Rahimi, Sina et al. (2011) International Worm Meeting "Mapping and Characterization of Caenorhabditis elegans Mutant Defective in Sperm Function."

Singson, Andrew, Singarevelu, Gunasekaran, Rahimi, Sina

[International Worm Meeting, 2011]

The molecular identities of the proteins participating in sperm and egg interactions during fertilization are unraveled by the isolation and mapping of mutants defective in the process. Here we report our preliminary results on mapping and characterization of as38, a non-conditional sperm function defective mutant that we isolated from a recent screen induced by Ethyl Methyl Sulfonate (EMS). Sperm function is defined as the ability of a normal, developed sperm to bind and enter the oocyte to execute fertilization. We have placed as38 in "spe-9 class" of mutants which are a group of mutants known for defective sperm function. Gonad morphology and the other processes of gamete development and differentiation occur normally in as38. Although leaky, hermaphrodites of as38 show significantly lower brood size in comparison to wild type. As38 shows temperature dependent decline in brood size. Examination of the oocytes that exit spermatheca indicates the lack of sperm entry and hence the unfertilized oocytes become endomitotic. The sperm from as38 male can successfully migrate into the spermatheca of recipient hermaphrodite. However, the fertility is significantly compromised in as38 males. Sperms from dissected males show normal morphology and undergo normal in vitro activation in response to Pronase E treatment. Using two factor mapping, we have placed as38 on the right arm of the Linkage Group IV near unc-26. We are currently performing three factor mapping to clone the as38 allele.

Singaravelu, Gunasekaran et al. (2011) International Worm Meeting "Regulation of the trafficking of TRP-3 channel by a novel protein during spermiogenesis in Caenorhabditis elegans."

Chatterjee, Indrani, Druzhinina, Marina, Singaravelu, Gunasekaran, Rahimi, Sina, Singson, Andrew

[International Worm Meeting, 2011]

Despite undergoing normal development and acquiring normal morphology, mutation in spe-38 or trp-3/spe-41 causes identical phenotype in C. elegans - the mutant sperm fails to fertilize oocyte. SPE-38 is a nematode-specific novel protein and TRP-3/SPE-41 is a Ca2+ channel. Localization of both of these proteins is confined to the membranous organelle in the undifferentiated spermatids. In mature spermatozoa, SPE-38 is localized to psedopod and TRP-3/SPE-41 is localized to the plasma membrane. Dynamic redistribution of TRP-3/SPE-41 from membranous organelle to plasma membrane is dependent on SPE-38, since in spe-38 mutant spermatozoa, TRP-3/SPE-41 is trapped within the membranous organelle and fails to reach cell surface. Split-ubiquitin yeast two hybrid analyses revealed the segments of SPE-38 and TRP-3/SPE-41 required for the regulation of the dynamic physical interaction between these proteins. The correlation between the ability or inability of the various forms of SPE-38 to rescue spe-38 mutant and the required segments of SPE-38 for its physical interaction with TRP-3/SPE-41 suggest the in vivo relevance of the interaction between SPE-38 and TRP-3/SPE-41.

Singaravelu, Gunasekaran et al. (2015) International Worm Meeting "A genetic screen for fertilization mutants identifies an Izumo-like gene."

Singaravelu, Gunasekaran, Dharia, Sunny, Smith, Harold, Shakes, Diane, Golden, Andy, Krauchunas, Amber, Rizvi, Anam, Rahimi, Sina, Singson, Andrew

[International Worm Meeting, 2015]

Fertilization is a conserved process in all sexually reproducing organisms whereby sperm binds and fuses with oocytes. Despite the acknowledgement of the importance of sperm-oocyte interactions in fertilization, the molecular underpinnings are still not well understood. Here we describe a novel genetic screen to isolate fertilization mutants in Caenorhabditis elegans. We cloned and characterization spe-45, one of the genes identified in our screen. SPE-45 is an Izumo-like protein and represents an ancient sperm function that is also required for mammalian fertility. This observation holds the promise that characterization of additional mutants from our screen will be useful to discover conserved components in fertility pathways.

Luiza Ghila et al. (2006) European Worm Meeting "Lunapark: A Link between Synaptogenesis and the Ubiquitin Proteasome System?"

Marie Gomez, Luiza Ghila, Ludovic Baillon, Patrizia Latorre

[European Worm Meeting, 2006]

?. Recently, new mechanisms involving protein degradation through the ubiquitin-proteasome system (UPS) have been shown to be involved in synaptic regulation. Regulation of neuronal function involving UPS encompasses a large number of molecular processes: synapse development, synapse size, vesicle cycling, neurotransmitter release, receptor trafficking, spine size, synaptic plasticity and downstream signalling. A new mouse gene coding for a protein named Lnp (Limb and neural pattern) was identified at the vicinity of the Evx2 and the Hoxd cluster. In vertebrates lnp is expressed, during embryogenesis, in limbs, eyes, heart, genitalia, and in the nervous system. Moreover its expression in neuronal structures, such as the neural tube, coincides with that of Evx transcription factors that play an important role in the interneuron identity in the spinal cord. In contrast to evx2 and hoxd genes, the function of lnp is completely unknown.. To elucidate a possible role of lnp in the nervous system we have developed a model system using C.elegans. We have analysed lnp loss-of-function mutants and found that they showed an increase in aldicarb resistance, resulting probably from either a presynaptic or/and postsynaptic defect. In a yeast two hybrid screen we have identified a putative interaction partner, the ortholog of the Drosophila seven in absentia (SINA) protein. Sina encodes a RING/E3 ubiquitin ligase. Studies in mammals and drosophila suggest that SIAH regulates neuronal development and function by mediating the ubiquitin-dependent degradation of a number of neuronal target proteins.. In C. elegans we have shown that lnp expression pattern is related to that of siah (sina homologue). Broad and probably ubiquitous expression is first visible during gastrulation. Postembryonically, LNP and SIAH reporters are expressed in numerous cells bodies along the ventral cord, around the pharynx and tail and most of them are neuronal cells. Moreover, immunohistochemistry in NGF-differentiated PC12 cells revealed significant colocalisation of these two proteins with synaptophaysin (synaptic vesicle protein) in a punctuate staining pattern in cell bodies and neuritic processes suggesting that LNP and SIAH are associated with vesicular structures like endosomes and synaptic-like microvesicles. We have performed in vitro ubiquitination experiments and we have showed that LNP enhances SIAH E3 ubiquitin ligase activity.. Together this suggests that LNP together with SIAH might be part of multisubunit RING finger complexes involved in synaptogenesis.

Luiza Ghila et al. (2006) European Worm Meeting "Lnp and Siah/Ring E3 Ubiquitin Ligase: Partners Associated to Multisubunit Ring Finger Complexes involved in Synaptogenesis?"

Luiza Ghila, Marie Gomez, Patrizia Latorre

[European Worm Meeting, 2006]

Lnp and Siah/Ring E3 Ubiquitin Ligase: Partners Associated to Multisubunit Ring Finger Complexes involved in Synaptogenesis? Luiza Ghila, Patrizia Latorre and Marie Gomez In C. elegans, two multisubunit Ring E3 complexes, the anaphase promoting complex/cyclosome (APC/C) and the Skp1/Cullin/Lin-23 complex (SCFLIN-23) are involved in the regulation of the GLR-1 (glutamate receptors) abundance in ventral cord nerve cells. The E3 ubiquitin ligase SIAH (the mammalian homolog of the drosophila seven in absentia/SINA) binds to the C-terminal part of APC and interacts with SCF through the Siah-interacting protein SIP. Moreover it has been shown that Siah interacts with the C-terminal part of the GLR-1 and the ubiquitin ligase activity of Siah through this interaction is required for GLR-1 degradation.. In C. elegans, a gene coding for a new protein named LNP was shown to interact in a yeast two hybrid screen with SIAH, the C. elegans homolog of SINA. lnp expression pattern is highly related to that of siah. They are widely expressed during embryogenesis and in later stages, their expression seems to be enriched in neural tissues, e.g. the ventral nerve cord and the nerve ring. We have shown that LNP enhances Siah E3 ubiquitin ligase activity. Two lnp loss-of-function mutants showed a significant increase in the resistance to the aldicarb, an acetylcholine esterase inhibitor, having egg laying defects and shorter life span. These suggest that Lnp has a function in the nervous system, probably connected to synaptogenesis. These three major E3 ligases (APC/C, SCF complexes and Siah) are also involved in the cell cycle regulation, being responsible for the periodic proteolysis of many cell-cycle regulators. We noticed that Lnp cellular localisation is regulated during cell cycle. During interphase, LNP protein presents a vesicular cytoplasmic pattern in all cell lines we tested. During M phase, LNP concentrates on not yet characterized structures underlying the plasma membrane reminiscent to cell adhesion structures. LNP is over-expressed all along the M phase, but in confluent monolayer cells (mitotic inactive) its expression is drastically reduced. We do not know yet the functional aspect of this regulation during cell cycle. We are currently investigating whether in worms SIAH together with LNP are required for the regulation of the GLR-1 receptors and/or for the regulation of the cell cycle and what are the connections of LNP and SIAH with the APC/C and the SCF complexes in these two biological processes.

Luiza Ghila et al. (2005) International Worm Meeting "A novel gene involved in neurogenesis? Functional analysis of lnp in C. elegans."

Ana Caratsch, Marie Gomez, Luiza Ghila

[International Worm Meeting, 2005]

We are interested in identifying new molecules involved in nervous system development and function. Recently, a mouse gene coding for a protein named LNP (limb and neural pattern; Spitz et al. Cell, 2003) was identified at the vicinity of the Evx2 and the Hoxd cluster. Lnp is expressed, during embryogenesis, in limbs, eyes, heart, genitalia, and in the nervous system. This expression pattern is related, at least partially, to that of Evx2 and Hoxd13 to Hoxd10 genes, which encode for transcription factors that are crucial players during morphogenesis and highly conserved throughout the animal kingdom. In contrast to Evx2 and Hoxd genes, the function of lnp is totally unknown. To elucidate a possible role of lnp in the nervous system we use C. elegans as a model system. We have first looked at the lnp expression during C. elegans embryogenesis and we could show that indeed lnp is expressed throughout embryonic development. Its expression seems to be enriched in neural tissues, specifically in some motor neurons of the ventral nerve cord. Vab-7, the C. elegans Evx ortholog, is also expressed in motor neurons of the ventral cord, suggesting that co-expression of these two genes in neuronal tissues might be conserved during evolution. Several approaches to understand the function of lnp have been initiated. First, we have analyzed two lnp loss-of-function mutants obtained from the National Bioresource Project for the Experimental Animal C. elegans. No obvious phenotypes were observed in both alleles but we are currently performing a more detailed analysis of these mutants. Second, in a yeast two hybrid screen we have identified a putative interaction partner, coding for the ortholog protein of the Drososophila seven in absentia (SINA) protein, which has been shown to play an important role in neuronal cell fate determination. We are currently testing this interaction in vivo. We have also investigated the sub-cellular localization of LNP protein in mammalian cell cultures using specific antibodies raised against the mouse LNP. Preliminary results suggested that LNP cellular localization is variable but linked to the cell cycle. Indeed, during interphase, LNP presents a vesicular cytoplasmic pattern in all cell lines we tested. During M phase, it concentrates on structures underlying the plasma membrane reminiscent to cell adhesion structures. In light of the different results we have obtained, implication of lnp in neurogenesis will be discuss.

John K Everett et al. (2001) International C. elegans Meeting "Elaborating the composition and structure of a touch-transducing complex: towards the determination of the structure of the MEC-4 N-terminal intracellular domain and characterization of 4 proteins that interact with this domain"

Nektarios Tavernarakis, ShiLiang Wang, Monica Driscoll, Guy Montelione, John K Everett, Nikos Kyrpides

[International C. elegans Meeting, 2001]

One of the looming mysteries in signal transduction today is the question of how mechanical signals, such as pressure or force delivered to a cell, are interpreted to direct biological responses. Elegant genetic and molecular studies by Chalfie and colleagues have identified several proteins thought to form a touch-transducing complex that mediates the response to body touch. At the core of this complex is a mechanically-gated ion channel made up of MEC-4 and MEC-10 subunits. This channel is homologous to the epithelial amiloride Na+ channel (ENaCs) and the acid sensing channels (ASICS)—the DEG/ENaC superfamily. The C. elegans channel subunits have been called degenerins because specific mutant forms of the channel can induce neurodegeneration. Understanding how the MEC-4/MEC-10 channel functions in touch transduction is a major question in the field of mechanical signaling. These channel subunits are positioned in the plasma membrane with N and C terminal domains inside the cell and a large extracellular domain projecting into a specialized extrcellular matrix. It is thought that specific proteins interact with the intracellular and extracellular domains to exert gating tension on the channel. We are currently focusing on the N-terminal domain to gain insight into mechanisms of channel function. The MEC-4 N-terminal domain includes an 87 amino acid stretch that is not highly conserved and a highly conserved domain close to the first transmembrane domain that has similarity to a half-site of thiol protease active sites. The conserved domain has been implicated in protein interactions and all known N-terminal mutations affect this subdomain. In order to gain greater insight into the nature of the MEC-4 amino terminal domain, we have created a homology model of this domain MEC-4{N}. Our model is based on sequence identity and similarity to the pro domains of proteases of the cathepsin protease family. Our homology model depicts MEC-4 {N} as a globular domain which is predominantly alpha helical in nature aside from an extended span of residues which comprises the hydrophobic core of the model. The model presents several interesting structural features, including small lipophilic surfaces (potential protein binding sites) and exposed tyrosines (potential phosphorylation site). Efforts are currently underway to solve the structure of MEC-4 {N}and MEC-10 {N} via NMR spectroscopy. We are testing highlighted residues for importance in channel function using site-directed mutagenesis. To complement genetic studies that identified candidate proteins of the touch transducing complex, we performed a two-hybrid screen for MEC-4 N-interacting proteins. We used the N-terminal domain of MEC-4 as bait to screen a C. elegans yeast two-hybrid cDNA library. We obtained 81 positive clones from a screen of 3 million primary yeast clones. Among these, four candidate interacting proteins were identified that interact in both two-hybrid and GST pulldown experiments. All the candidate interacting proteins are expressed in the touch receptor neurons and can interact with the non-conserved N-terminal domain of MEC-4. Two interacting proteins are probably involved in channel turnover: 2 isolates encode a AAA ATPase homolog located in F23F1.8 (B70326) and 4 isolates are of a them are SINA (seven-in-absentia) homolog located in Y37E.11 (U89792). The AAA ATPase is most likely a subunit of the proteosome required for MEC-4 degradation. RNAi directed against both genes suggests an essential role very early in embryogenesis. Two other candidates identify novel proteins. We obtained 61 isolates corresponding to an ORF located in C15G7.4 (U08022), and 3 isolates corresponding to ORF Y11D7A.12 (A215876). RNAi against these two proteins induces touch abnormalities and thus these are candidate proteins for regulation or function of the MEC-4 channel.