Simon Tuck

Umea University; Umea, Sweden

Simon Tuck et al. (2002) European Worm Meeting "A positive role for lin-1 in C. elegans vulval development?"

Simon Tuck, Eva Jonsson

[European Worm Meeting, 2002]

Cells in the vulval equivalence group in the C. elegans hermaphrodite can adopt one of three different fates, 1o, 2o or 3o. The fates of these 6 cells, termed collectively the VPCs, are thought to be influenced by at least three different signalling events. A cell in the somatic gonad, the anchor cell induces P6.p to adopt the 1o cell fate. Lateral signalling between adjacent VPCs results in P5.p and P7.p adopting the 2o fate. The remaining cells, P3.p, P4.p and P8.p, adopt the 3o fate, it is thought because they do not receive sufficiently high levels of either the inductive or lateral signals to overcome an inhibitory signal from the neighbouring syncytial hypodermal cell hyp7. Signalling between the anchor cell and P6.p is mediated by a signal transduction pathway involving let-60 Ras and mpk-1 MAP kinase. Previous work has shown that one likely target of this pathway is the ETS domain transcription factor LIN-1. Loss-of-function mutations in lin-1 cause the Multivulva phenotype: P3.p, P4.p and P8.p are induced to adopt a vulval (1o or 2o) fate. Since the Multivulva phenotype caused by lin-1(lf) is epistatic to the Vulvaless phenotype caused by loss-of-function mutations in mpk-1 MAP kinase, and since MPK-1 can directly phosphorylate LIN-1 in vitro, the current model is that the Ras/MAP kinase pathway mediates the induction of the 1o fate in part by phosphorylating and inactivating LIN-1, a negative regulator of this fate. One molecular marker for the 1o fate is an FGF-family ligand EGL-17. GFP under the control of egl-17 promoter sequences is expressed in P6.p and this expression is dependent upon let-60 Ras. Activation of let-60 in other VPCs causes ectopic expression of the reporter. Since let-60 Ras appears to function in part by inactivating LIN-1, if lin-1 regulated egl-17, then a reasonable expectation is that inactivation of lin-1 might, like activation of let-60, cause increased expression of the EGL-17::GFP reporter. We have found, however, that lin-1 null mutations have the converse effect: they abolish egl-17::gfp expression in P6.p. Thus lin-1 is required positively for the expression of at least one marker for the 1o fate. We reasoned that one possible explanation for this observation might be that loss of lin-1 function might cause constitutive activation of the lateral signalling pathway which promotes the 2o fate. Consistent with a role for lateral signalling in negatively regulating egl-17::gfp expression in P6.p, expression of the marker was consistently higher than in wild type in worms homozygous for n676n930, a hypomorphic mutation in lin-12, which encodes the receptor for this pathway. However, the marker was not expressed in lin-12(n676n930); lin-1(0) double mutant hermaphrodites. lin-1(0) also suppressed the ectopic expression of egl-17::gfp caused by constitutive activation of let-60 Ras. Together, these observations suggest that the role for LIN-1 in the induction of vulval fates might be more complicated than previously thought. By comparing the sequence of the C. elegans egl-17 promoter fragment with the analogous sequence from C. briggsae, we have identified 3 elements that show strong sequence conservation between the two species. We are presently assaying these and other fragments for their ability to direct expression of gfp in P6.p, and for their ability to be bound and regulated by LIN-1. We are also examining the effect of lin-1 mutations on other markers for the 1o fate such as downregulation of LIN-12::GFP, and downregulation of lip-1::gfp.

Simon Tuck et al. (2003) International Worm Meeting "A positive role for lin-1 during VPC fate specification."

Simon Tuck, Teresa Tiensuu, Emma Vernersson

[International Worm Meeting, 2003]

During vulval development, P6.p is induced to adopt the 1o cell fate by a cell in the somatic gonad, the anchor cell. This signaling event is mediated by a receptor-type tyrosine kinase, LET-23 that functions by activating a conserved Ras/MAP kinase signal transduction pathway. Previous work has suggested that the pathway functions in part by phosphorylating and inactivating LIN-1, an ETS protein 1, 2. lin-1 loss of function mutations cause a Multivulva phenotype similar to that caused by constitutive activation of let-60 ras. We have obtained evidence that the role of lin-1 in the induction of the 1o fate may be more complicated than previously thought. Specifically we find that, contrary to expectation, lin-1 is required positively for the expression of several markers for the 1o fate. For example whereas in WT, Ras/MAP kinase signaling induces the expression of egl-17::gfp in P6.p, expression of the marker is abolished in lin-1(0). The requirement for lin-1 for expression of the marker is downstream of or in parallel to let-60 ras since lin-1(0) let-60(gf) and lin-1(0); lin-15(0) double mutants fail to express the marker in P6.p. Furthermore, the effect of lin-1 on egl-17::gfp expression appears not to be caused by inappropriate activation or expression of lin-12, lip-1 or mab-5, three genes that each can inhibit the 1o vulval cell fate. Analysis of egl-17::gfp expression in the background of lin-1 mutations that affect a MAP kinase docking site in the protein suggest that LIN-1 might require modification by MAPK-1 Map kinase in order to function positively. A positive role for lin-1 in the expression of markers for the 1o fate is consistent with the SynVul phenotype seen in lin-1 double mutants with eor-1 or eor-23. In the regulation of egl-17::gfp lin-1 appears to function independently of lin-31(0), another target of the pathway, since expression is not affected in lin-31(0) but is abolished in lin31(0); lin-1(0) double mutants. In order to investigate lin-1's positive role further we have identified conserved elements in the egl-17 gene promoter that mediate induction of expression in P6.p. We are presently testing for binding to these elements. We are also carrying out experiments to determine the focus of lin-1 with respect to the expression of markers for the 1o fate. (1) Beitel, G. J., S. Tuck, I. Greenwald, and H. R. Horvitz (1995). Genes & Dev. 9: 3149-3162; (2) Jacobs, D., G. J. Beitel, S. G. Clark, H. R. Horvitz, and K. Kornfeld (1998). Genetics 149: 1809-1822; (3) Horward, R. and M. V. Sundaram (2002). Genes & Dev. 16: 1815-1827.

Simon P Tuck et al. (2005) International Worm Meeting "LIN-1 has both positive and negative functions in specifying multiple cell fates in C. elegans induced by Ras/MAP kinase signaling."

Morten Krog Larsen, Teresa Tiensuu, Emma Vernersson, Simon P Tuck

[International Worm Meeting, 2005]

lin-1 encodes an ETS domain transcription factor that functions downstream of a Ras/MAP kinase pathway that mediates induction of the 1o cell fate during vulval development in the C. elegans hermaphrodite. Mutants lacking lin-1 activity display a phenotype similar to that caused by mutations that constitutively activate let-60 Ras consistent with a model in which lin-1 is a repressor of the 1o fate whose activity is inhibited by phosphorylation by MPK-1 MAP kinase. Here we show that, contrary to the current model, lin-1 is required positively for the proper expression of several genes regulated by the pathway in cells adopting the 1o cell fate. We show that the positive requirement for lin-1 is downstream of let-60 Ras and mpk-1 MAP kinase, and that it appears to have a focus in P6.p. lin-1 alleles encoding proteins lacking a docking site for MPK-1 MAP kinase are defective in the positive funtion. We also show that lin-1 apparently has both positive and negative functions during the specification of the fates of other cells in the worm that require Ras/MAP kinase signaling.

Matthews LR et al. (1994) Worm Breeder's Gazette "A Potential C. elegans E2F Homolog: Is it zyg-9?"

Galas S, Matthews LR

[Worm Breeder's Gazette, 1994]

A potential C. elegans E2F homolog: Is it zyg-9? Lisa Matthews and Simon Galas, CRBM, CNRS Montpellier, France

Berger S et al. (2018) Lab Chip "Correction: Long-term C. elegans immobilization enables high resolution developmental studies in vivo."

Aegerter-Wilmsen T, DeMello A, Berger S, Casadevall I Solvas X, Hengartner M, Hajnal A, Lattmann E

[Lab Chip, 2018]

Correction for 'Long-term C. elegans immobilization enables high resolution developmental studies in vivo' by Simon Berger et al., Lab Chip, 2018, 18, 1359-1368.

Rahmani, Shapour et al. (2021) MicroPubl Biol

Tuck, Simon, Rahmani, Shapour

[MicroPubl Biol, 2021]

C. elegans males that have come into close proximity of hermaphrodites initiate copulatory behavior comprising at least five different steps termed response, turning, location of vulva, spicule insertion and sperm transfer (Loer and Kenyon 1993, Liu and Sternberg 1995, Chute and Srinivasan 2014). Mutations specifically affecting different steps have been isolated and characterized (Barr and Sternberg 1999, Hajdu-Cronin et al. 2017, Liu et al. 2017). However, our understanding of the molecular mechanisms acting in the neurons controlling copulation is far from complete. During the response step, males that have sensed the presence of a hermaphrodite move backwards in such a way that the males tail fan glides along the surface of the hermaphrodite until the tail reaches the vulva (or head or tail) (Loer and Kenyon 1993, Liu and Sternberg 1995, Sherlekar and Lints 2014). Response behavior is regulated by ciliated neurons in the tail whose dendrites lie in sensory rays within the fan (Liu and Sternberg 1995). If a male reaches the end of the hermaphrodite without having found the vulva, it executes a turn during which the tail bends tightly ventrally so that contact is established between the ventral surface of the fan and the other side of the intended mate (Loer and Kenyon 1993, Liu and Sternberg 1995). The ability to execute turns efficiently is dependent upon serotonergic neurons in the posterior ventral nerve cord (the CP neurons) and on their ability to produce serotonin (Loer and Kenyon 1993, Carnell et al. 2005). Serotonin stimulates the diagonal muscles in the tail to induce curling ventrally by stimulating a serotonin receptor, SER-1 (Loer and Kenyon 1993, Carnell et al. 2005). However, how serotonin affects diagonal muscles and ventral turning is not fully understood.

Rahmani, Shapour et al. (2021) International Worm Meeting "Investigating the function of TAT proteins in lipid transport within ciliated neurons."

Rahmani, Shapour, Tuck, Simon

[International Worm Meeting, 2021]

Cilia have protein and lipid compositions that are distinct from that of the cells from which they protrude. Although a clear picture exists of how protein transport occurs in cilia, the mechanisms regulating lipid composition are not presently understood. Furthermore, much remains to be learned about how changes in lipid distribution affect cilium function. The major phospholipids in plasma membranes are phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylchoine (PC) and phosphatidylinositol (PI). These phospholipids are not symmetrically distributed in the leaflets that make up membrane bilayers: in particular, PC is largely confined to the outer (exofacial) leaflet while PE, PS and PI are mostly confined to the inner. These asymmetries are maintained by the movement of phospholipids through the cell membranes catalyzed by transamphipathic aminophospholipid translocases belonging to the P4 family of transmembrane ATPases. C. elegans has six translocase genes, tat-1 - tat-6. We have found that tat-6 is specifically expressed in a subset of C. elegans ciliated neurons and that TAT-6 protein accumulates in cilia. To study the function of TAT-6 and that other translocases in lipid transport in C. elegans ciliated neurons, we developed a technique to allow labelling of cilia with lipids. For the first time in C. elegans ciliary research we used fusogenic liposomes to deliver fluorescently labelled lipids to the membranes of ciliated neurons. We have used this technique to study the roles of all the TAT proteins in this organism in maintaining the distinct lipid composition and lipid asymmetry in ciliary membranes. Our experiments revealed that TAT-5 and TAT-1 translocase activities primarily promote the transport of phosphatidylethanolamine (PE) and phosphatidylserine (PS) respectively whereas TAT-6 has an overlapping function in transporting both PS and PE. Mutants lacking pad-1, which encodes a cofactor for TAT-5, also show defects in PE transport. tat-5 and tat-6 mutants display defects in behaviours mediated by ciliated neurons. Overall, our investigation indicates that the regulation of lipid asymmetry and phospholipid transport is required for cilia to function properly in C. elegans.

BC12750

Caenorhabditis elegans

Nilsson, Lars et al. (2021) MicroPubl Biol

Tuck, Simon, Rahmani, Shapour, Nilsson, Lars

[MicroPubl Biol, 2021]

In healthy eukaryotic cells, the two leaflets that make up plasma membranes are highly asymmetric with respect to the lipids they contain. In both unicellular eukaryotes and metazoans, the asymmetry in the distribution of aminophospholipids is maintained by P4-family transmembrane ATPases, which catalyze the movement of selected phospholipids from the outer leaflet to the inner. C. elegans has six P4-family ATPases, TAT-1 - TAT-6. TAT-1 - TAT-5 are expressed in many tissues and cells. Here we report that, in contrast, TAT-6 is much less broadly expressed and that, within the somatic gonad, expression of TAT-6 reporters is restricted to the spermathecal-uterine core cell (sujc) cells.