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.

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 H-P et al. (1992) Worm Breeder's Gazette "Expression and Localization of the cha-1 and unc-17 Gene Products"

Rand JB, Duerr JS, Han H-P

[Worm Breeder's Gazette, 1992]

EXPRESSION AND LOCALIZATION OF THE cha-l AND unc-17 GENE PRODUCTS He-ping Han, Janet Duerr, and Jim Rand, Oklahoma Medlcal Research Foundation, Oklahoma Clty, OK 73104

Cui, Mingxue et al. (2009) International Worm Meeting "Roles of Rb and its related genes in regulating RNA interference."

Han, Min, Cui, Mingxue

[International Worm Meeting, 2009]

Extensive genetic and biochemical analysis revealed the essential functions of many factors such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases in the double-stranded RNA-mediated interference (RNAi) process. These factors are important for either the generation of small interfering RNA (siRNA) or the reorganization/degradation of the target mRNA. Some of the SynMuv B class genes, including lin-35/Rb (C. elegans ortholog of the RB tumor suppressor) and its related genes, have been shown to play roles in RNA interference, but the mechanism of their functions remains unclear. Since these synMuv B genes encode proteins that are either transcription factors or chromatin regulators, we hypothesized that they regulate the expression of genes that play a critical role in RNAi. By analyzing available microarray data, we first noticed that the expressions of several genes with known functions in exogenous RNAi were significantly up-regulated in the lin-35/Rb and lin-15B mutants. Quantitative RT-PCR analysis confirmed that these genes were up-regulated in the SynMuv B mutants with enhanced RNAi sensitivity, but not in the SynMuv B mutants that display wild type RNAi efficiency. This result suggests a functional link between the expression of these candidate Rb target genes and RNAi functions. We then found that loss of function mutations in three of these genes suppressed the enhanced RNAi efficiency in the lin-15B mutant, consistent with the idea that these genes mediate the regulation of SynMuv B genes on RNAi. Enhanced sensitivity to exogenous RNAi could be due to a disruption of endogenous RNAi, such as in the rrf-3 mutant that is defective in endogenous siRNA generation. To exclude the possibility that the up-regulation of these candidate genes was the result of endogenous RNAi reduction, we determined that expression of these candidate genes did not increase in the mutants of rrf-3. To further investigate the possibility that Rb and its related genes are involved in the production of endogenous siRNAs, we analyzed the expression of nrde-3::GFP in the mutants of these genes. The argonaute protein NRDE-3 has been shown to translocate into the nucleus when endogenous siRNAs are present, and remains in the cytoplasm when endogenous siRNAs synthesis is blocked in eri-1 and rrf-3 mutants. Our experimental results showed that NRDE-3::GFP was normally distributed in the mutants of Rb and several other SynMuv B genes, suggesting that endogenous siRNAs were normally produced. These data suggest that Rb and related SynMuv B genes likely affect RNAi by regulating the transcription level of genes required for exogenous RNAi. Future work to test this model is being carried out.

TANG, Hongyun et al. (2017) International Worm Meeting "Nutrient-mediated environmental sex determination: fatty acids regulate germline sex-determination and germ cell fate through ACS-4-dependent myristoylation."

Han, Min, TANG, Hongyun

[International Worm Meeting, 2017]

Fat metabolism has been linked to fertility and reproductive adaptation in animals and humans, but cellular and molecular mechanisms involved in the response of reproductive development to fat levels, including a potential role of lipids in environmental sex-determination, remain to be explored. Hypothesizing that a regulatory mechanism perceives and interprets lipid cues to regulate reproductive development, we investigated whether and how FA metabolism impacts the specification of germ cell fate. We first observed that somatic fatty acid metabolism is critically involved in the impact of food availability on germ cell fate. Subsequent screen and analysis identified ACS-4, an acyl-CoA synthetase, and its FA-CoA product, as key germline factors that mediate the role of FA in promoting oocyte fate. We next demonstrated that ACS-4-dependent protein myristoylation in the germ line can regulate germ cell fate by sensing the change of FA levels. Further genetic and molecular analyses indicate that this ACS-4-dependent protein myristoylation regulates the sex-determination pathway, and MPK-1/MAPK plays a critical role in mediating this modulation. Finally, by showing alternation in germ cell fate in C remanei female with acs-4 knocked down, we obtained evidence that this FA impact on sex-dertermination and germ cell fate specification is conserved in male/female species. Our findings uncover a likely conserved mechanism by which FA, an environmental factor, regulates sex determination. This mechanism translates nutrient availability into instructive cue for reproductive development.

Loer CM et al. (1988) Worm Breeder's Gazette "erratum for the embryonic cell lineage."

Waring DA, Kenyon CJ, Loer CM, Sulston JE

[Worm Breeder's Gazette, 1988]

In examining embryonic lineages that give rise to the dorsal and lateral hypodermis, we have found a clerical error in the embryonic cell lineage paper (Sulston et al. (1983), Dev. Biol. 100, 64-119). We have used the lineages of Caa and Cpa as positional markers in following the ABarpp lineage (which gives rise to, among others, V1, V2, V4 and V6 right and left) from 100 min to 260 min and have found that the positions of the descendants of Caaa and Cpaa are reversed in Figures 7a (260 min), 10 (290 min), 13 (L1) and 14 (L1). At 260 min, the four nuclei descended from Caaa are to be found on the left of the dorsal midline and those from Cpaa on the right. At about 310 min of embryogenesis, most of the dorsal hypodermal nuclei migrate across the midline to reside on the side opposite to that of their origin (Fig. 10). Thus, after this stage, the four descendants of Cpaa reside on the left side and Caaa on the right side (Figs. 13 and 14a). Note that Fig. 5 is correct with Caa to the left (and anterior) of Cpa. [See Figures 1 & 2]