Ding, Mei et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A Wnt-Frz/Ror-Dsh pathway regulates neurite outgrowth in C. elegans"

Song, Song, Zhang, Bo, Huang, Xun, Ding, Mei

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

One of the challenges to understanding the organization of nervous system has been to establish how axon guidance molecules govern the axon outgrowth. Through an unbiased genetic screen, we identified a conserved Wnt pathway composed of Wnt ligand CWN-2, Frizzled receptors CFZ-2 and MIG-1, co-receptor CAM-1/Ror and downstream component DSH-1, which is crucial for RME neurite anterior-posterior (A/P) outgrowth in C. elegans. Among them, CWN-2 acts as a local attractive cue for neurite outgrowth and its activity can be substituted with other Wnts, suggesting the spatial distribution is the key for the functional specificity of Wnts. As a co-receptor, CAM-1 functions cell-autonomously on neurons and together with CFZ-2 and MIG-1, transmits Wnt signal to downstream. Yeast two-hybrid screen identified DSH-1 as a binding partner for CAM-1, indicating that CAM-1 may convey CWN-2/Wnt signaling to DSH-1 through physical association. Since mig-2/Rho and ced-10/Rac mutants exhibit phenotype similar to cfz-2 mutant, implying some small G proteins may be specifically required for RME neurite outgrowth. Together, our study revealed an important role of Wnt-Frz/Ror-Dsh pathway in regulating neurite A/P outgrowth.

Mei Ding (2008) "An E3 ubiquitin ligase contributes to precise synaptic connectivity through selective synapse elimination"

Mei Ding

[2008]

"Synapse elimination is a developmental hallmark of neural circuit refinement. However, the molecular mechanisms that selectively eliminate certain synapses while sustaining others are poorly understood. We investigated synapse elimination in the egg-laying motor neuron of C. elegans, HSNL. In adult animals, HSNL connects to its targets, the vulval muscle and VC motor neurons, via a cluster of synapses localized exclusively to the vulval region, the primary synapse region (PSR). At early stage of development, we observed that additional synapses formed immediately anterior to the vulva, the secondary synapse region (SSR). In syg-1 mutant, synapses at the SSR failed to be eliminated. syg-1 encodes a immunoglobulin superfamily protein, which localizes specifically to the PSR. How does SYG-1 function in eliminating synapse at the SSR then? We found that SYG-1 may do so through SCF/SEL-10-complex-mediated protein degradation pathway. SCFSEL-10 complex is an E3 ubiquitin ligase, a Skp1-cullin-F-box (SCF) complex composed of SKR-1 and the F-box protein SEL-10. SYG-1 bound to SKR-1 and inhibited assembly of the SCF complex, thereby protecting nearby synapses. This ensures that synapses are stabilized at appropriate sites but removed from inappropriate sites. Currently, we are investigating the specific substrates of SCF/SEL-10 complex and seeking other E3 ligases, which function in parallel with SCFSEL-10 complex in synapse elimination. Progress will be reported in this meeting. "

Mei Ding et al. (2005) International Worm Meeting "Dissecting the synaptic specificity mediated by SYG-1/SYG-2 pathway"

Mei Ding, Kang Shen

[International Worm Meeting, 2005]

The intricate patterns of synaptic connections in a nervous system underlie nearly all aspects of neuronal function, but how each neuron distinguishes its appropriate synaptic targets from other non-target cells during synapse formation is not well understood. Recent studies have shown that in the absence of members of the Ig Superfamily, SYG-1 or SYG-2 protein, HSNL axon fails to recognize its normal targets and forms morphologically normal synapse onto abnormal target cells. The observation of presynaptic specialization being formed onto abnormal target cells in syg-1 /syg-2 mutant animals leads us to hypothesize that synaptic target choices of HSNL are flexible and maybe even reversible and the competition between the potential targets determines the preference of target selection, which results in synaptic specificity.In order to understand molecular mechanism of how SYG-1/SYG-2 dominates the synaptic competition and determines target specificity in HSNL synaptogenesis, we took two strategies:1. Identify proteins that interact with the cytoplasmic domain of SYG-1 using yeast two-hybrid approach. From this screen, we identified a set of SYG-1 potential binding partners, including a PDZ domain containing protein, which plays a role in selecting and transporting transmembrane proteins to the cell surface and an SCF ubiquitin ligase complex component, which has been shown interacting with presynaptic protein. Currently, we are examining the expression pattern and loss of function phenotype of SYG-1 interacting molecules, and the progress will be presented at the meeting.2. Enhancer/suppressor screen to identify genes involved in synaptic choice competition. In syg-1/syg-2 mutant animals, HSNL neuron still forms synapses but onto abnormal targets. In order to understand the molecular mechanisms underlying synapse formation at those abnormal targets we have carried out a direct genetic screen for second-site mutations with abnormal (enhancer) or wild type (suppressor) pattern of synaptic vesicle marker in a syg-1 mutant background. From a pilot screen, we isolated the wy43 mutation. Homozygous wy43 mutants appear wild type. In wy4; syg-1 double mutants the ectopic synaptic vesicle clustering of syg-1 is suppressed. We mapped wy43 to chromosome I. We are currently performing fine mapping and germline transformation to determine the identity of wy43. Through these approaches, we hope to understand how SYG-1 and SYG-2 determine synaptic choice by interacting with other synaptic specificity molecules.

Mei Ding et al. (2008) C.elegans Neuronal Development Meeting "Spatial Regulation of an E3 Ubiquitin Ligase Directs Selective Synapse Elimination"

George Wang, Kang Shen, Dan Chao, Mei Ding

[C.elegans Neuronal Development Meeting, 2008]

Synapse elimination is a developmental hallmark of neural circuit refinement. However, the molecular mechanisms that selectively eliminate certain synapses while sustaining others are poorly understood. We investigated synapse elimination in the egg-laying motor neuron of C. elegans, HSNL. In adult animals, HSNL connects to its targets, the vulval muscle and VC motor neurons, via a cluster of synapses localized exclusively to the vulval region, the primary synapse region (PSR). At early stage of development, we observed that additional synapses formed immediately anterior to the vulva, the secondary synapse region (SSR). In syg-1 mutant, synapses at the SSR failed to be eliminated. syg-1 encodes a immunoglobulin superfamily protein, which localizes specifically to the PSR. How does SYG-1 function in eliminating synapse at the SSR then? We found that SYG-1 may do so through inhibiting SCF-complex-mediated protein degradation pathway. SCF complex is an E3 ubiquitin ligase, a Skp1-cullin-F-box (SCF) complex composed of SKR-1 and the F-box protein SEL-10. SYG-1 bound to SKR-1 and inhibited assembly of the SCF complex, thereby protecting nearby synapses. This ensures that synapses are stabilized at appropriate sites but removed from inappropriate sites. Currently, we are investigating the specific substrates of SCF complex and seeking other E3 ligases, which function in parallel with SCF complex in synapse elimination. Progress will be reported in this meeting.

Mei Ding et al. (2002) West Coast Worm Meeting "VAB-19, a novel conserved protein involved in epidermal elongation in C. elegans"

Mei Ding, Andrew D Chisholm, Wei-Meng Woo

[West Coast Worm Meeting, 2002]

Trans-epidermal attachments (also known as fibrous organelles) are composed of intermediate filaments and hemidesmosome-like structures and are important for epidermal elongation in C. elegans. However, their exact function in controlling cell shape is not well understood. Previously, we reported that the gene vab19 is required for normal epidermal elongation and encodes a novel conserved protein. We further explore the function of vab-19 in epidermal elongation as reported here.

Mei Ding et al. (2001) International C. elegans Meeting "vab-19 functions in epidermal elongation and encodes an ankyrin repeat containing protein."

Mei Ding, Andrew CHISHOLM

[International C. elegans Meeting, 2001]

Epidermal elongation requires the coordination of the morphogenesis between epidermis and underlying body wall muscles. This process is mediated in part by hemidesmosome-like structures, called fibrous organelles, found in regions of the epidermis adjacent to muscle. We report here that the vab-19 gene functions in epidermal elongation and encodes a novel conserved, ankyrin repeat containing protein that appear to be associated with fibrous organelles. vab-19 is defined by a single cold-sensitive allele, e1036 (isolated by Jim Lewis). At 15deg C almost all of e1036 animals arrest during embryogenesis. At 22.5 degC 90% of the e1036 animals survive to adulthood and show variable notched-head and notched-tail morphological phenotypes (Vab). Four-dimensional Nomarski microscopy analysis shows that vab-19 mutants are defective in elongation and have no obvious defects in earlier embryogenesis. Preliminary results of vab-19 temperature shift experiments also suggest that vab-19 is required in elongation. vab-19 maps to chromosome II. We rescued the mutant phenotypes with an 11 kb DNA fragment containing a gene that encodes a novel protein with four ankyrin (ANK) repeats in the C-terminus. The e1036 mutation results in a stop codon, creating a truncated protein lacking the ANK repeats. VAB-19 homologs, of unknown function, exist in flies, mice, and humans. A rescuing VAB-19::GFP transgene is expressed in epidermal cells, within which it is localized to or close to fibrous organelles. The C-terminal ANK repeats are not essential for this sub-cellular localization. The function of these repeats in VAB-19 is thus not yet clear. To understand how vab-19 acts in embryonic morphogenesis, we have begun to examine genetic interactions with vab-19 by constructing double mutants with other known elongation defective mutations. We found that loss of function in the b H -spectrin sma-1 specifically suppresses vab-19 Vab and cold sensitive phenotypes. The suppression of vab-19 by sma-1 suggests that VAB-19 and SMA-1 may play antagonistic roles in the epidermal cytoskeleton.

Mei Ding et al. (2004) East Asia Worm Meeting "The cell signaling protein EPS-8 is required for embryonic epidermal morphogenesis and interacts with VAB-19"

Ryan King, Pier Paolo Di Fiore, Assunta Croce, Jeff Hardin, Andrew CHISHOLM, Mei Ding

[East Asia Worm Meeting, 2004]

Elongation of the C. elegans epidermis involves changes in epithelial cell shape. These cell shape changes require coordinated function of the actin, microtubule, and intermediate filament cytoskeletons. Disruption of epidermal intermediate filaments or IF-associated proteins causes elongation to arrest (Bosher et al 2003 JCB; Woo et al. 2004 Dev. Biol), reflecting an essential function for epidermal IF-containing structures known as trans-epidermal attachments. vab-19 mutants display phenotypes similar to those seen in IF mutants. VAB-19 encodes an ankyrin repeat protein that localizes to epidermal attachment structures (Ding et al., 2003 Development). A human homolog of VAB-19 known as Kank acts as a tumor suppressor in renal cancer. The ankyrin repeats of VAB-19 are essential for its function but not for its localization. To identify interacting partners of VAB-19 we screened a yeast two hybrid library (kindly provided by Z. Zhou and H.R. Horvitz) using the VAB-19 ankyrin repeat domain as bait. We found that VAB-19 ankyrin repeats interact with EPS-8. EPS-8 family proteins play roles in receptor tyrosine kinase signaling and in Rac-mediated remodeling of the actin cytoskeleton. RNA interference of eps-8 resulted in highly penetrant embryonic arrest during elongation. Some eps-8(RNAi) animals arrest during larval development due to defects in the intestine (Croce et al, 2003 International worm meeting abstract 761). We isolated a deletion mutation, eps-8(jc36), and found that these mutants display a fully penetrant arrest in embryonic elongation resembling the strongest RNAi phenotypes. A functional EPS-8::GFP is localized to trans-epidermal attachment structures and colocalizes with VAB-19. VAB-19::GFP transgenes can partly rescue the embryonic defects of an eps-8 null mutant, but the converse is not the case, suggesting that eps-8 functions upstream of vab-19 in a linear pathway. The EPS-8/VAB-19 pathway may be a conserved interaction in epithelial cells.

Lu C et al. (2000) West Coast Worm Meeting "A b -tubulin gene, tbb-2, functions as an activator of mei-1 and mei-2 in female meiotic spindle formation in Caenorhabditis elegans."

Mains PE, Srayko MA, Lu C

[West Coast Worm Meeting, 2000]

C. elegans female meiosis requires two meiotic-specific genes, mei-1 and mei-2. Loss of mei-1 or mei-2 function blocks female meiotic spindle formation while the subsequent mitotic cleavages are normal. In a mei-1 gain-of-function mutant, however, the mitotic cleavages are disrupted after normal meiotic divisions. Wild-type MEI-1 and MEI-2 localize to the meiotic, but not mitotic spindle.1,2 However, MEI-1(gf) and MEI-2 ectopically localize to mitotic spindles in mei-1(gf) embryos. mei-1 and mei-2 encode homologs of p60 and p80 subunits of the sea urchin microtubule severing protein katanin, respectively, and MEI-1 and MEI-2 together disassemble interphase microtubules in a HeLa cell system.2 We propose that MEI-1 and MEI-2 form a complex in meiosis and regulate meiotic spindle formation by katanin-like activities. In a mei-1(gf) suppressor screen, we recovered three extragenic suppressors. One of them, sb26, was found to be a missense mutation in a b -tubulin gene, tbb-2. tbb-2(sb26) also enhances a weak mei-2(lf) allele. Together, these results suggest that tbb-2 is required for the function of mei-1 and mei-2. Antibody staining shows that TBB-2 is widely expressed during worm development. Neither tbb-2 nor tbb-1 (another b -tubulin highly similar to tbb-2) RNAi has severe effects during early development. However, tbb-2 and tbb-1 double RNAi results in 100% dead eggs indicating that they act redundantly during embryogenesis. We are doing experiments to examine the interactions of MEI-1/MEI-2 with tbb-2 and tbb-1. 1. Clark-Maguire, S. and Mains, P.E. (1994). J. Cell Biol. 126, 199-209. 2. Srayko, M., Buster, D.W., Bazirgan O.A., McNally, F.J., and Mains, P.E. (2000). Genes Dev. 14 (9).

Chenggang Lu et al. (2002) West Coast Worm Meeting "C. elegans tubulin genes tbb-2 and tba-2 are required for microtubule severing of the MEI-1/MEI-2 katanin complex during meiotic spindle formation."

Chenggang Lu, Paul E Mains, Francis J McNally

[West Coast Worm Meeting, 2002]

Female meiotic spindle formation requires two meiosis-specific genes, mei-1 and mei-2, which encode the C. elegans subunits of the microtubule severing complex katanin. In addition to their sequence similarities to katanin, MEI-1 and MEI-2 disassemble interphase microtubules when coexpressed in Hela cells.1 In wild-type embryos, MEI-1 and MEI-2 localize to the female meiotic spindle and are inactivated after meiosis, likely through a UBQ-like Nedd-8 pathway.2 MEI-1 and MEI-2 likely restrict the length of meiotic spindle fibres to maintain the unique morphology of the anteriorly-localized, barrel-shaped meiotic spindle. Therefore, the ectopic persistence of MEI-1/MEI-2 activity into the following mitosis is disastrous. This is seen in a mei-1 gain-of-function (gf) mutant where MEI-1 and MEI-2 localize ectopically to mitotic spindles resulting in small, misoriented spindles similar to those seen after treating embryos with microtubule destabilizing drug, nocadazole.3 This is consistent with our idea that an ectopic microtubule severing activity is present in the mutant mitosis.

MA Srayko et al. (1999) International C. elegans Meeting "Genes Involved in Meiotic and Mitotic Spindle Formation"

MA Srayko, MR Dow, PE Mains

[International C. elegans Meeting, 1999]

The C. elegans oocyte cytoplasm supports the formation of both meiotic and mitotic spindles. However, these two structures differ greatly in their size, structural morphology, and position within the embryo. We have genetically identified genes that are involved in the assembly of the meiotic spindle and the transition to mitotic spindle formation. mei-1 , a gene encoding a member of the AAA family of ATPases, is required for the assembly of the meiotic spindle. However, mei-1 must be inactivated prior to mitosis, otherwise mitotic spindles are small and misoriented. mei-2 and tbb-2 are genetic activators of mei-1 . mei-2 encodes a novel protein that, like MEI-1, localizes to the meiotic spindle and polar bodies, but unlike MEI-1, also strongly stains condensed meiotic chromatin and the condensed sperm pronucleus. Despite these differences in immunolocalization, the entire MEI-2 staining pattern is dependent on mei-1 (+). MEI-1 and MEI-2 staining disappears at the completion of anaphase II. A mutation in a b -tubulin gene, tbb-2 , was identified as a suppressor of the mitotic-defective mei-1 (gf). tbb-2(sb26) has a missense mutation within the isotype-specific C-terminus. tbb-2(sb26) homozygotes are wild-type, suggesting that this sequence alteration specifically lowers, but does not eliminate, the activity of mei-1 . tbb-2(sb26) enhances the weak meiotic defects associated with mei-1(ct103) and mei-2(ct98) mutations, also indicating a role in meiotic spindle assembly. mel-26 is the post-meiotic inhibitor of mei-1 . Mutations in mel-26 result in mitotic spindle defects due to ectopic mei-1 activity during mitosis. The function of mel-26(+) seems very specific for turning off mei-1 since it is not required in the absence of mei-1 : mitosis is normal in mei-1(0);mel-26 double mutants (although meiosis is abnormal). Ectopic MEI-1 and MEI-2 are concentrated at the mitotic centrosomes in mel-26 mutants. MEL-26(+) localizes to the centrosomes during wild-type mitosis, suggesting a possible role in protecting mitotic spindles from mei-1 / mei-2 activity after meiosis is complete.