Regulation of Intraflagellar Transport in the sensory cilia of Caenorhabditis Elegans

Cilia zijn kleine uitstulpingen op het celoppervlakte. Ze zijn belangrijk bij de beweging van cellen, zoals bijvoorbeeld bij sperma cellen, maar hebben daarnaast ook een sensorische functie. Wij hebben voor ons cilia onderzoek gekozen voor het model organisme Caenorhabditis elegans, aangezien cilia zeer geconserveerd zijn tijdens de evolutie en defecten in cilia niet lethaal zijn in dit organisme in tegenstelling tot vele andere dieren. Alle cilia hebben een vergelijkbare opbouw. Het bestaat uit verschillende buisvormige filamenten omgeven door het celmembraan. Het begin van de cilia wordt de transitie zone genoemd, waarna het eerste deel van de cilia, het middel segment en het uitMany environmental signals are detected by specialized sensory neurons, which have cilia extending from the cell surface as long appendices and exposed to the environment. Cilia consist of a microtubular axonemal core surrounded by a membrane and are anchored in the cell by the basal body. The end of the basal body and the beginning of the axoneme are called the transition zone. Since cilia do not have the capacity to synthesize proteins, all components, both structural and signaling molecules, need to be transported into and out of the cilia. This is probably achieved by a process called intraflagellar transport (IFT) and is driven by three motors in Caenorhabditis elegans. Two motors, kinesin-II and OSM-3 kinesin, are used for anterograde transport from the base of the cilium to the distal tip. Both these kinesin motor complexes are involved in the transport in the first 4 µm of the cilium, called the middle segment, whereas only OSM-3 kinesin is required for the transport in the last 2,5 µm of the cilium until the distal tip, called the distal segment. Retrograde transport from the distal tip back to the basal body is dependent on the dynein motor complex. IFT is not only responsible for the transport of structural components but recently three signalling molecules have been described to be transported in the cilia i.e. OSM-9, a transient receptor potential vanilloid channel involved in sensory signal transduction, a PKD associated protein called qilin and Smoothened, a plasma membrane protein involved in hedgehog signaling

A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation

Previously, a promoter was identified in Lactococcus lactis that is specifically induced by chloride. Here, we describe the nucleotide sequence and functional analysis of two genes transcribed from this promoter, gadC and gadB. GadC is homologous to putative glutamate-γ-aminobutyrate antiporters of Escherichia coli and Shigella flexneri and contains 12 putative membrane-spanning domains. GadB shows similarity to glutamate decarboxylases. A L. lactis gadB mutant and a strain that is unable to express both gadB and gadC was more sensitive to low pH than the wild type when NaCl and glutamate were present. Expression of gadCB in L. lactis in the presence of chloride was increased when the culture pH was allowed to decrease to low levels by omitting buffer from the medium, while glutamate also stimulated gadCB expression. Apparently, these genes encode a glutamate-dependent acid resistance mechanism of L. lactis that is optimally active under conditions in which it is needed to maintain viability. Immediately upstream of the chloride-dependent gadCB promoter Pgad, a third gene encodes a protein (GadR) that is homologous to the activator Rgg from Streptococcus gordonii. gadR expression is chloride and glutamate independent. A gadR mutant did not produce the 3 kb gadCB mRNA that is found in wild-type cells in the presence of NaCl, indicating that GadR is an activator of the gadCB operon.

Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections

Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot immunoassay (DBI) was compared to a commercially available DEN antigen detection kit (denKEY Blue kit; Globio Co., Beverly, Mass.) and a reverse transcription-PCR (RT-PCR) kit. Serial serum or plasma samples (n = 181) obtained from 55 acute DEN-infected patients were used. In samples obtained from 32 of these 55 DEN-infected patients, viral RNA could be detected by RT-PCR. DEN antigen was detected in only 10 of these 55 patient samples by using the denKEY kit. When these samples were treated with acid to release the immune-complex-associated NS-1 antigen for detection by DBI, 43 of these 55 patients were found to be positive for DEN NS-1 antigen. In nondiss

Ciliary dyslexia candidate genes DYX1C1 and DCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

DYX1C1, DCDC2, and KIAA0319 are three of the most replicated dyslexia candidate genes (DCGs). Recently, these DCGs were implicated in functions at the cilium. Here, we investigate the regulation of these DCGs by Regulatory Factor X transcription factors (RFX TFs), a gene family known for transcriptionally regulating ciliary genes. We identify conserved X-box motifs in the promoter regions of DYX1C1, DCDC2, and KIAA0319 and demonstrate their functionality, as well as the ability to recruit RFX TFs using reporter gene and electrophoretic mobility shift assays. Furthermore, we uncover a complex regulation pattern between RFX1, RFX2, and RFX3 and their significant effect on modifying the endogenous expression of DYX1C1 and DCDC2 in a human retinal pigmented epithelial cell line immortalized with hTERT (hTERT-RPE1). In addition, induction of ciliogenesis increases the expression of RFX TFs and DCGs. At the protein level, we show that endogenous DYX1C1 localizes to the base of the cilium, whereas DCDC2 localizes along the entire axoneme of the cilium, thereby validating earlier localization studies using overexpression models. Our results corroborate the emerging role of DCGs in ciliary function and characterize functional noncoding elements, X-box promoter motifs, in DCG promoter regions, which thus can be targeted for mutation screening in dyslexia and ciliopathies associated with these genes.Peer reviewe

Increased Expression of the Dyslexia Candidate Gene DCDC2 Affects Length and Signaling of Primary Cilia in Neurons

Peer reviewe

SQL-1, homologue of the Golgi protein GMAP210, modulates intraflagellar transport in C. elegans

Primary cilia are microtubule-based organelles that have important sensory functions. For their function, cilia rely on the delivery of specific proteins, both by intracellular trafficking and intraflagellar transport (IFT). In the cilia of Caenorhabditis elegans, anterograde IFT is mediated by kinesin-II and OSM-3. Previously, we have shown that expression of a dominant active G protein a subunit (GPA- 3QL) in amphid channel neurons affects the coordination of kinesin-II and OSM-3 and also affects cilia length, suggesting that environmental signals can modulate these processes. Here, we show that loss-of-function of sql-1 (suppressor of gpa-3QL 1), which encodes the homologue of the mammalian Golgi protein GMAP210, suppresses the gpa-3QL cilia length phenotype. SQL-1 localizes to the Golgi apparatus, where it contributes to maintaining Golgi organization. Loss of sql-1 by itself does not affect cilia length, whereas overexpression of sql-1 results in longer cilia. Using live imaging of fluorescently tagged IFT proteins, we show that in sql-1 mutants OSM-3 moves faster, kinesin-II moves slower and that some complex A and B proteins move at an intermediate velocity, while others move at the same velocity as OSM-3. This indicates that mutation of sql-1 destabilizes the IFT complex. Finally, we show that simultaneous inactivation of sql-1 and activation of gpa-3QL affects the velocity of OSM-3. In summary, we show that in C. elegans the Golgin protein SQL-1 plays an important role in maintaining the stability of the IFT complex

Lifespan decrease in a Caenorhabditis elegans mutant lacking TRX-1, a thioredoxin expressed in ASJ sensory neurons

22 páginas, 4 figuras.Thioredoxins are a class of small proteins that play a key role in regulating many cellular redox processes. We report here the characterization of the first member of the thioredoxin family in metazoans that is mainly associated with neurons. The Caenorhabditis elegans gene B0228.5 encodes a thioredoxin (TRX-1) that is expressed in ASJ ciliated sensory neurons, and to some extent also in the posterior-most intestinal cells. TRX-1 is active at reducing protein disulfides in the presence of a heterologous thioredoxin reductase. A mutant worm strain carrying a null allele of the trx-1 gene displays a reproducible decrease in both mean and maximum lifespan when compared to wild-type. The identification and characterization of TRX-1 paves the way to use C. elegans as an in vivo model to study the role of thioredoxins in lifespan and nervous system physiology and pathology.This work was supported by a Ramón y Cajal contract from the Spanish Ministerio de Educación y Ciencia to A.M.-V., by grants from the Swedish Research Council and from the Swedish Foundation for Strategic Research (SSF) to P.S. and by a fellowship from the Austrian Academy of Sciences to G.G. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR), and by the C. elegans Community Gene Knock-Out Consortium.Peer reviewe

Antagonistic sensory cues generate gustatory plasticity in Caenorhabditis elegans

Caenorhabditis elegans shows chemoattraction to 0.1–200 mM NaCl, avoidance of higher NaCl concentrations, and avoidance of otherwise attractive NaCl concentrations after prolonged exposure to NaCl (gustatory plasticity). Previous studies have shown that the ASE and ASH sensory neurons primarily mediate attraction and avoidance of NaCl, respectively. Here we show that balances between at least four sensory cell types, ASE, ASI, ASH, ADF and perhaps ADL, modulate the response to NaCl. Our results suggest that two NaCl-attraction signalling pathways exist, one of which uses Ca(2+)/cGMP signalling. In addition, we provide evidence that attraction to NaCl is antagonised by G-protein signalling in the ASH neurons, which is desensitised by the G-protein-coupled receptor kinase GRK-2. Finally, the response to NaCl is modulated by G-protein signalling in the ASI and ADF neurons, a second G-protein pathway in ASH and cGMP signalling in neurons exposed to the body fluid