Singaravelu, Gunasekaran (2013) Worm Breeder's Gazette "CTGC - a portal for distributing transgenic worms"

Singaravelu, Gunasekaran

[Worm Breeder's Gazette, 2013]

Singaravelu, Gunasekaran et al. (2009) International Worm Meeting "Isolation of Caenorhabditis elegans mutants defective in sperm function."

Shakes, Diane, Singaravelu, Gunasekaran, Singson, Andrew

[International Worm Meeting, 2009]

The functional components required during fertilization can be conveniently studied in Caenorhabditis elegans. Here we describe a screening strategy for isolation of C. elegans mutants defective in sperm function (Spe) or egg function (Egg). We mutagenized the hermaphrodite population of the genotype sem-2(n1343) Is[Pelt-7::gfp + rol-6 (su 1006)] I using Ethyl methyl sulfonate (EMS) and screened for the self sterile mutants that could be rescued by allowing them to mate with wild type males. The sem-2(n1343) genetic background prevents the worm from laying eggs and the progeny are hatched inside the body. The transgene, Pelt-7::gfp, drives the expression of gfp under elt-7 promoter, which is active in intestine. Thus, amid the mutagenized population, sterile mutants can be readily identified by the absence of fluorescing eggs and/or larvae in their uterus. In our most recent screen, we isolated twenty-four non-conditional sterile Spe mutants and eleven temperature sensitive Spe mutants. Our preliminary characterization suggests that various aspects of C. elegans reproductive biology such as sperm development, differentiation and/or functions are compromised in these mutants. A slightly modified strategy can be used to identify mutants that cause defective oogenesis or oocyte function. We are currently mapping and further characterizing these mutants with the aim of unraveling the genetic determinants behind the normal, reproductively competent sperm.

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.

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.

Gunasekaran Singaravelu et al. (2005) International Worm Meeting "Characterization of Calumenin"

Ahnn Joohong, Gunasekaran Singaravelu

[International Worm Meeting, 2005]

Calumenin is a Ca2+ binding, EF-hand protein localized in the secretory pathway. Apparent homologues of calumenin have been identified from worm to vertebrates. Caenorhabditis elegans calumenin shows 50% identity and 67% similarity with its human counterpart. Various high-throughput experiments reveal the casual association of calumenin with drug resistance, metastatic potential, and atherosclerosis. However, the precise role of calumenin is unknown. We are currently investigating the functional importance of C. elegans calumenin utilizing the deletion mutant recently isolated from National BioResource Project, Japan. Weather the mutation is a molecular-null or not will be analyzed by combination of RT-PCR and western blot. We then plan to probe into the detail of the phase of C. elegans development at which functional calumenin is necessary. Next, by utilizing promoter-GFP fusion and immunostaining, we are interested in understanding how this gene is regulated both spatially and temporally.

Gunasekaran Singaravelu et al. (2008) ""Pleiotropic roles of a Calcium binding protein, Calumenin in Caenorhabditis elegans""

Do Han Kim, Gunasekaran Singaravelu, Soonjae Kwon, Hyun-Ok Song, Weixun Li, Joohong Ahnn

[2008]

"Calumenin is calcium binding protein containing EF hand motif. It is localized in the ER. Perturbed level of calumenin is casually associated with several diseases such as cancer, atherosclerosis and drug resistance. However, the physiological role of calumenin is largely unknown. Caenorhabditis elegans calumenin shows 45% identity and 62% similarity with its human counterpart. We have characterized a deletion mutant of C. elegans calumenin, calu-1(tm1783), in which approximately 200bp of 5 upstream region followed by the initial two exons are deleted. Western blot and RT-PCR showed that it is a loss-of-function mutant. Around 37% of the mutants showed bursted vulva phenotype during the transition from L4 to adult molt. Furthermore, mutants exhibit pleiotropic defects such as decreased brood size, sluggish locomotion, small body size, slow growth, suggesting calumenin is important for normal development in C. elegans. We found that, in male, calumenin has strong expression in pharynx, spicules."

Gunasekaran Singaravelu et al. (2004) East Asia Worm Meeting "Troponin C: a novel binding partner of Calcineurin A in C elegans."

Changhoon Jee, Hiroaki Kagawa, Joohong Ahnn, Bijaya Kumar Dhakal, Gunasekaran Singaravelu

[East Asia Worm Meeting, 2004]

Calcinerin is a widely distributed serine/threonine phosphatase that is well conserved from yeast to human. Troponin C ( pat-10 ) is a calcium binding protein with EF hand motif and has been shown to play essential roles in muscle cells of C elegans . When the catalytic subunit of Calcineurin (Calcineurin A) was utilized as a bait in yeast two-hybrid screening, many interacting proteins were identified; one of the proteins is Troponin C-1 ( pat-10 ). Surprisingly, Calcineurin A does not bind with Troponin C-1 when bacterially expressed proteins were utilized in vitro . However, when worm lysate were utilized, either of the protein found to interact with each other, as monitored by pull-down assay. This study indicates the novel mode of interaction between Calcineurin A and Troponin C in C elegans . Currently we are investigating the biological meaning of this novel interaction.

Gunasekaran Singaravelu et al. (2006) East Asia C. elegans Meeting "Localization of Calumenin to the Secretory Pathway is Indispensable for Maintaining Cuticle integrity, Locomotion and Growth rate in Caenorhabditis elegans."

Joohong Ahnn, Gunasekaran Singaravelu

[East Asia C. elegans Meeting, 2006]

Calumenin is a Ca<SUP>2+</SUP> binding protein containing EF-hand motif, localized to the secretory pathway. Perturbed level of calumenin is casually associated with several diseases such as cancer, atherosclerosis and drug resistance. However, the physiological role of calumenin is largely unknown. Caenorhabditis elegans calumenin shows 45% identity and 64% similarity with its human counterpart. We are characterizing the C. elegans deletion mutant, tm1783, in which approximately 200bp of 5 prime upstream region followed by the initial two exons of calumenin homolog, M03F4.7 are deleted. Combination of RT-PCR and western blot experiments reveal that tm1783 is a loss-of-function mutant. Around 37% of the mutants burst at vulva during the transition from L4 to adult molt. Further more, tm1783 exhibit pleiotropic defects such as decreased brood size, reduced pace of locomotion and slow growth. We are currently investigating its anatomical focus of action utilizing targeted expression of calumenin into hypodermis, body wall muscle or pharynx of tm1783. To our knowledge, this is the first report of genetic analysis of calumenin.

Gunasekaran Singaravelu et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Physical And Genetic Interaction Between tax-6 and kin-29 In Modulating Hypodermal, Muscular And Neuronal Functions Of Caenorhabditis elegans."

Joohong Ahnn, Jihye Lee, Changhoon Jee, Gunasekaran Singaravelu

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Calcineurin is a Ca2+ /Calmodulin activated Ser/Thr phosphatase that is well conserved from yeast to human. Functional calcineurin consists of two subunits; catalytic subunit and regulatory subunit. In Caenorhabditis elegans, tax-6 and cnb-1 encodes catalytic and regulatory subunits of calcineurin, respectively. The activity of mammalian calcineurin has been shown to be modulated by variety of means such as, proteolysis, phosphorylation and lipid binding. In order to isolate novel proteins that interact with TAX-6, we performed yeast two hybrid screening using tax-6 as bait. Among the several candidates, KIN-29 is one of them. KIN-29 is a Ser/Thr kinase, involved in regulating str-1 expression. TAX-6 and KIN-29 are expressed in hypodermis, muscles and neurons. Specifically, AFD, AWA, AWC, ASH, AIY, AIZ neurons express both tax-6 and kin-29, suggesting the possible consequence(s) of their interaction might be to facilitate or modulate the functions of these neurons. Interestingly, kin-29(oy38 lf), tax-6(p675 lf) and cnb-1(jh103 null) mutants exhibit similar phenotype, namely small body size and slow growth. We plan to investigate the genetic interaction between tax-6 and kin-29, with regard to body size, growth rate and locomotion. Further more, we plan to examine behavioral phenotype relevant to the neurons wherein both tax-6 and kin-29 are expressed, such as thermotaxis, chemotaxis, osmotic avoidance and mechanosensation.

Marcello, Matthew R et al. (2011) International Worm Meeting "Development of a C. elegans Spermiogenesis and Sperm Function Protein Interaction Network."

Marcello, Matthew R, Singson, Andrew, Singaravelu, Gunasekaran, Druzhinina, Marina

[International Worm Meeting, 2011]

The proper temporal and spatial organization of proteins during the spermatid to spermatozoa transition is necessary for the spermatozoa to function properly during fertilization. However, the protein-protein interactions necessary for ensuring spermiogenesis and sperm function during fertilization are not well understood. Sperm-enriched genes necessary for these processes are underrepresented in high throughput protein-protein interaction studies because of the low abundance of these proteins relative to the entire worm proteome. The purpose of this study was to generate a protein-protein interaction network for the proteins that are necessary for spermiogenesis and sperm function in C. elegans. The development of this protein interaction network will allow for the identification of novel interactions between proteins necessary for these processes and will also further our understanding of how spermiogenesis and sperm function are coordinated. To identify interacting proteins, we performed pair-wise split-ubiquitin yeast two-hybrid analysis of the full-length gene products of genes that have a known function in spermiogenesis (fer-1, spe-4, spe-6, spe-8, spe-12, spe-19, spe-27, and spe-29) or sperm function during fertilization (fer-14, spe-9, spe-13, spe-38, spe-41, and spe-42). Our results indicate that for some genes that that interact genetically there are physical interactions between their gene products. For example, spe-6 is known to interact with spe-19 and spe-29 genetically; our results indicate that SPE-6 also physically interacts with SPE-19 and SPE-29. We also identified novel protein-protein interactions, including interactions between SPE-8 and SPE-19 and SPE-9 and SPE-19. We represented the network topology using Power Graphs analysis rather than standard representations so as to remove redundancy and simplify network visualization. A future goal is to develop an integrated network model that incorporates additional datasets, including genetic interactions, expression profiles, localization data, and additional protein interaction data, along with the protein-protein interaction network we have generated. The integrated network of spermiogenesis and sperm function proteins will provide a robust source for generating hypotheses about how to assemble functional spermatozoa capable of fertilization.