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

Phosporylation of androgen receptor isoforms

Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser-Ala substitution mutant S650A (Ser-650-->Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed

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

Dauer pheromone and G-protein signaling modulate the coordination of intraflagellar transport kinesin motor proteins in C. elegans

Cilia length and function are dynamically regulated by modulation of intraflagellar transport (IFT). The cilia of C. elegans amphid channel neurons provide an excellent model to study this process, since they use two different kinesins for anterograde transport: kinesin-II and OSM-3 kinesin together in the cilia middle segments, but only OSM-3 in the distal segments. To address whether sensory signaling modulates the coordination of the kinesins, we studied IFT protein motility in gpa-3 mutant animals, since dominant active mutation of this sensory Gα protein GPA-3QL) affects cilia length. In addition, we examined animals exposed to dauer pheromone, since dauer formation, which involves gpa-3, induces changes in cilia morphology. Live imaging of fluorescently tagged IFT proteins showed that in gpa-3 mutants and in larvae exposed to dauer pheromone, kinesin-II speed is decreased and OSM-3 speed is increased, whereas structural IFT proteins move at an intermediate speed. These results indicate that mutation of gpa-3 and exposure to dauer pheromone partially uncouple the two kinesins. We propose a model in which GPA-3-regulated docking of kinesin-II and/or OSM-3 determines entry of IFT particles into the cilia subdomains, allowing structural and functional plasticity of cilia in response to environmental cues