Sung Min Han et al. (2008) Development & Evolution Meeting "MSP Domains Are Evolutionarily Conserved Ligands For Eph Receptors"

Sung Min Han, Kriti Mohan, Anthony Zoghbi, Yong Lin, Claire Haueter, Hiroshi Tsuda, Youfeng Yang, Hugo Bellen, Michael Miller, Yadollah Harati, Justin Kwan, Chao Tong

[Development & Evolution Meeting, 2008]

The C. elegans MSPs are multi-functional proteins that are abundantly expressed in sperm. MSPs function as intracellular cytoskeletal proteins that drive sperm motility and extracellular signaling proteins. Secreted MSPs bind to the VAB-1 Eph receptor tyrosine kinase expressed on oocytes and ovarian sheath cells to induce oocyte maturation and sheath contraction. C. elegans VPR-1, human VAPB/ALS8, and Drosophila VAP33 are homologous proteins (collectively called VAPs) with an N-terminal MSP domain and a C-terminal transmembrane domain. A missense mutation (P56S) in the VAPB/ALS8 MSP domain is associated with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Here, we report that VAP MSP domains are cleaved from their transmembrane domains and secreted into the extracellular environment, where they directly bind to cell surface Eph receptors and other receptors. We show that VAPs and Eph receptors function in common genetic pathways during C. elegans and Drosophila development. The P56S mutation causes intracellular VAP aggregation and a failure to secrete the MSP domain. Secretion of MSPs and VAP MSP domains is likely unconventional, as these proteins lack signal sequences. Our results show that VAP MSP domains are secreted ligands for Eph receptors, the largest class of receptor tyrosine kinases in the human genome. Furthermore, our studies provide insight into potential mechanisms that may impact the pathogenesis of ALS and SMA.

Sung Min Han et al. (2007) International Worm Meeting "Identifying receptors that interact with major sperm protein in Caenorhabditis elegans."

Michael A. Miller, Sung Min Han

[International Worm Meeting, 2007]

An evolutionary conserved feature of oogenesis is that oocytes arrest in meiotic prophase. Oocytes resume meiosis in response to extracellular signals by a process called oocyte meiotic maturation, which prepares the oocyte for fertilization and embryogenesis. The Caenorhabditis elegans major sperm protein (MSP) is the most abundant protein in sperm. It is exported by a budding mechanism from the sperm cytoplasm to the extracellular environment, where it induces oocyte meiotic maturation and ovulation. During evolution, MSP has acquired extracellular signaling and intracellular cytoskeletal functions. Proteins with MSP-related domains have been found in diverse metazoans, raising the possibility that MSP has a conserved signaling function. Previous work using an in situ MSP-FITC binding assay identified the VAB-1 Eph receptor as an MSP receptor. However, vab-1 null gonads still bind to and respond to MSP. These results suggest that MSP binds to multiple receptors that act redundantly to induce oocyte maturation and sheath contraction. To identify additional MSP receptors, I conducted an RNAi-based screened of 58 receptor candidates whose mRNAs are expressed during oogenesis, but not spermatogenesis. Five receptor candidates have been identified that are required for MSP binding. These encode members of evolutionarily conserved receptor classes. Currently, I am testing whether any of five candidates directly binds to MSP and whether mutations in the candidates cause defects in MSP signaling.

Sung Min Han et al. (2003) International Worm Meeting "The Caenorhabditis elegans DICE-1 homologue is required for germ-line development and embryogenesis in Caenorhabditis elegans"

Sung Min Han, Hyeon-Sook Koo, Sung-Shin Ryu

[International Worm Meeting, 2003]

The protein encoded by the open reading frame F08B4.1 was found to interact with a RecQ family DNA helicase (RecQ5) encoded by E03A3.2 in yeast two-hybrid assay. The gene product of F08b4.1 is homologous to human DICE-1 protein, which was first identified in a human lung carcinoma cell as a candidate tumor suppressor. Although human DICE-1 (deleted in cancer 1) is presumed to be a tumor suppressor, its detailed molecular function is unknown. We have examined the functions and expression pattern of Ce-Dice-1 by RNA interference and GFP reporter expression. When the Ce-dice-1 was inhibited by double-stranded RNA microinjection, complete embryonic lethality was observed. In order to examine the functions of Ce-Dice-1 at postembryonic stage, Ce-dice-1 gene expression was inhibited by feeding Caenorhabditis elegans worms with the double-stranded RNA from the L1 stage. Protruding vulva, growth arrest at the L4 stage, and pleiotropic phenotypes in the germ-line such as undersized gonads, abnormal gonad migration, defective oocyte development, endomitotic oocytes, and increased apoptosis appeared. GFP reporter gene expression induced by the promoter sequence was observed in hypodermal cells after the L4 stage. The gene expression in hypodermal cells probably is associated with the phenotypes of protruding vulva and growth arrest at the L4 stage. Ce-dice-1 is probably also expressed in the germ-line and early embryos, as deduced from the RNAi phenotypes. Although, Ce-Dice-1 interacts with RecQ5 protein in yeast two-hybrid assay, no genetic interaction has been detected in double RNAi of the two genes.

Sung Min Han et al. (2005) International Worm Meeting "Intracellular localization and functions of Caenorhabditis elegans DIC-1, a homologue of human DICE-1(Deleted In Cancer 1)"

Tae Hoon Lee, Moon Jeong Kim, Se-Jin Lee, Ekaterini Kritikou, Michael Hengartner, Hyeon-Sook Koo, Sung Min Han

[International Worm Meeting, 2005]

The protein encoded by the open reading frame F08B4.1 in C. elegans is homologous to human DICE-1(Deleted In Cancer 1) protein. Although human DICE-1 was first identified in a human lung carcinoma cell line as a tumor suppressor candidate, its detailed molecular function largely remains unknown. We set out to elucidate the functions and expression pattern of DIC-1. RNA interference of the dic-1 expression resulted in a complete embryonic lethality accompanied by abnormal morphogenesis and increased apoptosis. When RNA interference was performed from the L1 stage, pleiotropic phenotypes appeared, including undersized gonads, defective oogenesis, small body size, reduced motility, abnormal mitochondrial morphology, and resistance to oxidative stress. The increased germline apoptosis was found to be mediated by CED-3/CED-4 caspase in the main apoptosis pathway of C. elegans. In a deletion mutant of dic-1(tm1615) generated by National Bioresource Project, homozygous embryos from a heterozygous mother hatched but arrested at the L3 stage, suggesting maternal rescue of the embryonic lethality and an essential role of DIC-1 during larval development as well. DIC-1 was immuno-localized in the cytoplasm of germ cells, oocytes, and embryos, in a similar expression pattern to that of the Cytochrome C observed at the mitochondria. Our results suggest that DIC-1 plays a critical role for normal mitochondrial functions in C. elegans such as energy production and regulation of cell death. We are currently investigating the possibility that DIC-1 is involved in one of the biological pathways such as mitochondrial protein folding, translocation, interaction with cytoskeletons, and oxidative phosphorylation.

Sung Min Han et al. (2006) East Asia C. elegans Meeting "Deleted in cancer 1 (DICE1) is an essential protein controlling the topology of the inner mitochondrial membrane in C. elegans"

Se-Jin Lee, Michael O. Hengartner, Tae Hoon Lee, Ekaterini Kritikou, Hyeon-Sook Koo, Ji Young Mun, Sung Min Han, Moon Jeong Kim, Sung Sik Han

[East Asia C. elegans Meeting, 2006]

DICE1 (deleted in cancer 1), first identified in human lung carcinoma cell lines, is a candidate tumor suppressor, but the details of its activity remain largely unknown. We have found that RNA interference of its C. elegans homolog (DIC-1) produced inviable embryos with increased apoptosis, cavities in cells and abnormal morphogenesis. In the dic-1(RNAi)germ line, ced-3-dependent apoptosis increased, and cell cavities appeared at the late-pachytene/oogenic stage, leading to defective oogenesis. Immunofluorescence microscopy of DIC-1 revealed its ubiquitous expression in the form of cytoplasmic foci, and cryoelectron microscopy narrowed down the location of the foci to the inner membrane of mitochondria. After dic-1 RNAi, mitochondria had an irregular morphology and contained numerous internal vesicles. Homozygous embryos from a heterozygous dic-1 mother arrested at the L3 larval stage, in agreement with the essential role of DIC-1 in mitochondria. In summary, C. elegans DIC-1 plays a crucial role in the formation of normal morphology of the mitochondrial cristae/inner membrane. Our results suggest that human DICE1 may have several functions in multiple intracellular locations.

Han, Sung Min et al. (2009) International Worm Meeting "VAP MSP domains are secreted metabolic factors that bind to Eph and Roundabout receptors."

Tsuda, Hiroshi, Yang, Youfeng, Tong, Chao, Miller, Michael A., Bellen, Hugo J., Han, Sung Min, Haueter, Claire

[International Worm Meeting, 2009]

In C. elegans, MSPs function as secreted hormones that bind to Eph receptors and unidentified receptors to trigger oocyte maturation. Human VAPB, Drosophila VAP33, and C. elegans VPR-1 are homologous proteins called VAPs with an N-terminal MSP domain and a C-terminal transmembrane domain. A missense mutation (P56S) in the VAPB MSP domain is associated with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), but the function of VAP proteins is not understood. Recently, we showed that VAP MSP domains are cleaved and secreted ligands for Eph receptors (Tsuda et al. 2008). The human VAPB MSP domain is present in blood serum and the P56S mutation prevents secretion in fly cells. These data suggest that VAPB proteins have a signaling function that is relevant to ALS pathogenesis. There is increasing evidence that ALS is a systemic disorder, as patients and mouse models can exhibit mitochondrial abnormalities inside and outside of the CNS, insulin resistance, dyslipidemia, and defective energy homeostasis. We show that in C. elegans and Drosophila loss of VAPs causes muscle mitochondrial defects, which can be rescued by VAP expression specifically in neurons. Genetic, biochemical, and molecular studies demonstrate that mitochondria from vap mutants have impaired respiration. These data support the hypothesis that VAP proteins are secreted signals that regulate mitochondrial function. However, Eph receptor mutants do not have mitochondrial defects. To identify new MSP domain receptors, we used a fluorescent binding assay. We show that MSP and VAP MSP domains bind to Roundabout (Robo) receptors, which are broadly expressed in neurons, muscle, and likely oocytes. sax-3 Robo is required for muscle mitochondrial function and vpr-1 and sax-3 act in the same genetic pathway. In addition to mitochondrial defects, loss of C. elegans vpr-1 causes defects consistent with impaired insulin signaling, including fat accumulation in the intestine and body wall muscle that is dependent on DAF-16/FOXO activity. The increased DAF-16 activity in vpr-1 mutants appears to be part of a compensatory mechanism. Loss of the vab-1 Eph receptor, a VPR-1 receptor, causes mild fat metabolism defects. Finally, vpr-1 mutants are sterile and neuronal overexpression can partially rescue these defects in some animals and cause germline tumors in others. Collectively, our data support a model in which VAPs are components of a neuroendocrine signaling mechanism that controls metabolism and energy homeostasis.

Moon Jeong Kim et al. (2004) European Worm Meeting "DEFICIENCY OF A DICE-1 HOMOLOGUE IN CAENORHABDITIS ELEGANS INTERFERES WITH OOGENESIS AND EMBRYONIC MORPHOGENESIS WITH ENHANCEMENT OF APOPTOSIS"

Moon Jeong Kim, Sung Min Han, Hyeon-Sook Koo

[European Worm Meeting, 2004]

The protein encoded by the open reading frame F08b4.1 is homologous to human DICE-1 protein, which was first identified in a human lung carcinoma cell as a candidate tumor suppressor. Although human DICE-1 (deleted in cancer 1) is presumed to be a tumor suppressor, its detailed molecular function is unknown. We have examined the functions and expression pattern of Ce-DIC-1 by RNA interference and immunostaining. Ce-DIC-1 was localized by immunostaining in the endoplasmic reticulum (ER) of germ cells from the pachytene stage, oocytes, and embryonic cells. RNAi of the Ce-dic-1 expression at the young adult stage resulted in complete embryonic lethality. When RNAi was performed from the L1 stage, pleiotropic phenotypes in the germ line such as undersized gonads, abnormal gonad migration, defective oocyte development, endomitotic oocytes, increased apoptosis, and protruding vulva appeared. dic-1(RNAi) embryos showed increased number of cell corpses and morphological defects, followed by developmental arrests at a late embryonic stage. The morphological defects induced by RNAi of dic-1 were observed in pharynx, gut, hypodermis, and muscle using several GFP-expressing strains. These RNAi phenotypes showed that the Ce-DIC-1 is required for oocyte formation and embryonic morphogenesis in Caenorhabditis elegans. Especially, increased apoptosis by the RNAi together with the intracellular localization in ER suggest the possibility that the DIC-1 protein may be involved in the regulation of apoptosis.

Myon Hee Lee et al. (2003) International Worm Meeting "C. elegans dna2 is involved in the germ-line development and DNA repair"

Ji Won Han, Sung Min Han, Myon Hee Lee, Joohong Ahnn, Yun Mi Kim, Hyeon-Sook Koo

[International Worm Meeting, 2003]

Dna2 is a highly conserved helicase-nuclease essential for Okazaki fragments processing of the lagging strand in yeast DNA replication. To determine the in vivo function of C. elegans Dna2 (CeDna2) in the germ-line development, we investigated phenotypes in the germ cells and oocytes of a C. elegans mutant. The first homozygous mutants appeared normal, but their progeny showed severe phenotypes in the germ cell proliferation and development, including defective homologous chromosome pairing in oocytes. When germ cells were treated with DNA-damaging agents such as -ray and camptothecin, Cedna2 mutants showed a higher incidence of aberrant chromosomes in oocytes compared with the wild type and resultantly a higher embryonic lethality. Deficiency of Cedna2 combined with RNA interference of mre11 gene synergistically aggravated the germ-line development, especially oocyte formation. Based on the Cedna2 mutant phenotypes in the germ-line development and its hypersensitivity to DNA damages, CeDna2 is thought to be involved in DNA repair in association with or in parallel to mre11.

Tiwary, E. et al. (2019) International Worm Meeting "In vitro production of prostaglandins and their effects on sperm motility in C. elegans."

Tiwary, E., Hu, M., Miller, M., Prasain, J.

[International Worm Meeting, 2019]

Prostaglandins are lipid signaling molecules involved in various physiological and pathological process. Conventionally, these PGs are synthesized by cyclooxeganses (a.k.a Cox) or free radicle pathway from polyunsaturated fatty acids (PUFAs). Our earlier study in C. elegans showed that a non cox- pathway also exists which synthesizes F2- series PGs and had critical role in sperm migration and motility. To further delineate this pathway, we developed an in vitro assay using arachidonic acid (AA) as substrate and worm lysate as source of enzyme. We further characterized PGs by tandem mass spectrometry using MRM and MS/MS method. Using MRM with mass transition m/z 353/193 we found that four major peaks eluting at eluting at Rt 11.2, 11.5, 11.7 and 11.9 min and were higher in soluble fraction compare to insoluble fraction of worm lysate. Among them peaks eluted at 11.2, 11.5 and 11.7 min matched with the chemically synthesized 8 iso PGF2?, ( plus or minus ) 5 iPF2 VI and PGF2 ?, respectively. Next, we confirmed synthesis of PGFs replacing AA with 5C13 using high resolution triple TOF mass spectrometer. Extracted ion chromatogram with mass transition m/z 358.24, showed four peaks eluted at Rt 10.3, 10.5, 10.7 and 10.9 min. Peaks eluted at 10.3, 10.5 and 10.7 min had characteristic F2 class PGs fragment ion corresponds to chemically synthesized 8 iso PGF2?, ( plus or minus ) 5 iPF2 VI and PGF2 ?, respectively. The peak eluted at 10.9 min was unidentified as its MS/MS profile didn't match with available PG standards. These PGFs are synthesized rapidly and not inhibited by anti-oxidant and specific cox inhibitors, suggesting that their synthesis in C. elegans is neither cox dependent nor ROS under in vitro condition. Our detailed study suggested that this alternative pathway had a signature F2-series PG profile and this can be a characteristic feature of PG synthesis biochemical pathway in C. elegans. Studies evaluating sperm motility effects of PGs is underway in our lab.

Taffoni, C. et al. (2017) International Worm Meeting "Complex dynamic subcellular patterning underlies the innate immune response."

Marguet, D., Taffoni, C., Ewbank, J., Pujol, N.

[International Worm Meeting, 2017]

Sterile wounding or infection by the fungus D. coniospora leads to a rapid increase in the transcription of antimicrobial peptide (AMP) genes in the worm epidermis. Forward and reverse genetic screens identified many players involved in this immune response [1] including the GPCR DCAR-1 that acts upstream of a conserved p38 MAPK pathway. Its activation by HPLA, a tyrosine derivative, leads to the expression of AMPs through the STAT-like transcription factor STA-2. STA-2 physically interacts with vesicle-bound SNF-12, a member of the SLC6 family of bioamine transporters [2]. To characterise further the response of the epidermal cell to damage, we are monitoring in vivo the dynamics of these signaling proteins upon wounding, together with that of membrane and cytoskeletal components. We operate a laser wound under a spinning disc microscope. As previously reported, this produces an immediate calcium wave (in less than 1 sec) and the formation of an actin ring (in 20 min) [3]. In between these 2 events, using a CAAX::GFP reporter, we observe a rapid (1 min) reorganization of membranes, forming a barrier for cytoplasmic diffusion, maintained for up to 20 min. It is followed by the accumulation of both highly motile RAB-11 and RAB-5 vesicles (10 min). In the main epidermis, the microtubules (MT) are found either circumferentially orientated in parallel bundles over the muscle quadrants, or randomly orientated in the main lateral cytoplasm. They are highly dynamic as revealed by the rapid movement of the MT + end binding EBP-2 protein. Upon wounding, EBP-2 is recruited (2 min) in a highly directional way to form a ring closely juxtaposed inside the actin ring. These rings progressively close the wound (20 min). Our observations reveal a rapid stabilization of MT at the wound site and an unexpected association with the actin ring. The DCAR-1 receptor, which has a dotted pattern at the plasma membrane, accumulates at the wound site within 10-15 min. Remarkably, SNF-12 that localizes in apical vesicles disappears within 30 sec after wounding. This could be either due to vesicle internalization, or release of SNF-12 from these vesicles. The SNF-12 vesicles progressively reappear apically and subsequently are recruited around the wound. We could show that inactivating genes encoding specific tubulins (TBA-2 and TBA-4), while not affecting the development of the worm, destabilizes the epidermal MTs, impacts SNF-12 vesicle dynamics and prevents the induction of AMP. We thus propose a direct link between the reorganization of the cytoskeleton upon wounding and the activation of the innate immune response. 1. Taffoni, C. and N. Pujol,. Tissue Barriers, 2015. 2. Dierking, K., et al., Cell Host Microbe, 2011. 3. Xu, S. and A.D. Chisholm,. Curr Biol, 2011.