Lamkin, E.R. et al. (2017) International Worm Meeting "Morphogenesis of a neuron-glia attachment that shapes dendrite extension."

Lamkin, E.R., McLachlan, I.G., Heiman, M.G.

[International Worm Meeting, 2017]

The function of the nervous system depends on precise connections among neighboring cells, including specialized contacts between neurons and glia. In vivo studies of neuron-glia contacts have been hindered by the challenge of visualizing individual contacts amidst the vast number of cells in the vertebrate nervous system. To study the development of neuron-glia contacts at the single-cell level, we have turned to C. elegans. Two classes of neuron-glia interactions have been studied in C. elegans: (1) glial wrapping of axons in the nerve ring and (2) epithelial-like interactions between sensory dendrite endings and their ensheathing glia. We have focused on a third class of neuron-glia interactions reminiscent of contacts between Bergmann glia and Purkinje cell dendrites in the mammalian cerebellum. The most striking example within this class is the interaction between the BAG sensory neuron and its neighboring glial cell, in which the BAG dendrite ending precisely wraps a protrusion from the glial cell. This interaction was first seen by electron microscopy over four decades ago but has not been further studied. We used super-resolution optical microscopy to visualize the BAG-glia contact in living animals. We also performed forward genetic screens to identify factors required for BAG dendrite development. We showed that the nascent dendrite ending attaches to the glial cell and is pulled to its final length during embryo elongation. Our genetic screens revealed that this attachment requires the cytoskeletal adaptor protein GRDN-1/Girdin, which acts non-cell autonomously in the glial cell to promote dendrite extension in the neuron, and the adhesion molecule SAX-7/L1CAM, which is localized via GRDN-1 to the glial ending where the attachment forms. Based on the partial penetrance of these mutants, we reasoned that additional factors may contribute to the specification and morphogenesis of the BAG-glia contact. Using unbiased and candidate screens for modifiers of the sax-7 dendrite defect we identified roles for the adaptor protein AFD-1/afadin, which is known to localize to cell junctions, the membrane-associated guanylate kinase MAGI-1, and the fibroblast growth factor receptor EGL-15. Our working model is that these molecules form an adhesion complex that specifies the site of the neuron-glia contact. All the adhesion factors we have identified are conserved in mammals, suggesting that mechanisms of neuron-glia contact formation may be shared across systems. We anticipate that, in addition to adhesion factors, other molecules facilitate communication between the neuron and the glial cell to coordinate cell shape changes and generate the precisely interlocking morphologies of the mature contact. The BAG-glia contact thus provides an innovative model for studying the development of a special class of neuron-glia contacts at single-cell resolution.

Alicea, Bradly et al. (2015) International Worm Meeting "An experimental evolution model of C. Elegans adaptability."

Schroeder, Nathan, Androwski, Rebecca, Alicea, Bradly

[International Worm Meeting, 2015]

While the genetic architecture of C. elegans is well-studied, the adaptability of various strains and their reproductive fitness is less understood. To address this, an experimental-based model will allow for characterization of C. elegans adaptability amongst strains which may exhibit low reproductive potential and/or survivability under selection. Two experimental approaches representing within- and between-generation survivability (preconditioning and experimental evolution, respectively) will allow survivability to be assessed. Preconditioning [1] assesses adaptability within a generation using environmental selection, while experimental evolution [2] assesses adaptability via cross-generational artificial selection. For a given strain, populations grown from single founder individuals are repeatedly subject to genetic drift and then selected for fecundity (largest brood size). These results demonstrate how specific genotypes exhibit robustness in the face of adaptive constraints.Biological adaptability involves both survivability and reproductive fitness. Survivability is the degree to which a single founder worm can survive and produce viable clones (e.g. the number of generations constituting a single genealogy). Reproductive fitness is the mean population size for a given genealogy. In strains with high survivability, founder populations with high reproductive fitness should also generate subsequent founder populations with a high degree of survivability. Strains with high reproductive fitness but low overall progeny sizes may also exhibit high adaptability if selection results enhances adaptation to varying environmental conditions. While this model does not control for stochastic effects and demographic constraints [3], it does allow for genealogies with consistently high survivability [4].1. Calabrese, E.J. et.al, Tox App Pharm, 222(1), 122-128 (2007).2. Kawecki, T.J. et.al, Trends Ecol Evol, 27(10), 547-560 (2012).3. Szendro, I.G. et.al, PNAS, 110(2), 571-576 (2012).4. Wahl, L.M., Krakauer, D.C., Genetics, 156(3), 1437-1448 (2000).

Tuck SP et al. (1995) International C. elegans Meeting "MULTIPLE TARGETS FOR THE VULVAL INDUCTION PATHWAY?"

Greenwald IS, Horvitz HR, Tuck SP, Beitel GJ

[International C. elegans Meeting, 1995]

Induction of vulval fates by the anchor cell is mediated by a conserved signal transduction pathway involving let-60 Ras and the kinases lin-45 Raf and mpk-1/sur-1 MAP kinase. However, how this cascade causes cells to adopt vulval fates is not yet clear. Besides the kinases, three genes have been shown to function downstream of let-60 in the genetic pathway for vulval induction, lin-1, lin-25 and lin-31. lin-31 encodes a putative transcription factor of the forkhead/HNF3 family. We have shown that lin-25 encodes a novel protein (S.T and I.G.) and that lin-1 encodes a putative nuclear protein with an ETS DNA binding domain (G.J.B. and H.R.H.). To order lin-1, lin-25 and lin-31 with respect to one another in the pathway we have conducted genetic epistasis tests using null alleles of each gene. lin-1 encodes a negative regulator of vulval induction, lin-25 is a positive regulator and lin-31 is required for the correct selection of vulval and non-vulval fates. In lin-1 null mutants, all six VPCs adopt vulval fates. Lineage analysis of double mutants indicates that lin-1 is epistatic to both lin-25 and lin-31. Since LIN-1 contains a number of potential MAP kinase phosphorylation sites and ETS-domain-containing proteins are known targets of MAP kinases in other organisms, it seems likely that LIN-1 is a direct target of MPK-1. Therefore, a simple interpretation of the epistasis results, that lin-25 and lin-31 act between mpk-1/sur-1 and lin-1, is unlikely to be correct. An alternative explanation is that lin-25 and lin-31 act in parallel to lin-1. Our analysis of the pattern of VPC fates in lin-1 null mutants suggests that lin-1 does not mediate all the gonad-to-VPC signalling. Although all six VPCs adopt vulval fates in lin-1 animals, the fate adopted by P6.p is still affected by the presence or absence of the gonad and anchor cell. Therefore gonad-to-vulva signalling cannot be a simple linear pathway. We are currently examining double mutants between lin-1 and Ras pathway mutants to determine whether this non-linearity arises from branches in the Ras pathway, or whether multiple signalling events between the gonad and the VPCs are involved. We have also examined the interactions between lin-1 and mutations in lin-12, the receptor for the lateral signalling pathway. lin-1 is epistatic to lin-12(d)/+ but not to lin-12(d)/lin-12(d) suggesting that the different vulval fates are determined by the relative rather than absolute levels of the outputs from the inductive and lateral signalling pathways.

Hoppe PE et al. (1998) Midwest Worm Meeting "THE SEMIDOMINANT ASSEMBLY DEFECTS OBSERVED IN THE PARAMYOSIN ALLELE e73 RESULT FROM ALTERATION OF THE CHARGE PATTERN ON THE SURFACE OF THE COILED COIL."

Waterston RH, Hoppe PE

[Midwest Worm Meeting, 1998]

We are interested in the characteristics of coiled coil important for assembly of myosin and paramyosin into the highly-ordered thick filament. In site-directed mutagenesis experiments we have generated intragenic suppressors of the semidominant missense allele of paramyosin, e73. Our results suggest that the severe assembly defects in this mutant which accumulates normal levels of protein, are primarily due to disruption of the conserved charge pattern on the coil surface, as proposed by Genyo-Ando and Kagawa, 1991. Proteins that form coiled coils exhibit a heptad repeat (a-g) where positions a and d contain nonpolar residues that form a hydrophobic seam between the 2 strands. Positions e and g often contain charged residues that form interhelical salt bridges to further stabilize the dimer (McLachlan and Stewart, 1975), although their contribution is debated (Lumb and Kim, 1995). The sequence of e73 and its intragenic suppressors led to a model (Gengyo-Ando and Kagawa, 1991) which in part proposed that the e73 mutation (E to K) affected dimer-dimer charge-charge interactions critical for assembly of the paramyosin dimers. Our examination of myosin and paramyosin sequences suggests that e73 affects a highly conserved 1g-2e' interhelical salt bridge, changing a favorable charge pair (K-E) to an unfavorable pair (K-K). This raised the possibility that the assembly defects in e73 mutants might reflect a local disruption in shape or stability of the coiled coil, rather than a simple charge change. Both a construct containing negative charges at both positions of the potential salt bridge (E-E) and a construct in which the residues are reversed (E-K) act as dominant suppressors when injected into e73 animals, suggesting the 2 residues of the potential salt bridge do interact in vivo. Further, the reversed salt bridge construct (E-K), which pairs the mutant e73 lysine residue with an introduced negative charge and thus recreates the potential salt bridge in opposite orientation, restores motility in animals homozygous for the paramyosin null mutation, e1214. Constructs containing other charge alterations in the potential salt bridge residues (K-Q and Q-K) can restore motility in e73 animals, suggesting that concentration of positive charge is required to cause the semidominant e73 phenotype. In preliminary experiments we have found that a construct containing excess negative charge at the e73 site (E-E) can restore motility in a paramyosin null background, indicating that neither gain of negative charge nor loss of the interhelical salt bridge per se can phenocopy the e73 defects. The C-terminal location of point mutations (m208 and m209) that produce charge changes and act as intragenic e73 suppressors led to a model for the axial stagger at which paramyosin dimers associate in parallel during thick filament assembly (Gengyo-Ando and Kagawa, 1991). Sequencing of another intragenic e73 suppressor isolated in the Riddle lab (MR25) has identifed a premature stop codon just C-terminal to the m208/m209 region believed to interact with the e73 site. This result suggests that loss-of-function mutations which probably impair the ability of paramyosin to assemble act as suppressors of e73, without altering the region that may directly interact with the e73 site. The paramyosin molecule is homologous to the C-terminal two-thirds of myosin heavy chain. The interhelical salt bridge at the e73 site is conserved in all striated muscle myosins. We introduced the e73 mutation into the homologous site of myosin heavy chain A (MHC A), and found that this construct rescues both the lethality of myo-3 (MHC A) and paralysis of unc-54 (MHC B) mutations. The lack of semidominant defects suggests that the homologous regions in the two proteins may play different roles during assembly.