Han M et al. (2000) West Coast Worm Meeting "sur-7, a gene that suppresses activated ras"

Hague E, Han M

[West Coast Worm Meeting, 2000]

Suppressors of let-60 (n1046) ras have proven to be important in the regulation of the ras signal transduction pathway. The sur (suppressor of activated ras) genes include sur-1/mpk-1, sur-2 (downstream of mpk-1), sur-3/ksr-1, sur-6 (a PP2A regulatory subunit) and sur-8 (a leucine rich repeat containing protein). ksr-1, sur-6 and sur-8 appear to act positively between ras and raf. I am attempting to clone sur-7using single nucleotide polymorphisms (SNP's). sur-7 has no phenotype alone, but suppresses n1046 better than 99%. sur-7 maps between sdc-1 and sup-10 on LGX.

Han M et al. (2000) West Coast Worm Meeting "sur-9 a Suppressor of Activated let-60(n1046) in the C.elegans Vulva."

Eastburn D, Han M

[West Coast Worm Meeting, 2000]

The adult C. elegans vulva is induced by an EGF like signal which activates the Ras/MAPK pathway. Constitutively active alleles of ras result in a multivulva (Muv) phenotype where numerous pseudovulvae are formed from vulval precursor cells (VPCs) that are normally not induced in a wild-type background. By initiating suppressor screens of activated let-60 ras, many previously unknown components of this pathway have been identified. One gene, sur-9, has been isolated from such a screen and is currently being cloned and characterized. sur-9(ku258) has been mapped to chromosome III and is currently being fine mapped. The ku258 allele can suppress the Muv phenotype of let-60(n1046) from 80% to approximately 1%. In addition, ku258 is dominant with respect to its suppression. Cloning and elucidation of sur-9's molecular identity could contribute to our knowledge of a highly conserved and important biological pathway.

Han M et al. (1993) International C. elegans Meeting "Isolation and analysis of suppressors of activated let-60 ras mutation."

Hara M, Han M, Herrmann G, Singh N, Han Y

[International C. elegans Meeting, 1993]

Han M et al. (1989) International C. elegans Meeting "the dov-1 locus and its interaction with lin-15 during vulval induction."

Aroian RV, Holboke A, Han M, Sternberg PW

[International C. elegans Meeting, 1989]

Han M et al. (2000) West Coast Worm Meeting "Mutations in cyclin E reveal coordination between cell-cycle control and vulval development."

Han M, Fay DS

[West Coast Worm Meeting, 2000]

In screens for mutations affecting vulval development, we have identified strong loss-of-function mutations in the C. elegans cyclin E gene, cye-1. Mutations in cye-1 lead to the under-proliferation of many postembryonic blast lineages as well as defects in fertility and gut-cell endoreduplication. In addition, cye-1 is required maternally, but not zygotically for embryonic development. Our analysis of vulval development in cye-1 mutants suggests that a timing mechanism may control the onset of vulval cell terminal differentiation: once induced, these cells appear to differentiate after a set amount of time, rather than a specific number of division cycles. cye-1 mutants also show an increase in the percentage of vulval precursor cells (VPCs) that adopt vulval cell fates, indicating that cell-cycle length can play a role in the proper patterning of vulval cells. By analyzing cul-1 mutants, we further demonstrate that vulval cell terminal differentiation can be uncoupled from associated changes in vulval cell division planes.

Han M et al. (1991) International C. elegans Meeting "ANALYSIS OF DOMINANT NEGATIVE MUTATIONS OF let-60: MUTIPLE RAS MOLECULES MAY FUNCTION IN A COMPLEX"

Han M, Sternberg PW

[International C. elegans Meeting, 1991]

Genetic analysis of the let-60 gene has shown that its activity controls the decision between vulval and hypodermal cell fates for the six vulval precursor cells (P3.p-P8.p, or VPCs). The activity of let- 60, which normally is subject to the regulation by the inductive signal from anchor cell, can be forced to constitutively high or constitutively low levels by mutations within lel-60 gene or by misexpression of ectopic wild type gene. Iet-60 encodes a protein highly similar to mammalian ras proteins. Iet-60 appears to act after lin-3 (growth factor like molecule) and let-23 (EGF receptor like molecule) but before lin-l in the signal transduction pathway. Dominant negative mutations of let-60 [let-60(dn)] cause a dominant vulvaless phenotype, and all but one allele (sy93) also cause a recessive lethal phenotype. Determination of the molecular lesions of these let-60(dn) mutations, as well as of two intragenic revertants suggests that all the let-60(dn) mutations likely inactivate the protein by disrupting the binding ability of the ras protein to guanine nucleotides. Further genetic and molecular analysis of let-60(dn) has been carried out to study the mechanism of the dominant negative effect and the mechanism of ras protein function in the pathways. Four of the let- 60(dn mutant genes were introduced back into hermaphrodites in the form of extrachromosomal arrays. Our results suggest that: (l) the dominant negative effect is comman to all let-60-mediated signal transduction pathways, and the pathway involved in vulval development is more sensitive to alteration of let-60 activity than is the pathway( s) during early larval growth; (2) let-60(dn) mutant proteins may interfere with wild type ras activity prior to formation of the ras- GTP complex; (3) let-60 ras protein may interact with each other directly or indirectly to form a complex containing multiple ras molecules. The dominant negative effect may be caused by inactivation of a protein complex containing both let60(dn) and let-60(+) proteins.

Han M et al. (2000) West Coast Worm Meeting "Genetic and phenotypic characterization of evl-14 and evl-20, genes involved in C. elegans vulva development"

Han M, Antoshechkin I

[West Coast Worm Meeting, 2000]

In an attempt to identify new genes that participate in C. elegans vulva development, we carried out a screen for sterile mutants with protruding vulva. The mutants were then examined using Nomarski optics for abnormal vulva morphology at the "Christmas tree" stage. Several of isolated mutations were alleles of evl genes previously identified by Geraldine Seydoux . Here we report genetic and phenotypic characterization of two such genes, evl-14 and evl-20. Both mutations are completely recessive and result in a 100 % sterile phenotype. Their vulva structures are variably abnormal and are missing anywhere from 0 to 10 cells suggesting either under induction or cell cycle/division defect. In order to distinguish between these possibilities we are making doubles with members of Ras signaling pathway and GFP reporters for vulval cell fates. We are also performing detailed lineage analysis of the mutants. evl-20 also displays gonad migration defect not seen in evl-14. evl-14 maps to linkage group III just left of pal-1. evl-20 is located on chromosome II next to rol-6. We have injected cosmids from these regions and identified cosmid pools that rescue both evl-14 and evl-20. We are in the process of injecting single cosmids and making subclones to identify single open reading frames that will rescue the mutants.

Han M et al. (2000) East Coast Worm Meeting "Cytoplasmic dynein light-intermediate chain is required for distinct aspects of cell division in C. elegans"

Han M, Yoder JH

[East Coast Worm Meeting, 2000]

We have cloned the C. elegans homologue of cytoplasmic dynein light intermediate chain (dli-2) and shown through RNAi analysis that the gene product is required for several aspects of cell divisions in C. elegans. Cytoplasmic dynein is a multisubunit complex consisting of two heavy chain motor proteins associated with subunits categorized as light, light-intermediate or intermediate chains. Cytoplasmic dynein has been shown to be required for distinct aspects of mitosis and this activity requires association with another multisubunit complex, dynactin. Previous RNAi analysis of dynein heavy chain (dhc-1) and two dynactin components (dnc-1 and dnc-2) revealed a variety of mitotic defects (Gnczy et al., 1999). Particularly, RNAi against each of these three genes shows early embryonic defects including failed pronuclear migration and failed centrosome separation in the one cell stage embryo. Interestingly, RNAi against dli-2 reveals similar but distinct phenotypes. Pronuclear migrations are slowed but not prevented while centrosome separation occurs but proper spindle alignment fails (a phenotype also observed when dynein heavy chain function is diminished only partially with RNAi). Cytoplasmic dynein light intermediate chain (LIC) was only recently identified as a component of the dynein complex. Rat LIC-2 was found to directly interact with pericentrin, a conserved component of the centrosome but a proposed function has yet to be ascribed. It is possible, based on the observed differences in RNAi phenotypes, that dynein light intermediate chain may function in a regulatory or targeting capacity for the dynein complex and therefore be required only for a subset of dynein function. Sequence analysis of LIC-2 and DLI-2 reveal a region of extended sequence similarity with the nucleotide binding P-loop domain with strongest similarity to this region in members of the ABC transporter family (ATPases). To investigate the requirement of nucleotide binding for DLI-2 function we are currently generating point mutations in conserved residues within its P-loop domain and testing these constructs for their ability to rescue the phenotype in a null background.

Julie-Anne Fritz et al. (2005) International Worm Meeting "Investigation of the role of sterols in moulting and the clear phenotype."

Carolyn Behm, Julie-Anne Fritz

[International Worm Meeting, 2005]

When C. elegans are starved of food, they appear small and pale (clear) due to the absence of dark intestinal granules. A number of different types of intestinal granules exist in C. elegans. One class of granules is stained by Sudan Black or Nile Red, which indicates that they contain lipid stores. In this study we are assessing the function of a gene of unknown function. When the expression of this gene is knocked down by RNAi, worms take on a starved appearance. A large proportion arrest at the L2 stage, with some failing to shed old cuticle. When these animals are examined for Nile Red staining, the lipid-containing granules are still abundant, indicating that total lipid storage is not drastically altered. Depleting worms of cholesterol affects moulting and development in a similar way (Yochem et al., 1999). We are investigating the role of sterols in the RNAi phenotype and are undertaking microarray analyses to identify potential pathways that contribute to the phenotype. The results of these experiments will be presented.Yochem J, Tuck S, Greenwald I, Han M (1999) Development 126:597-606

Malone CJ et al. (2000) Midwest Worm Meeting "A genetic approach to study nuclear migration and anchorage"

Han M, Malone CJ, Starr DA

[Midwest Worm Meeting, 2000]

We have undertaken a molecular analysis of three genes to better understand the basic processes of nuclear migration in the context of cellular migrations during development. Mutations in two of these genes, unc-84 and unc-83, block the nuclear migrations of hypodermal P cells, which normally migrate from a lateral position to the ventral cord, and of hyp7 precursor cells, which normally migrate circumferentially across the dorsal midline (2, 3). Mutations in unc-84 and anc-1 disrupt the proper anchorage of hypodermal nuclei (1, 2). Both unc-83 and unc-84 have recently been cloned in our laboratory in collaboration with Robert Horvitz. unc-84 encodes for a novel protein with a predicted trans-membrane domain and a C-terminal domain with high similarity to the S. pombe spindle pole body component Sad1 which is also conserved in humans. An UNC-84:GFP protein is detected at the nuclear envelope (3). UNC-84 has also been localized to the nuclear envelope with an antibody raised in collaboration with Yossef Greuenbaum. unc-83 encodes for a completely novel protein with a predicted trans-membrane domain. There are two unc-83 transcripts. One is required for the hyp-7 cell migration, and the longer transcript is required for the P-cell migration. These results have been confirmed by RNAi analysis and identification of lesions in 17 mutant alleles. We are currently raising monoclonal antibodies against the unc-83 gene product. Finally, we are in the process of cloning anc-1. We have mapped it between two very close SNPs on chromosome I. Unfortunately, cosmids and YACs in this region have failed to rescue anc-1 mutant alleles. We therefore plan to clone the gene by further SNP mapping. 1. Hedgecock, E. M., and J. N. Thomson. 1982. Cell. 30:321-330. 2. Malone, C. J., W. D. Fixsen, H. R. Horvitz, and M. Han. 1999. Submitted to Development. 3. Sulston, J., and H. R. Horvitz. 1981. Dev. Biol. 82:41-55.