Katherine Erickson et al. (2005) International Worm Meeting "Novel Domains of the Muscle Regulatory Protein Troponin T and Their Roles"

Sarah Pilzer, Taylor Allen, Katherine Erickson, Malcolm Hoshi

[International Worm Meeting, 2005]

Among the components of the calcium-switch for contraction in striated muscle, troponin T (TnT) is the most enigmatic. To uncover its in vivo roles, we are studying a deletion allele of tnt-3/esp-1, as well as a suppressor of tnt-1/mup-2 (e2346ts). Transcripts of both TnT genes are expressed in embryonic body wall muscle and hermaphroditic gonad; tnt-3 is expressed also in the pharyngeal terminal bulb.Alternative mRNA splicing of tnt-3 generates four isoforms, three of which depart in major ways from the archetypal design of TnT. Most dramatic is an N-terminal insertion of 834-residues bearing resemblance to few known proteins. Similarity was detected to the WH2 domain (Wiskott Aldrich syndrome protein, or WASP, homology domain 2) and flanking proline-rich region found in proteins thought to control actin dynamics. Another comprised numerous local matches to the PEVK (proline, glutamate, valine, and lysine) domain of elastic proteins. For example, residues 482-758 of TnT-3 isoforms b, c, and d display 42% similarity to residues 5605-5881 of crayfish I-connectin (or titin; gi:15425681). In this region, TnT-3 contains 13% Pro, 7% Glu, 4% Val, and 9% Lys, and I-connectin has 16% Pro, 19% Glu, 5% Val, and 20% Lys.The gk170 allele of tnt-3, made available by the C. elegans Reverse Genetics Core Facility at U.B.C., evidences a deletion of 909 bp (A14563 to G13655 of cosmid C14F5, coding for Ser101 to Ala403). Surprisingly, gk170 homozygotes were indistinguishable from wild types in appearance (e.g., following rhodamine-phalloidin staining to visualize filamentous actin) and in frequency of body wall contractile waves for adult homozygotes swimming in S-buffer at 23 C. Growth of the mutant on difficult-to-eat bacteria (DA837) failed to highlight eating defects. Absence of a mutant phenotype may stem from preservation of WH2-like and PEVK-like domains in the deletion allele.The product of tnt-1/mup-2 resembles the archetypal TnT, save for an extension of ~125 residues at the C-terminus, which is truncated by half by the e2346ts mutation. A suppressor of e2346ts, obtained following mutagenesis with N-ethyl-N-nitrosourea, displayed normal structure and placement of body wall muscle, as well as absence of the MUP and STE phenotypes. Current work is directed toward mapping the suppressor and thereby potentially identifying interactions made by the extension. Supported by NSF grant IBN-9985315 (TA).

Anne Middleton et al. (2005) International Worm Meeting "Design and Efficacy of Two Investigative, Physiological Projects Using C. elegans"

Anne Middleton, Sarah Pilzer, Taylor Allen, Rachel Decker, Katherine Erickson

[International Worm Meeting, 2005]

A growing body of pedagogical writing argues for use of investigative laboratory projects to promote learning. Assessment of the efficacy of such projects is limited, but if it were more plentiful, it could guide choice of projects and offer benchmarks for evaluating their adaptation. We designed, implemented, and assessed two multi-week investigative, physiological projects using nematodes. Both projects aimed to boost within the context of an animal physiology course students (i) mastery of concepts, (ii) engagement with science, and (iii) understanding of the nature of science.In one project (named adopt-a-protein and described by Priest et al., 2001 International C. elegans Meeting), students adopted a disease linked to a mutant transporter, channel, or contractile protein and then used computational tools to identify a candidate homologue in C. elegans, to analyze domain architecture, and to formulate an hypothesis concerning the physiological role of the protein. Students tested their hypothesis by eliminating the candidates expression through RNA interference. For the other project (herb an legends), students identified on basis of herbal medical lore (e.g., Balds Leech Book; Culpepers English Physitian, and Chinese materia medica) and biomedical research an herb worthy of physiological examination. Students then formulated and tested an hypothesis concerning the functional alterations that should arise following growth of worms on the herb.The adopt-a-protein project was used in the fall 2003 offering of an intermediate-level physiology course, and herb an legends in the fall 2004 offering. Assessment of students attitudes at completion of the projects revealed for the two cases comparable levels of sense of reward (4.5 on scale from 1=not rewarding to 5=most rewarding) and sense of connection to science (4.5). Comparison of attitudes pre- and post-project showed no change associated with herb an legends, but an increase in feelings of strain, insecurity, and hesitation associated with adopt-a-protein. This unexpected increase might have stemmed from placement of the project toward the end of semester, when pressures of examinations weighed heavily (herb an legends occurred during first half of semester). Neither project shifted perceptions on the nature of science. Arguing against the notion that multi-week projects are insufficient to prompt a shift, assessment of an investigative project based on human cardio-respiratory function and used in the 2004 course revealed gains in sophistication of students perceptions.

Iwasa H et al. (2013) Exp Cell Res "Characterization of RSF-1, the Caenorhabditis elegans homolog of the Ras-association domain family protein 1."

Hata Y, Ikeda M, Iwasa H, Kuroyanagi H, Nakagawa K, Maimaiti S

[Exp Cell Res, 2013]

Mammals have 10 RASSF proteins, which are characterized by the Ras-association (RA) domain. Among them, RASSF1 to RASSF6 have the Salvador/RASSF/Hippo (SARAH) domain and form the subclass C-terminal RASSF proteins. Drosophila genome has a single C-terminal RASSF, dRASSF. All these RASSF proteins are related to the tumor suppressive Hippo pathway, and are considered to function as tumor suppressors. Caenorhabditis elegans T24F1.3 encodes a protein with the RA and the SARAH domains. The amino acid sequences are 40% and 55% similar to those of RASSF1 in the RA and the SARAH domains, respectively. We have characterized T24F1.3 gene product and named it RSF-1 as RASSF1 homolog. RSF-1 is widely expressed in epithelial cells. About 14% rsf-1 mutants exhibit defects in embryonal morphogenesis and the actin disorganization. The combinatorial synthetic lethal analysis demonstrates that the lethality is enhanced to more than 80% in rsf-1 mutants with the WASP-family verprolin homologous protein complex-related gene depletions and corroborates the implication of RSF-1 in the regulation of actin cytoskeleton. In rsf-1 mutants, the structures of muscle actin are preserved, but the swimming ability is impaired. Although we could not detect the direct physical interaction of LET-60 with RSF-1, rsf-1 mutants suppress the multivulva phenotype of the active let-60 mutants, suggesting that rsf-1 genetically interacts with the Ras signaling.

Ali Khan L et al. (1996) Worm Breeder's Gazette "emb-8 mislocalizes Glp-1 and P-granules"

Siddiqui SS, Ali Khan L

[Worm Breeder's Gazette, 1996]

We have previously reported the transformation rescue of the emb-8(hc69) mutant with a cosmid containing the gene cej-1(C. elegans Meeting 1995, abstract 310). emb-8 is an embryonic lethal mutant at nonpermissive temperature (25oC). Cell division arrest between one cell to 50 cells stage. The egg shell is osmotically sensitive. Recently we have come to know from Sarah Crittenden( Judith Kimble Lab.) that emb-8 mislocalizes Glp-1 & P-granules specific antigens. We have earlier observed symmetric distribution of P-granules in a fraction of embryos using a monoclonal antibody specific to germ cells. These observation suggest a role of emb-8 in early cell divisions of the embryos.

Ali Khan L et al. (1997) International C. elegans Meeting "MATERNAL GENE EMB-8 IS NECESSARY FOR ASYMMETRIC DISTRIBUTION OF DEVELOPMENTAL POTENTIALS IN EARLY C. ELEGANS EMBRYOS"

Siddiqui SS, Ali Khan L, Ali MY, Miwa J, Kikuno R, Kohara Y, Siddiqui ZK

[International C. elegans Meeting, 1997]

A number of observations suggest that emb-8 gene plays critical roles in C. elegans development. At non-permissive temperature, emb-8 embryos divides symmetrically; in 50% the first division produces two equal sized cells, and in the second cleavage both of these cells divide symmetrically again to produce four equal sized cells. In some embryos, these four cells fuse togather and produce two asymmetric cells. emb-8 variably mislocalizes P-granules, they are uniformly distributed in 1-cell embryos, are localized on both anterior AB, and posterior P1 cells of the 2-cell embryos, and are found in all four blastomeres of the 4-cell embryos. Crittenden et al., (1996) also observed mislocalization of the P-granules and Glp-1 in emb-8 embryos. emb-8 also appears to control the orientation of early mitotic spindles, similar to the par-3 muatnts (K. Kemphues and others). However complementation tests show that emb-8 and par-3 are different genes. We thank Sarah Crittenden, Judith Kimble and Ken Kemphues for sharing unpublished results.

Dayong Wang (2003) International Worm Meeting "Exploring the roles of hlb-1, homologue of iprin protein SYD-2"

Dayong Wang

[International Worm Meeting, 2003]

Exploring the Roles of hlb-1, homologue of liprin protein SYD-2 Dayong Wang and Mei Zhen Samuel Lunenfeld Research Institute, Toronto, ON, Canada M5G 1X5 At synaptic junctions, the active zone, an electron-dense membrane structure, functions in vesicle docking and neurotransmitter release. Signaling mechanism that control the formation of the active zone and how active zone components regulate the neurotransmitter release, are not fully understood. SYD-2, a liprin family protein, localizes at the active zones, and restricts the area of active zones in C. elegans (Zhen and Jin, Nature 401:371-375, 1999). SYD-2 homologue in Drosophila may recruit receptor type tyrosine phosphatases to define areas for active zone development (Kaufmann et al., Neuron 34:27-38, 2002). It was also shown in vertebrates that a liprin protein directly interacts with RIM, another active zone component required for vesicle fusion (Schoch et al., Nature 415: 321-326, 2002). Despite its interaction with molecules essential for synaptic development and synaptic functions, the behavior phenotypes of syd-2 null animals are very mild. For example, active zones only show a 2-fold increase in size, and neurotransmission, as indicated by aldicarb tests, is only moderately defective. It is therefore possible other liprin homologue in C. elegans plays redundant roles. hlb-1(homologue of liprin-beta) encodes the only other liprin protein in C. elegans. HLB-1 protein has three coiled-coil domains, a putative trans-membrane domain and a SAM domain. Its SAM domain at the carboxy terminus is similar to that of SYD-2 protein, but its coiled-coil domain at the amino terminus is more divergent from that of SYD-2. We have generated a mutant in hlb-1 gene with deletion of the carboxy terminal region. The hlb-1 mutant animals exhibit a mild decrease in the thrashing frequency, moderate aldicarb resistance, and transient egg laying defects, suggesting that hlb-1 may be required for neuronal function. In the nervous system, we did not find axonal abnormalities, whereas there might be a more diffuse SNB-1::GFP vesicle marker distribution. Furthermore, the syd-2hlb-1 double mutant has more severe vesicle marker diffusion and acute aldicarb resistance than the syd-2 null mutant, or the hlb-1 mutants. We are analyzing the subcellular localization of HLB-1 protein, and if SYD-2 and HLB-1 functions together in presynaptic termini to regulate the development and function of active zones. (We acknowledge Dr. Joe Culotti and Sarah Bajurny for the deletion library and initial screen for hlb-1 mutants)