggests that the stress responsible for the production of bending is also trapped by the fixation procedure. Longitudinal sections of these well-preserved flagella were used to document variations in the relative axonemal diameter. Sections aligned to the plane of bending, showing both the central pair microtubules and outer doublets, were examined for this purpose. Micrographs were selected that continuously showed both the outer doublets and the central pair from a straight region to a curved region of the flagellum. Axoneme diameters measured from these select micrographs showed an increase in relative diameter that averaged 39 nm greater at the crest of the bent region. This constituted a 24% increase in the axoneme diameter in the bends. The transverse stress acting across the axoneme during bending was calculated from the Geometric Clutch computer model for a simulated Chlamydomonas-like flagellar beat. If we assume that this is representative of the transverse stress acting in a real flagellum, then the Young's modulus of the intact axoneme is approximately 0.02 MPa. The possibility that the distortion of the axoneme during the beat could play a significant role in regulating dynein function is discussed.     

Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

Background

Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.

Methodology/Principal Findings

We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.

Conclusions/Significance

Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.     

The proteome of Populus nigra woody root: response to bending

Background and Aims

Morphological and biomechanical alterations occurring in woody roots of many plant species in response to mechanical stresses are well documented; however, little is known about the molecular mechanisms regulating these important alterations. The first forest tree genome to be decoded is that of Populus, thereby providing a tool with which to investigate the mechanisms controlling adaptation of woody roots to changing environments. The aim of this study was to use a proteomic approach to investigate the response of Populus nigra woody taproot to mechanical stress.

Methods

To simulate mechanical perturbations, the taproots of 30 one-year-old seedlings were bent to an angle of 90 ° using a steel net. A spatial and temporal two-dimensional proteome map of the taproot axis was obtained. We compared the events occurring in the above-bending, central bending and below-bending sectors of the taproot.

Key Results

The first poplar woody taproot proteome map is reported here; a total of 207 proteins were identified. Spatial and temporal proteomic analysis revealed that factors involved in plant defence, metabolism, reaction wood formation and lateral root development were differentially expressed in the various sectors of bent vs. control roots, seemingly in relation to the distribution of mechanical forces along the stressed woody taproots. A complex interplay among different signal transduction pathways involving reactive oxygen species appears to modulate these responses.

Conclusions

Poplar woody root uses different temporal and spatial mechanisms to respond to mechanical stress. Long-term bending treatment seem to reinforce the defence machinery, thereby enabling the taproot to better overcome winter and to be ready to resume growth earlier than controls.     

Diesel Exhaust Particles Induce Cysteine Oxidation and S-Glutathionylation in House Dust Mite Induced Murine Asthma

Background

Diesel exhaust particle (DEP) exposure enhances allergic inflammation and has been linked to the incidence of asthma. Oxidative stress on the thiol molecules cysteine (Cys) and glutathione (GSH) can promote inflammatory host responses. The effect of DEP on the thiol oxidation/reduction (redox) state in the asthmatic lung is unknown.

Objective

To determine if DEP exposure alters the Cys or GSH redox state in the asthmatic airway.

Methods

Bronchoalveolar lavage fluid was obtained from a house dust mite (HDM) induced murine asthma model exposed to DEP. GSH, glutathione disulfide (GSSG), Cys, cystine (CySS), and s-glutathionylated cysteine (CySSG) were determined by high pressure liquid chromatography.

Results

DEP co-administered with HDM, but not DEP or HDM alone, decreased total Cys, increased CySS, and increased CySSG without significantly altering GSH or GSSG.

Conclusions

DEP exposure promotes oxidation and S-glutathionylation of cysteine amino acids in the asthmatic airway, suggesting a novel mechanism by which DEP may enhance allergic inflammatory responses.     

Characterization of engineered actin binding proteins that control filament assembly and structure

Background

Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate.

Methodology/Principal Findings

We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs), are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel “reduced genetic code” phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively.

Conclusions/Significance

From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.     

Determining the Precise Cerebral Response to Acupuncture: An Improved fMRI Study

Background

In acupuncture brain imaging trials, there are many non-acupuncture factors confounding the neuronal mapping. The modality of the placebo, subjects’ psychological attitude to acupuncture and their physical state are the three most confounding factors.

Objective

To obtain more precise and accurate cerebral fMRI mapping of acupuncture.

Design and Setting

A 2×2 randomized, controlled, participant-blinded cross-over factorial acupuncture trial was conducted at Xuanwu Hospital in Beijing, China.

Participants

Forty-one college students with myopia were recruited to participate in our study and were allocated randomly to four groups, Group A, Group B, Group C and Group D.

Interventions

Group A received real acupuncture (RA) and treatment instruction (TI); Group B received RA and non-treatment instruction (NI); Group C received sham acupuncture (SA) and TI; Group D received SA and NI.

Results

Stimulation at LR3 activated some areas of the visual cortex, and the cerebral response to non-acupuncture factors was complex and occurred in multiple areas.

Conclusions

The results provide more evidence regarding the credibility of acupuncture therapy and suggest that more precise experimental designs are needed to eliminate sources of bias in acupuncture controlled trials and to obtain sound results.     

Postural Abnormality as a Risk Marker for Leg Deep Venous Thrombosis in Parkinson’s Disease

Background

Pulmonary thromboembolism is a common cause of death in patients with autopsy-confirmed Parkinsonism. This study investigated the incidence of leg deep vein thrombosis in Parkinson’s disease and relationships between deep vein thrombosis and clinical/laboratory findings, including postural abnormalities as assessed by photographic measurements.

Methods

This cross-sectional study assessed the presence of deep vein thrombosis using bilateral leg Doppler ultrasonography in 114 asymptomatic outpatients with Parkinson’s disease.

Results

Deep vein thrombosis was detected in 23 patients (20%) with Parkinson’s disease. Deep vein thrombosis was located in the distal portion in 18 patients and in the proximal portion in 5 patients. No significant differences in age, sex, body mass index, disease duration, Hoehn-Yahr stage, anti-Parkinson’s drugs, or daily levodopa-equivalent dose were seen between deep vein thrombosis-positive and -negative groups. Univariate analysis for developing deep vein thrombosis in patients with Parkinson’s disease identified the following markers: long-term wheelchair use, bent knee, bent spine, and D-dimer elevation. Bending angles were significantly greater in the deep vein thrombosis-positive group at the knee and spine than in the deep vein thrombosis-negative group. Half of Parkinson’s disease patients with camptocormia had deep vein thrombosis. Among diabetes mellitus cases, long-term wheelchair use, bent knee over 15°, camptocormia, D-dimer elevation, the more risk markers were associated with a higher incidence of DVT. The presence of risk markers contributed to the development of deep vein thrombosis. On multivariate logistic regression analysis, a bent knee posture was strongly associated with an increased risk of deep vein thrombosis.

Conclusion

Presence of leg deep vein thrombosis correlated with postural abnormalities in Parkinson’s disease. We recommend non-invasive ultrasonographic screening for leg deep vein thrombosis in these high-risk patients with Parkinson’s disease.     

Hypoxia Transiently Sequesters Mps1 and Polo to Collagenase-Sensitive Filaments in Drosophila Prometaphase Oocytes

Background

The protein kinases Mps1 and Polo, which are required for proper cell cycle regulation in meiosis and mitosis, localize to numerous ooplasmic filaments during prometaphase in Drosophila oocytes. These filaments first appear throughout the oocyte at the end of prophase and are disassembled after egg activation.

Methodology/Principal Findings

We showed here that Mps1 and Polo proteins undergo dynamic and reversible localization to static ooplasmic filaments as part of an oocyte-specific response to hypoxia. The observation that Mps1- and Polo-associated filaments reappear in the same locations through multiple cycles of oxygen deprivation demonstrates that underlying structural components of the filaments must still be present during normoxic conditions. Using immuno-electron microscopy, we observed triple-helical binding of Mps1 to numerous electron-dense filaments, with the gold label wrapped around the outside of the filaments like a garland. In addition, we showed that in live oocytes the relocalization of Mps1 and Polo to filaments is sensitive to injection of collagenase, suggesting that the structural components of the filaments are composed of collagen-like fibrils. However, the collagen-like genes we have been able to test so far (vkg and CG42453) did not appear to be associated with the filaments, demonstrating that the collagenase-sensitive component of the filaments is one of a number of other Drosophila proteins bearing a collagenase cleavage site. Finally, as hypoxia is known to cause Mps1 protein to accumulate at kinetochores in syncytial embryos, we also show that GFP-Polo accumulates at both kinetochores and centrosomes in hypoxic syncytial embryos.

Conclusions/Significance

These findings identify both a novel cellular structure (the ooplasmic filaments) as well as a new localization pattern for Mps1 and Polo and demonstrate that hypoxia affects Polo localization in Drosophila.