Suzanne Rademakers

Erasmus Medical Centre; Rotterdam, Netherlands

Rademakers, Suzanne et al. (2021) International Worm Meeting "Identifying novel interactors of the guanylate cyclase GCY-22 involved in NaCl chemotaxis"

Rademakers, Suzanne, Jansen, Gert

[International Worm Meeting, 2021]

C. elegans senses salts in their environment using the ASE neurons. Detection of salts occurs in sensory organelles, called primary cilia. cGMP signalling plays an important role in salt detection. The receptor-type guanylate cyclase (rGC) GCY-22 is involved in the response to NaCl in the environment. We generated a full-length GFP knock-in the gcy-22 gene. GCY-22::GFP shows unique localization to the ciliary tip and periciliary membrane compartment (PCMC) of one ciliated neuron, ASER. Our goal is to understand the molecular mechanisms that regulate its trafficking and unique localization. To identify proteins that physically interact with GCY-22, we performed mass spectrometry after immunoprecipitation to identify proteins bound to GFP-tagged GCY-22. Next, we study where the identified candidate interacting proteins are expressed and localized. Mutants are used to investigate their role in salt detection, GCY-22::GFP trafficking towards the cilium and localization to the ciliary tip. The most prominent candidate interacting protein is GCY-19. GCY-19::GFP is expressed in ASER and colocalized with GCY-22. Loss-of-function of gcy-19 resulted in lower levels of GCY-22::GFP at the ciliary tip. Similarly, gcy-22 loss-of-function animals showed lower levels of GCY-19::GFP at the tip of the ASER cilium . We also found GCY-4 and GCY-5 as possible interactors of GCY-22. As rGCs are thought to act as dimers, these findings suggests that GCY-22 might be a common subunit for heterodimeric complexes possibly to achieve ion-selectivity. In addition, we identified DAF-25 in our GCY-22::GFP IP-MS experiments. DAF-25 is the ortholog of the mammalian ankyrin repeat and Mynd domain containing protein Ankmy2. DAF-25 has been reported previously to be important for rGC transport. Mutants lacking DAF-25 show no ciliary tip localization of GCY-22::GFP and do not respond to NaCl. Other candidate genes are currently being investigated. This work will allow us to gain insight in the molecular mechanisms that regulate ciliary tip localization of GCY-22.

Rademakers, Suzanne et al. (2011) International Worm Meeting "The GMAP210 homologue SQL-1 modulates Intraflagellar Transport in C. elegans."

Dekkers, Martijn, Broekhuis, Joost, Rademakers, Suzanne, Burghoorn, Jan, Jansen, Gert

[International Worm Meeting, 2011]

The cilia of C. elegans' amphid channel neurons can be divided into a middle and distal segment. Anterograde intraflagellar transport (IFT) in these cilia is mediated by two kinesin-2 complexes, kinesin II and OSM-3. In the middle segment OSM-3 and kinesin II move together at a speed of 0.7 mm/s, and in the distal segments OSM-3 moves alone at 1.2 mm/s. In the absence of osm-3 kinesin II moves alone at 0.5 mm/s. The architecture of C. elegans' cilia suggests that cilia length and function can be dynamically regulated. We have previously shown that the expression of a dominant active G protein a subunit (GPA-3QL) in amphid channel neurons affects the coordination of OSM-3 and kinesin-2 and cilia morphology, resulting in a dye filling defect. We performed a genetic screen to identify mutants that suppress the gpa-3QL Dyf phenotype, and identified sql-1, which encodes the homologue of the mammalian Golgi protein GMAP-210. Using immunofluorescence we showed that SQL-1 also localizes to the Golgi in C. elegans. In sql-1(lf) animals we see no effects on cilia morphology, and also the Golgi appears normal. Overexpression of SQL-1 results in longer cilia. Speed measurements showed that in the middle segment of sql-1(lf) animals OSM-3 moves faster (0,85 mm/s) and kinesin II moves slower (0,6 mm/s), suggesting that the two kinesins are at least partially uncoupled. Both complex A and B proteins move at the same speed as OSM-3. This suggests that in sql-1(lf) animals IFT is predominantly mediated by OSM-3 kinesin. In the middle segment of gpa-3QL; sql-1 double mutants we observed similar velocities as in sql-1 single mutants, suggesting that in the middle segment the sql-1 mutation is epistatic to gpa-3QL, which is in line with the suppression of gpa-3QL. Surprisingly, in the distal segment of gpa-3QL; sql-1 double mutants OSM-3::GFP speed is significantly reduced. We are currently investigating if triple mutants can help us explain this result.

Suzanne Rademakers et al. (2009) European Worm Neurobiology Meeting "Dauer pheromone and G protein signaling regulate the coordination of intraflagellar transport motors."

Joost Broekhuis, Suzanne Rademakers, Martijn Dekkers, Gert Jansen, Jan Burghoorn

[European Worm Neurobiology Meeting, 2009]

Recent studies suggest that the structure of cilia and the sensory signaling machinery in the cilia is regulated by intraflagellar transport (IFT). In C. elegans this regulation may be achieved by a structural compartmentalization of the cilia in middle and distal segments and the coordination of two kinesin-2 motor complexes used for anterograde IFT: kinesin II and OSM-3. The kinesins are loaded with complex A and B particle proteins and cargo molecules. Both kinesins enter the cilia middle segments and move together, but only OSM-3 enters the distal segments. We study gpa-3 mutant animals, since dominant active mutation of this sensory G. protein (gpa-3QL) affects cilia length. Using EM and cilium specific GFP markers we show that in gpa-3QL animals the length of a subset of amphid cilia is reduced and that they are sometimes posteriorly displaced. Interestingly, IFT proteins are located normally in the cilia, but IFT is perturbed in both gpa-3(lf) and gpa-3QL mutants: kinesin II speed is reduced and OSM-3 speed is increased in the middle segments of the cilia. However, complex A/B speed is approximately wild type. We propose that GPA-3 can modulate the coordination of IFT in response to an environmental cue. Since GPA-3 plays a role in dauer development and since dauer formation results in changes in cilia structure we tested whether exposure to dauer pheromone affects IFT. Interestingly, exposure to dauer pheromone affected kinesin II and OSM-3 speeds, similar to mutation of gpa-3. The results indicate that mutation of gpa-3 and exposure to dauer pheromone partially uncouples the two kinesins, and suggests the presence of three types of IFT particles: those transported by OSM-3, those transported by kinesin II, and a subset transported by both kinesins. We propose a model in which GPA-3 regulated docking of either kinesin II, OSM-3 or both, determines entry of IFT particles into the cilia subdomains, allowing plasticity of cilia structure and function in response to environmental cues.

NL2105

Caenorhabditis elegans

Krpelanova, Eva et al. (2009) International Worm Meeting "Functional Analysis of Sp Transcription Factors in Caenorhabditis elegans ."

Jansen, Gert, Philipsen, Sjaak, Krpelanova, Eva, Rademakers, Suzanne

[International Worm Meeting, 2009]

Sp (specificity protein) transcription factors are important regulators of many cellular processes, such as cell cycle, metabolism and morphogenesis. The Sp family is united by a specific combination of three conserved Cys2His2 zinc fingers that form the DNA-binding domain and a Buttonhead (BTD) box CXCPXC, just N-terminal to the zinc fingers. There are nine Sp genes both in humans and in mice. C. elegans has three Sp-related transcription factors: sptf-1, sptf-2 and sptf-3. Our aim is to characterize the functions of these three transcription factors and to find some of their interacting partners. Therefore we have made GFP fusion constructs to determine their expression patterns and we set out to analyze the phenotypes of knockout, knockdown and transgenic animals carrying additional copies of a particular sptf-gene. The gene structure of spft-1 has been confirmed previously. sptf-1::GFP expression is first observed in late embryos; from L1 to adult GFP is expressed in the intestine, rectum, 3 neurons in the head and from L3 onwards in the vulva. sptf-1(tm784) knockout animals are homozygous viable, but with a low rate of survival until adulthood. We rescued the phenotype in knockout animals by injecting a low dose of sptf-1. RNAi experiments show no obvious phenotype. High level of overexpression of sptf-1, sptf-1XS, caused effects on movement and body morphology. sptf-2 gene structure has been confirmed. sptf-2 is expressed post embryonically in the intestine, body muscles, and two neurons in the head. sptf-2(tm1130) knockouts are viable, no obvious phenotype was observed. RNAi experiments show no obvious phenotype. High levels of sptf-2 overexpression is probably lethal since F1 sptf-2XS animals did not produce viable transgenic progeny. We have identified two splice forms of sptf-3, one confirms the prediction, the second splice variant has three additional upstream exons. sptf-3::GFP is expressed ubiquitously in early embryos and at the comma stage; from late embryos to adults GFP is restricted to the intestine, neurons of the ventral and dorsal nerve cords as well as two neurons in the head close to the posterior pharyngeal bulb. sptf-3(tm607) knockout animals are lethal, 20% embryonically, 77% as L1, 3% as L2. RNAi screens of sptf-3 show a spectrum of mutant phenotypes, comprising embryonic lethality, maternal sterility, abnormal both body morphology and locomotory behaviour. We have observed growth defects in sptf-3XS transgenic animals. Currently we are setting up a screen to identify suppressors of sptf-induced lethality.

van der Vaart, Aniek et al. (2015) International Worm Meeting "DLK-1/p38 MAP Kinase signaling controls cilium length by regulating RAB-5 mediated endocytosis."

Rademakers, Suzanne, Jansen, Gert, van der Vaart, Aniek

[International Worm Meeting, 2015]

Many cells in the vertebrate body use specialized sensory organelles to sense particular cues in their environment: primary cilia. Proteins that function in cilia are transported into and within cilia by a specialized transport machinery, called intraflagellar transport, IFT. The amphid channel cilia of C. elegans are a good model to study the regulation of cilium length and intraflagellar transport (IFT), since they consist of two segments where different kinesin complexes are used for IFT. Previously, we have shown that coordination of the kinesins is regulated by exposure to dauer pheromone and the heterotrimeric G protein alpha-subunit GPA-3. Furthermore, animals that carry a dominant active mutant gpa-3 (gpa-3QL) have shorter cilia.To find out how G protein signalling modulates cilium length, we screened for suppressors of the short cilium phenotype of gpa-3QL animals. This screen identified three components of the DLK/p38 MAP kinase pathway, the MAP3K dlk-1, the p38 MAPK pmk-3 and the ubiquitin-conjugating enzyme variant uev-3. Previous studies have shown that this pathway is important for axon development and regrowth and synapse formation. We show that DLK-1, PMK-3 and UEV-3 localize in cilia or at their base. Surprisingly, mutations in all three genes affect IFT, but these changes do not explain the suppression of the gpa-3QL induced cilium length defect.p38 MAP kinase has been shown to regulate endocytosis. Interestingly, endocytosis at the base of the cilium is important for proper cilium function. We found that blocking endocytosis by mutating rabx-5 or rme-6, GEFs for RAB-5, or chc-1, the clathrin heavy chain, suppressed the gpa-3QL induced short cilium phenotype. In addition, we found GFP::RAB-5 accumulation at the base of the cilium in pmk-3 and pmk-3; gpa-3QL animals. In contrast, gpa-3QL animals showed relatively low levels of GFP::RAB-5.Our results identify a new role for the DLK/p38 MAP kinase pathway in the control of cilium length. We propose that environmental signals mediated by GPA-3 and the DLK/MAPK pathway can modulate cilium function by regulating RAB-5 mediated endocytosis.

Broekhuis, Joost et al. (2009) International Worm Meeting "G protein signaling and the GMAP210 homologue SQL-1 modulate Intraflagellar Transport in C. elegans."

Broekhuis, Joost, Burghoorn, Jan, Dekkers, Martijn, Jansen, Gert, Rademakers, Suzanne

[International Worm Meeting, 2009]

The cilia of C. elegans'' amphid channel neurons can be divided into a middle and distal segment. Anterograde intraflagellar transport (IFT) in these cilia is mediated by two kinesin-2 complexes, kinesin II and OSM-3. In the middle segment OSM-3 and kinesin II move together at a speed of 0.7 mm/s, and in the distal segments OSM-3 moves alone at 1.2 mm/s. In the absence of osm-3 kinesin II moves alone at 0.5 mm/s. The architecture of C. elegans'' cilia suggests that cilia length and function can be dynamically regulated. To investigate whether sensory signals can modulate cilia or IFT we examined the cilia of gpa-3 mutant animals. GPA3 is a sensory Ga protein that is expressed in all amphid neurons and involved in various sensory processes. Loss of gpa-3 (lf) does not affect cilia morphology, while a dominant active (gpa-3QL) mutation results in shortened cilia. In addition, we examined animals exposed to dauer pheromone, since previous studies have shown that dauer formation changes in the morphology of some cilia. Furthermore, mutations of gpa-3 affect dauer formation. We found that in both gpa-3 (lf) and gpa-3QL mutants, as well as in animals exposed to dauer pheromone, kinesin II and OSM-3 are at least partially uncoupled, while structural IFT particle proteins move at speeds intermediate to the two kinesins. This suggests that the cilia of gpa-3 mutant animals contain two, possibly three, types of IFT particles: particles transported by OSM-3 or kinesin II alone, and perhaps a small subset transported by both kinesins. We propose a model in which GPA-3 regulated docking of either kinesin II, OSM-3 or both, determines entry of IFT particles into the cilia subdomains. This mechanism would allow plasticity of cilia structure and function. We performed a genetic screen for suppressors of gpa-3QL and identified sql-1, which encodes the homologue of the mammalian Golgi protein GMAP-210. sql-1(lf) suppresses the effect of gpa-3QL on cilia length, but does not seem to affect cilia morphology by itself. Speed measurements showed that in the middle segment of sql-1(lf) animals OSM-3 moves faster (0,85 mm/s) and kinesin II moves slower (0,6 mm/s), suggesting that the two kinesins are at least partially uncoupled. However, both complex A and B proteins move at the same speed as OSM-3. This suggests that in sql-1(lf) animals IFT is predominantly mediated by OSM-3 kinesin.

UMUERRI, OLUWATOROTI O et al. (2009) International Worm Meeting "MOLECULAR MECHANISM OF SALT TASTE."

Dekkers, Martijn, Jansen, Gert, UMUERRI, OLUWATOROTI O, Rademakers, Suzanne, Hukema, Renate

[International Worm Meeting, 2009]

NaCl is essential for homeostasis and physiological functions in many organisms. However, the molecular mechanism of NaCl detection is not well known. In mammals, the epithelial Na+ channel (ENaC) and the transient receptor potential ion channel of the vanilloid type 1 (TRPV1) have been shown to be involved in NaCl detection. Previous studies in C. elegans identified five genes involved in NaCl chemoattraction. These are tax-2 and tax-4 (cyclic nucleotide gated (CNG) channel subunits), tax-6 and cnb-1 (calcineurin A and B subunits) and ncs-1 (neuronal calcium sensor). Analysis of these mutants in our assay, in which we exposed the animals to a very steep NaCl gradient, showed reduced chemotaxis to NaCl. However we found that these mutants still showed significant attraction at higher NaCl concentrations. By analyzing the behaviour of double mutants, we found that chemotaxis to NaCl involves two genetic pathways. The first pathway involves two mitogen activated protein (MAP) kinases, nsy-1 and sek-1, and three genes that have been previously characterized, tax-2, tax-4 and tax-6. The second pathway involves tax-2, another CNG channel subunit, cng-3, the Ga protein odr-3, the TRPV channel subunit osm-9 and the guanylate cyclase gcy-35. We used cell specific rescue of the mutant genes, laser ablation of specific neurons and neuronal calcium imaging to find out where in the neuronal circuit of C. elegans these genes function. Thus far, the involvement of the main salt sensing neurons, ASE, has been confirmed. In addition, we found that the ADF neurons also play a role. We are currently performing a synthetic genetic screen to identify additional genes that play a role in NaCl chemotaxis. We are using odr-3 mutants to find mutants that affect the nsy-1/sek-1/tax-2/tax-4/tax-6 NaCl chemotaxis pathway.

van der Burght, Servaas et al. (2019) International Worm Meeting "Formation of a salt-sensing compartment at the tip of cilia."

Rademakers, Suzanne, van der Burght, Servaas, Li, Chunmei, Johnson, Jacque-Lynne, Jansen, Gert, Leroux, Michel

[International Worm Meeting, 2019]

Cells and organisms detect cues in their environment using sensory organelles, called primary cilia, in which signaling proteins exhibit specific localizations. The mechanisms that determine their localization within cilia, and the significance of these localizations, are largely unknown. We identify a unique ciliary localization for the receptor-type guanylate cyclase GCY-22, which plays a role in chemotaxis to NaCl, and unravel the mechanisms that control its ciliary localization. We generated a GFP knock-in of GCY-22 and discovered its prominent localization to the ciliary tip, and periciliary membrane compartment (PCMC) of one ciliated neuron, ASER. FRAP experiments showed that these two pools of GCY-22 are very stable but exhibit motility within the respective subcellular domains. Quantification experiments suggest the presence of more than 10,000 GCY-22::GFP molecules within the ciliary tip. To determine how GCY-22::GFP is transported to its ciliary destinations, we used time-lapse microscopy. We observed vesicular transport in the dendrite and particles coinciding with intraflagellar transport (IFT) in the cilium. Dual-color imaging suggested that IFT partially maintains GCY-22::GFP tip localization by targeting the protein to the tip. This localization is altered upon disruption of either BBS-8, an IFT cargo adaptor protein or OSM-3, a specialized IFT anterograde motor. Furthermore, we observed an abrupt collapse of the GCY-22::GFP ciliary tip compartment after treatment with sodium azide, indicating that an ATP-dependent process maintains this ciliary tip localization. Additionally, we investigated which proteins are involved in trafficking of GCY-22::GFP to the cilium. We found that GCY-22::GFP does not enter the cilium in a mutant lacking DAF-25, the ortholog of the poorly studied mammalian ankyrin repeat and MYND domain-containing protein Ankmy2. Interestingly, the daf-25 mutant animals do not respond to NaCl, but the localization and behavioral phenotype could be rescued by disrupting the transition zone protein MKS-5, a core component of a ciliary gate. Finally, GCY-22::GFP deletion and chimeric constructs showed that the C-terminal RD3-associated and dimerization domains are important for ciliary tip localization and that loss of tip localization results in reduced chemotaxis to lower NaCl concentrations. Our findings reveal that a cGMP-producing signaling protein functions within a distinct ciliary domain and that it uses several protein transport machineries to reach and maintain its singular subcellular localization.