Kaech SM et al. (1995) International C. elegans Meeting "CELLULAR JUNCTIONS AND LATERAL SIGNALING IN VULVAL DEVELOPMENT"

Kim SK, Kaech SM

[International C. elegans Meeting, 1995]

The vulva is formed by a series of cell-cell interactions. A set of six equipotential cells, the Pn.p cells, are induced to express one of three different cell fates (1 degree, 2 degree, or 3 degree). The 1 degree cell fate is specified by an anchor cell signal that activates the let-23 receptor tyrosine kinase. The 2 degree cell fate is specified by a lateral signal between the Pn.p cells that activates the lin-12 receptor. We are addressing two different questions of cell-cell signaling in the hermaphrodite vulva. Firstly, we are investigating how LIN-2, LIN-7, and LIN-10 function during vulval induction. The homology of lin-2 and lin-7 with proteins that are localized to cell junctions has lead to our current model that these three proteins are involved in localizing the receptor LET-23 to cellular junctions in the Pn.p cells. We are using the yeast two-hybrid system to analyze if these proteins physically interact. We will also pursue other protein-protein interactions by screening a two hybrid library of C. elegans cDNAs. Secondly, we are investigating whether the gene lag-2 may encode the lateral signal that specifies the 2 degree cell fate in vulval development. Iag-2 is a homolog of apx-1 in worms, and of delta and serrate in Drosophila. LAG-2 is the putative ligand for GLP- 1 and LIN-12 in non-vulval cell signaling events, and its role in vulval development is unknown due to early larval lethality in lag-2 mutants. The lin-12 receptor is required for specification of the 2 degree cell fate in vulval development, leading to the possibility that LAG-2 might be the ligand for LIN- 12 in the vulva as well. We are using mosaic analysis to determine if lag-2 is required in the vulval precursor cells for proper vulval formation.

Kaech SM et al. (1998) West Coast Worm Meeting "BASOLATERAL LOCALIZATION OF LET-23 RTK BY A LIN-2/LIN-7/LIN-10 PROTEIN COMPLEX IN VULVAL PRECURSOR CELLS"

Kim SK, Whitfield CW, Kaech SM

[West Coast Worm Meeting, 1998]

The establishment and maintenance of epithelial cell polarity is crucial for polarized cell functions, such as specific signaling to the basolateral sides of epithelial cells. Polarized epithelial cells are separated into apical and basolateral membrane domains that contain different subsets of transmembrane proteins, causing these domains to be biochemically and functionally distinct. To understand how cell polarity is generated, an important goal is to elucidate the mechanisms that target and retain transmembrane proteins to distinct membrane compartments. The LET-23 receptor tyrosine kinase is localized to the basolateral membranes of polarized vulval epithelial cells. lin-2, lin-7 and lin-10 are required for basolateral localization of LET-23, since LET-23 is mislocalized to the apical membrane in lin-2, lin-7 and lin-10 mutants. Yeast two hybrid, in vitro binding and in vivo co- immunoprecipitation experiments show that LIN-2, LIN-7 and LIN-10 form a ternary complex. Furthermore, compensatory mutations in lin-7 and let-23 exhibit allele-specific suppression of apical mislocalization and signaling defective phenotypes. These results present a mechanism for basolateral localization of LET-23 RTK by direct binding to the LIN- 2/LIN-7/LIN-10 complex. Each of the binding interactions within this complex is conserved, suggesting that this complex may also mediate basolateral localization in mammals.

Kaech SM et al. (1997) International C. elegans Meeting "LIN-2, LIN-7 and LET-23 Protein Interactions Provide a Mechanism for LET-23 Localization during Vulval Development"

Kim SK, Kaech SM

[International C. elegans Meeting, 1997]

The vulval precursor cells (VPCs) are polarized epithelial cells that adopt vulval cell fates in response to a signal (LIN-3) released by the anchor cell. Cellular junctions maintain the polarized structure of the VPCs and presumably, inhibit the passage of LIN-3 between the VPCs. We found that the receptor for the anchor cell signal, LET-23, is localized to the basal surface of the VPCs and that this subcellular localization is dependent on the three gene products LIN-2, LIN-7, and LIN-10. In lin-2, lin-7 or lin-10 mutant animals, the receptor is found localized to the apical membrane compartment. Therefore, in lin-2, lin-7 and lin-10 mutants, the vulvaless phenotype results from the inability of the receptor in the apical membrane domain to bind to LIN-3 in the basal extracellular space. We are interested in the mechanism of LET-23 localization, and tested whether there might be direct protein interactions between LET-23, LIN-2 or LIN-7 using the yeast two-hybrid system and in vitro binding experiments. The combination of data from these two approaches suggest that LET-23 can bind LIN-7 and that LIN-7 can bind LIN-2. We were able to delineate the regions of these proteins involved in the binding. The N-terminus of LIN-7 (amino acids 1-125) binds to the PDZ domain of LIN-2, and the PDZ domain in the C-terminus of LIN-7 (amino acids 171-297) binds to the last 50 amino acids of LET-23. Currently, we are investigating the requirement of the above protein interactions during vulval development. Our general strategy is to find an allele-specific mutation in one binding site that can interact with a specific mutation in the other. These allele-specific mutations would then restore protein binding in vitro and suppress the vulvaless phenotype in vivo. Using the PDZ domain crystal structure, we have identified a mutation in LIN-7(H259V) that is unable to bind to LET-23 in vitro and is unable to rescue the vulvaless phenotype in lin-7 mutants. We have also found a compensatory mutation in LET-23 (TCLstop to YFIstop) that restores the protein-protein interaction in vitro, and we will be testing to see if these mutant forms restore vulval development in vivo. We are currently using similar tactics to analyze LIN-2 and LIN-7 binding during vulval induction in vivo. In summary, our biochemical data suggests a mechanism for LET-23 localization to the basal membrane domain of the VPCs through direct protein interactions with LIN-7 and LIN-2.

Kaech SM et al. (1996) West Coast Worm Meeting "PROTEIN INTERACTIONS BETWEEN LIN-2, LIN-7 AND THE RECEPTOR TYROSINE KINASE LET-23: A POSSIBLE MECHANISM FOR LOCALIZING LET-23 IN THE PN.P CELLS DURING VULVAL INDUCTION"

Kaech SM, Kim SK

[West Coast Worm Meeting, 1996]

The activity of LIN-2, LIN-7 and the receptor tyrosine kinase LET-23 are required for the vulval precursor cell P6.p to receive and transduce the anchor cell signal that induces the primary vulval cell fate. Mutations in either lin-2, lin-7 or let-23 prevent vulval induction and result in a vulvaless phenotype. The vulval precursor cells are polarized epithelial cells; their basal surface faces the anchor cell and their apical surface faces the cuticle. In wild-type animals, a LET-23::GFP fusion protein is found in a lateral ring that is co-localized with and basal to the vulval precursor cell junctions. LIN-2 and LIN-7 are necessary for subcellular localization of LET-23 (the putative receptor for the inductive ligand), since LET-23::GFP is mislocalized and distributed across the apical surface of the Pn.p cells in lin-2 and lin-7 mutants (Simske et al., (1996) Cell 85, 195-204). We are interested in the mechanism of LET-23 localization, and are testing whether there might be direct protein interactions between LET-23 and LIN-2 or LIN-7. LIN-2 is a member of a family of proteins called MAGUKs (for Membrane-Associated GUanylate Kinase) (Hoskins et al., (1996) Development 122, 97-111). MAGUKs have been found to specifically localize to cellular junctions or to regions of cell-cell contact. MAGUKs contain at least one PDZ domain, which are domains known to mediate protein-protein interactions. LIN-7 contains a PDZ domain, but does not contain other domains typically found in MAGUKs. To test if LIN-2, LIN-7 or LET-23 interact with each other, we used the yeast two-hybrid system and in vitro binding experiments. The combination of data from these two approaches suggest that LET-23 can bind LIN-7 and that LIN-7 can bind LIN-2. We did not detect any protein interactions between LIN-2 and LET-23. There are two distinct regions of LIN-7 that mediate the interactions with LIN-2 and LET-23. The N-terminus of LIN-7 (amino acids 1-125) binds a 200 amino acid region spanning the PDZ of LIN-2, and the PDZ domain in the C-terminus of LIN-7 (amino acids 171-297) binds to the intracellular region of LET-23. We have not yet delineated the region of intracellular LET-23 that binds to the PDZ of LIN-7. The above data suggest that LIN-2, LIN-7 and LET-23 form a ternary complex. To test this idea, we used a yeast "three" hybrid system. We were unable to detect an interaction between LIN-2 and the intracellular region of LET-23 in the yeast two hybrid system. However, when we co-expressed LIN-7 (tagged with an SV40 nuclear localization signal) in the presence of LIN-2 and LET-23 in the yeast two-hybrid system, it appeared that the three proteins were able to interact. These data suggest that LIN-2 binds LIN-7 and LIN-7 binds LET-23 to form a ternary complex. Experiments are in progress to test if these interactions are required for vulval development in vivo.

Whitfield CW et al. (1998) West Coast Worm Meeting "BASOLATERAL LOCALIZATION OF THE EGF RECEPTOR IN EPITHELIAL CELLS BY THE PDZ PROTEIN LIN-10"

Whitfield CW, Benard C, Barnes TM, Kim SK, Hekimi S

[West Coast Worm Meeting, 1998]

The EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. We show here that lin-10 acts in LET-23 RTK basolateral localization, as lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 (ref. 5) was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a homologue of mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains, and is present in descendants of the vulval precursor cells and in neurons. LIN-10 acts with LIN-2 and LIN-7 in an evolutionarily conserved protein complex that directly interacts with LET-23 (S. M. Kaech, J. -P. Borg, S. Butz, pers. comm.). LIN-10 is also involved in localization of a neurotransmitter receptor at post-synaptic elements (C. Rongo, pers. comm.). Thus, LIN-10 is part of a common mechanism for the localization of transmembrane receptors in epithelia and neurons in C. elegans, and this mechanism is likely to be conserved in mammals.

Cadena del Castillo, C. et al. (2019) International Worm Meeting "PTC-3 is a cholesterol permease which regulates membrane structure and fatty acid composition."

Riezman , H., Cadena del Castillo, C., Hannich, J.T., Spang, A., Faergeman, N.F., Kaech, A.

[International Worm Meeting, 2019]

The Hedgehog (Hh) signaling pathway is essential for development and organ physiology. In the canonical pathway, the morphogen Hedgehog binds to the membrane cholesterol permease PTCH conducing to a relief in the inhibition of Smoothened (SMO), which in turn will activate a signalling cascade, required for proper development. There is increasing evidence pointing to the existence of Hh pathway independent of SMO activity, however the co-existence with the canonical pathway has undermined the efforts to characterize it. C. elegans lacks SMO, nevertheless the PTCH homolog PTC-3 is essential for the worm, thereby providing an excellent model to study Smo-independent Hh pathway. Here we show that PTC-3 mediates cholesterol efflux, thus showing that the function of PTCH is conserved in C. elegans. Moreover, by measuring sterol levels through mass spectrometry (MS) and in vivo cholesterol detection we demonstrated that ptc-3(RNAi) lead to accumulation of intracellular cholesterol. Further analysis of lipid metabolism using MS-lipidomics revealed a reduction in acyl chain length and desaturation suggesting structural defects upon the loss of PTC-3. Indeed, we observed through electron microscopy defects in ER structure and lipid droplet formation. In addition to a structural role, cholesterol is known to mediate signalling cascades, particularly through nuclear hormone receptors (NHR). We found that NHR-181 expression levels undergo a two-fold increase upon ptc-3(RNAi). Finally, we show that reduction of dietary cholesterol in ptc-3 (RNAi) animals rescued all described phenotypes consequently improving development and survival. In sum, we have uncovered cholesterol efflux as the essential role of PTC-3, at the same time we have shown how the Hh pathway can operate in a Smo-independent manner.

Escobar Restrepo, Juan M. et al. (2011) International Worm Meeting "An in vivo screen of localization and trafficking regulators of LET-23 EGFR."

M, Langouet, Escobar Restrepo, Juan M., Herrmann, Christina J., A, Hajnal

[International Worm Meeting, 2011]

Proper EGFR localization in polarized cells is required for cell fate determination. Aberrant localization and changed receptor dynamics can cause human disease including various types of cancer. In C. elegans, the conserved LIN-2, LIN-7 and LIN-10 complex retains LET-23 EGFR at the baso-lateral compartment of the Vulval Precursor Cells (VPCs), allowing the receptor to receive the inductive LIN-3 EGF signal secreted from the gonadal Anchor Cell. Loss-of-function mutations in components of the LIN-2, LIN-7 and LIN-10 complex cause apical mis-localization of LET-23 (Kaech et al 1996). As a result, the LIN-3 signal is not efficiently received, leading to a reduction in the number of induced VPCs and consequently a Vulvaless (Vul) phenotype. Several screens have been performed based on the Vul phenotype to identify new components required for proper activation and regulation of LET-23 EGFR. However, it has so far not been possible to observe LET-23 EGFR localization in vivo. Additionally, most of the knowledge of kinetics and trafficking of the EGFR comes from studies of mammalian cell cultures and not from whole organisms. We have therefore created a functional LET-23::GFP translational reporter to visualize the receptor in the VPCs of live animals. Using this reporter we are performing a forward RNAi screen for genes controlling LET-23 localization and trafficking during vulval development. We are currently screening 740 RNAi clones that have been annotated to give a Protruding vulva (Pvl) phenotype and 110 RNAi clones of candidate genes that are implicated in receptor regulation or trafficking. This approach allows us to identify new components of the LET-23 localization machinery and to investigate in live animals changes in receptor dynamics during vulval development.

D'Amico Laure-Anne et al. (2008) European Worm Meeting "AP-1 role in EGFR trafficking in C. elegans epithelial cells"

Michaux Gregoire, D'Amico Laure-Anne

[European Worm Meeting, 2008]

Epithelial growth factor EGF is a highly regulated developmental signal acting in cell. communication thanks to its receptor EGFR. During C. elegans vulval induction the anchor cell of. the gonad sends an EGF signal to the vulval precursor cells. These cells are polarized epithelial. cells which are part of the epidermis and are called Pn.p cells. Due to its position just below the. anchor cell the P6.p cell is induced first and adopts the primary cell fate. To receive this EGF signal,. the LET-23 EGFR must be localized at the basolateral membrane.. The complex LIN-2/LIN-7/LIN-10 has been shown to be essential for LET-23 targeting to the. basolateral membrane (Kaech et al. 1998). Mutants for these genes fail to form a vulva and, unable. to lay eggs, look like a ''bag of worms''. A similar phenotype is observed in the let-23(sy1) mutant. where the last 6aa are deleted. In these mutants let-23 is apical instead of basolateral.. Genetic data indicates the role of the AP-1 adaptor complex in this basolateral targeting of. LET-23. unc-101 encodes one of the two micro subunits of AP-1. Although the null unc-101 mutants do. not display any vulval phenotype, unc-101 mutations can suppress null mutations affecting lin-2, lin-. 7 and lin-10 as well as let-23(sy1). This demonstrates the role of unc-101 in LET-23 basolateral. targeting. We are using genetic and cellular approaches to investigate the role of AP-1 in LET-23. targeting.. To do this we are currently generating GFP tools in order to dynamically visualize LET-23. trafficking among the different stages of vulva formation. We plan to genetically test if AP-1 known. interactors and major trafficking proteins could be involved in the fine regulation of the process.

ME Grunwald et al. (1999) International C. elegans Meeting "Localization pathways of the glutamate-gated ion channels NMR-1 and GBR-2"

JM Kaplan, C Rongo, ME Grunwald

[International C. elegans Meeting, 1999]

Glutamate plays a major role in the signal transduction of ASH neurons. ASH neurons connect to interneurons involved in the response to nose touch, osmotic shock and volatile repellents (Hart et al. , 1995; Maricq et al. , 1995). The glutamate receptor GLR-1 has previously been shown to be expressed in synaptic target neurons of ASH and to mediate nose touch sensitivity (Hart et al. , 1995; Maricq et al. , 1995). We have recently shown that the PDZ protein LIN-10 is responsible for localizing GLR-1 to the synapses between ASH and interneurons (Rongo et al. , 1998). The role of LIN-10 is particularly interesting since this protein is also part of a complex of PDZ proteins required for the localization of the epithelial LET-23 receptor (Simske et al. , 1996; Kaech et al. , 1998). We are now interested whether the localization of other glutamate receptors is similarly controlled. We are focussing on the putative NMDA receptor subunit NMR-1 and glutamate-gated chloride channel subunits GBR-2a and GBR-2b encoded for by avr-14 . NMR-1 encodes a protein bearing 33 % identity to the vertebrate NMDA receptor subunit NR1. GBR-2a and GBR-2b encode proteins of 416 and 430 amino acids, respectively; both proteins are homologous to the previously identified a -subunit of a glutamate-gated ion channel which forms ivermectin-sensitive channels (Cully et al. , 1994). We have generated translational fusion proteins of NMR-1, GBR-2a and GBR-2b with GFP (expressed using the glr-1 promoter) and examined their subcellular localization in neurons. GBR-2a was restricted to cell bodies whereas GBR-2b was also localized in a punctate pattern in the ventral nerve cord. Current experiments aim to identify which synapses utilize GBR-2b and whether pathways similar to those for GLR-1 are involved.

Hajnal AF et al. (1996) West Coast Worm Meeting "PHYSICAL INTERACTIONS BETWEEN LIN-2 AND LIN-36: A POSSIBLE LINK BETWEEN THE ANCHOR CELL SIGNALING AND THE lin-15 PATHWAYS."

Kim SK, Hajnal AF

[West Coast Worm Meeting, 1996]

lin-2 is required for the induction of P6.p by the anchor cell at the onset of vulval differentiation. lin-2 encodes a Membrane-Associated Guanylate Kinase (MAGUK) with similarity to the product of the Drosophila tumor suppressor gene lethal(1)discs-large-1. lin-2 acts cell-autonomously in P6.p to allow the efficient activation of the receptor tyrosine kinase LET-23. LIN-2 localizes LET-23 to the basal side of the Pn.p cell junctions since loss of lin-2 function causes mislocalization of LET-23 to the apical compartment of the Pn.p cells. LIN-2 may form a complex with LET-23 and LIN-7 (see abstract of S. Kaech and S. K. Kim). We have used the yeast two hybrid system to isolate cDNAs encoding proteins that bind LIN-2 and found a specific interaction between LIN-2 and LIN-36, which is the product of a class B synthetic multivulva gene. Synthetic multivulva genes form two redundant signaling pathways (termed class A and B) that inhibit vulval differentiation. lin-36 encodes a novel protein that inhibits the induction of the Pn.p cells in a cell-autonomous fashion (Thomas and Horvitz, WBG 13.2, 33-34). In vitro binding studies with recombinant proteins confirmed the specificity of the interaction. Deletion analysis indicated that the C-terminus of LIN-2 encoded by amino acids 903-961 is both necessary and sufficient for binding to LIN-36. Two lin-2 alleles (e1453 and n105) cause C-terminal truncations of LIN-2 at amino acids 897 and 940, respectively. These alleles would be predicted to eliminate or reduce binding to LIN-36 mediated by the C-terminus of LIN-2, but should not affect the localization of LET-23 mediated through the interaction of LIN-2 with LIN-7. Both alleles display a hyperinduced phenotype in which the outer Pn.p cells P3.p, P4.p or P8.p are induced by the anchor cell in addition to P6.p. A failure to interact with LIN-36 could therefore be responsible for the hyperinduced phenotype. In summary, these findings may suggest a role for lin-2 in the inhibition of vulval development through interaction with lin-36.