Valenzuela MRL et al. (1991) International C. elegans Meeting "STRUCTURES AND FUNCTIONS OF THE REPEATING MOTIFS IN TWITCHIN."

Valenzuela MRL, Benian GM

[International C. elegans Meeting, 1991]

Twitchin, the 753,570 Da polypeptide encoded by the unc-22 gene, was the first member of a growing family of intracellular and mostly muscle proteins, found in diverse animals, composed of multiple copies of motif I (fibronectin type III domain-like) and motif II ( immunoglobulin C2 domain-like). To date, this family includes, smooth muscle and non-muscle myosin light chain kinases, titin, C-protein, 86 kDa protein, skelemin and probably insect projectin. The similarities to motifs found in extracellular and cell surface proteins engaged in recognition or adhesion suggests that motifs I and II of muscle proteins are also involved in binding--probably to myosin, other thick filament components, and also binding of these proteins to themselves and other family members. We hope to be able to demonstrate binding of small numbers of these motifs to thick filament components both in vitro and in vivo. Crystallographic structure determination will be done in collaboration with Pamela Bjorkman (Cal Tech). Using the pGEX- 2T vector, we have been able to express and purify large quantities of motif I, motif II and the predominant triplet I,I,II as glutathione-S- transferase (GST) fusion proteins in E. coli. We have succeeded with the thrombin cleavage of the motifs from GST and are trying ELISA-type binding assays with nematode and rabbit myosin. For in vivo 'binding' studies, we have cloned motif I, motif II, the trio I,I,II and the C- terminal 5 motif IIs into Andy Fire's pPD30.38 vector which directs body wall muscle expression. These constructs will be microinjected into unc-22 nulls and transgenics examined by immunofluorescence for co-localization to myosin.

Valenzuela MRL et al. (1993) International C. elegans Meeting "The association of twitchin with isolated nematode thick filaments."

Valenzuela MRL, Benian GM

[International C. elegans Meeting, 1993]

Benian GM et al. (1989) International C. elegans Meeting "the unc-22 polypeptide defines two highly conserved motifs found in several muscle proteins."

Benian GM, Neckelmann N, Valenzuela MRL

[International C. elegans Meeting, 1989]

Lei JY et al. (1991) International C. elegans Meeting "STUDIES ON THE PROTEIN KINASE DOMAIN OF TWITCHIN."

Benian GM, Valenzuela MRL, Lei JY

[International C. elegans Meeting, 1991]

Previously, we recognized that a portion of twitchin has convincing homology to protein kinase catalytic domains, especially to myosin light chain kinases (MLCK). Moreover, the organization of motifs I and II surrounding the catalytic domain is conserved among twitchin, smooth muscle and nonmuscle MLCK. This suggested that at least one function of twitchin is to phosphorylate a muscle protein, probably the regulatory myosin light chain (MLC). Since the phosphorylation of regulatory MLCs is necessary for initiating muscle contraction in vertebrate smooth muscle and non-muscle tissue, this might explain 'twitching' as improperly regulated contraction. We have now expressed the kinase domain plus the adjacent motif II in E. coli using the pGEX- 3X vector. This glutathione-Stransferase fusion protein has Ca+2- dependent kinase activity to several serine-threonine containing substrates in vi tro . In order to determine the substrates of the twitchin kinase in vivo, we are comparing wild type vs. unc-22 null actomyosin preparations. So far, on a 2D gel, we find several spots at 18,000 Da (based on Cummins and Anderson's DNA sequences the likely size of the regulatory MLCs) that in the unc-22 null, ct37, have shifted compared to wild type, to more positive isoelectric points consistent with decreased phosphorylation. Soon we will label worms with 32p and compare the state of phosphorylation of MLCs. To explore the functions of the motifs surrounding the catalytic domain, we have placed the kinase coding sequence alone and in combination with various numbers of motifs into Andy Fire's pPD30.38 vector which directs specific expression in body wall muscle. By creating transgenics in an unc-22 null background, we will test the hypothesis that the motifs localize the kinase domain to the myosin head where the presumed substrates reside and also find out whether such 'mini- twitchins' might rescue some aspect of the unc-22 phenotype.

McShea M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "The MRL signaling protein MIG-10/Lpd interacts with ABI-1 during guided migrations in C. elegans"

Ryder E F, Dubuke M, Sullivan-Keiser J, Grant C, McShea M, Stringham E G, Schmidt K L

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Neuronal and axonal migration are vital to the establishment of proper neuronal connections during development. The process of directed migration is dependent on reorganization of the actin cytoskeleton in response to external guidance cues. The MRL signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons during nervous system development (Quinn and Wadsworth, 2008). In C. elegans, mutations in the MRL family member mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or 'canals' of the excretory cell, which is required for osmoregulation in the worm. To determine more of the molecular partners of MIG-10, we conducted a yeast two hybrid screen using isoform MIG-10A as bait. ABI-1, a downstream target of ABL non-receptor tyrosine kinase, was independently isolated 6 times as a strong MIG-10 interactor. ABI-1 is known to be part of the WAVE complex involved in the initiation of actin polymerization. Single mutants for mig-10 and abi-1 display similar phenotypes of incomplete migration of the ALM neurons and the excretory cell process. Furthermore, abi-1(RNAi) enhanced the excretory canal truncation observed in mig-10 mutants. Using both the yeast two-hybrid and a co-immunoprecipitation system, we are identifying domains within MIG-10 and ABI-1 that are necessary for their interaction. Our results suggest that MIG-10 and ABI-1 act together to link cell surface signaling with polarized actin polymerization.

McShea, M. et al. (2011) International Worm Meeting "MIG-10/Lamellipodin and Abelson-interactor-1(ABI-1) act together to mediate directed migration and outgrowth in C. elegans."

Zhang, S., Kuhlwein, M., Hashmi, S., Ryder, E.F., McShea, M., Dubuke, M., Schmidt, K.L., Grant, C., Stringham, E.G.

[International Worm Meeting, 2011]

Neuronal and axonal migration are vital to the establishment of proper neuronal connections during development. The process of directed migration is dependent on reorganization of the actin cytoskeleton in response to external guidance cues. The MRL signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons during nervous system development (Quinn and Wadsworth, 2008). In C. elegans, mutations in the MRL family member mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or 'canals' of the excretory cell, which is required for osmoregulation in the worm. To determine more of the molecular partners of MIG-10, we conducted a yeast two hybrid screen using MIG-10 as bait. ABI-1, a downstream target of ABL non-receptor tyrosine kinase, was independently isolated 6 times as a strong MIG-10 interactor. ABI-1 is known to be part of the WAVE complex involved in the initiation of actin polymerization. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncation of the excretory canal. Furthermore, abi-1(RNAi) enhanced the excretory canal truncation observed in mig-10 mutants. Using both the yeast two-hybrid and a co-immunoprecipitation system, we are identifying domains within MIG-10 and ABI-1 that are necessary for their interaction. We have demonstrated that cell autonomous expression of MIG-10 in the excretory cell rescues the canal truncation phenotype, consistent with MIG-10 and ABI-1 functioning together in this cell. Our results suggest that MIG-10 and ABI-1 act together to link cell surface signaling with polarized actin polymerization.