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Cytoplasmic Adaptor Protein MIG-10 Interacts With Abelson Target ABI-1 During Neuronal Migration In C. ElegansFlaherty, Erin 01 May 2014 (has links)
Cellular migration is an essential process for establishing neural connections during development. The MIG-10/RIAM/Lamellipodin signaling proteins are thought to send positional information from guidance cues to actin polymerization machinery, promoting the polarized outgrowth of axons. In C. elegans, mutations in the gene mig-10 result in the truncation of the migration of the mechanosensory neurons. Biochemical analysis demonstrates that MIG-10 interacts with abelson-interactor protein 1 (ABI-1), and therefore investigation into whether these proteins work together in the neuron to promote migration was completed. To demonstrate MIG-10 cell autonomy in the neuron, transgenic strains with specific expression of mig-10 were created. mig-10 mutants were rescued in the mechanosensory, anterior lateral microtubule neuron (ALM) by neuron specific expression of mig-10 but not by epithelial expression, suggesting that MIG-10 is acting cell autonomously. To determine ABI-1 cell autonomy, transgenic strains with specific neuronal expression of abi-1 were compared to the wild type strain. abi-1 mutants were rescued by neuron specific expression of abi-1 in the ALM, suggesting that ABI-1 also functions cell autonomously in the ALM during this migration. Further investigation into the MIG-10/ABI-1 relationship was done by feeding RNAi of abi-1 in a mig-10(ct41) mutant strain. The ALM migration was not more severely truncated in the double mutant, suggesting that MIG-10 and ABI-1 work in the same pathway. Taken together, this evidence supports a model where MIG-10 and ABI-1 work together autonomously within the ALM to promote migration.
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Two genes, dig-1 and mig-10, involved in nervous system development in C. elegansBurket, Christopher T 15 November 2002 (has links)
"We are using genetic and molecular techniques to study a simple model organism, C. elegans, to determine the cues involved in the formation of the nervous system. Two molecules currently being studied in the laboratory play roles in the formation of the IL2 neurons, a class of sensory neurons in C. elegans. The first gene, dig-1, influences the sensory process or dendrite and is involved in adhesion as well as potentially providing directional information during development. The second gene, mig-10, influences the axon and may be involved in a cell signal cascade. Genetic screens of C. elegans using Ethyl methyl sulfonate (EMS) as a mutagen resulted in the isolation of mutants with defects in the IL2 sensory map; sensory processes followed aberrant paths, appearing to be defasciculated. Complementation tests showed that the mutations failed to complement n1321, a known allele of dig-1; thus, these new mutations were alleles of dig-1 (Ryder unpub. results). Several of these new alleles of dig-1, including nu336 and n1480, have been further studied to elucidate the role of this gene in sensory map formation. A dig-1 candidate gene was identified that encodes a protein that is a member of the immunoglobulin super-family (IgSF). The candidate gene is predicted to be a large gene, with a transcript of approximately 45Kb. The encoded protein contains three distinct regions and is similar to the hyalectan family of proteoglycans. N terminal region 1 contains immunoglobulin and fibronectin-like domains. Central region 2 is an area that is highly repeated with a potential to have GAGs attached. C-terminal region 3 contains domains associated with adhesion. Polymerase chain reaction (PCR) products from alleles nu336 and n1480 were amplified and sequenced from the candidate gene. The DNA lesion present in the candidate gene from both alleles fit the method for how that mutation was generated. The point mutation in allele nu336 removes a potential glycosylation site. The large re-arrangement in allele n1480 truncates the transcript, suggesting that the protein is also truncated. The sequencing results along with rescuing data (R. Proenca, personal communication) showed that the candidate gene for dig-1 was the gene of interest. Each of the alleles was further studied to determine how severe that allele was by looking at the neuronal process aspect and the brood size as well as displacement of the gonad. In general, alleles with severe defects in the nervous system also had severe gonad displacement, suggesting the gene functions similarly in the two tissues. To determine if the gene was expressed at the RNA level, reverse transcriptase polymerase chain reaction (RT-PCR) was used. Most of the RT-PCRs amplified a cDNA of the appropriate size that showed dig-1 was expressed at the RNA level. RT-PCR further suggested that all three regions were in one transcript as well as confirming part of the predicted exon structure to be correct. In addition, northern analysis showed the presence of a large transcript in wildtype worms as well as a smaller truncated transcript from allele n1480. To investigate developmental differences mixed stage of RNA and embryonic RNA from wildtype animals were compared using gene specific primers. The initial RT-PCR showed potential alternative splicing occurring at the 5? end of the gene during development. To examine expression at the protein level, two recombinant proteins from dig-1 were successfully made by cloning cDNA products from the 5?and 3? end of dig-1. The constructs were sequenced and shown to be in frame. The recombinant proteins (Ant1Con1 and Ant3Con3) were mass produced and sent to a commercial source for injection into pre-screened rabbits. Western analysis showed the presence of an antibody in the serum from two of the rabbits. These antibodies should prove useful in future determination of correctness of our models of DIG-1 function. IgSF members have been shown to have many roles in nervous system development. DIG-1 could act in either an attractive or a repellent role to position sensory processes during development. DIG-1 might also change its function over time; early in development DIG-1 could be adhesive and later become repellant as more sugars are added. The gene mig-10 is involved in sensory map formation. To localize MIG-10 expression, several transgenic animals were generated by injection of two constructs that should recombine in the worm to create a MIG-10::GFP fusion protein. Ten transgenic lines were generated and screened by PCR for the presence of the correct recombinant construct. If this construct makes functional, rescuing protein, the GFP expression should reflect the expression pattern of the MIG-10 protein."
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Neuronal Migration: Investigating Interactions of the Cytoplasmic Adaptor Protein MIG-10 in <i>C. elegans</i>Ficociello, Laura Faraco 09 January 2008 (has links)
Neuronal migration is an essential aspect of nervous system development; improper or incomplete neuronal migration can lead to debilitating disorders. The model organism Caenorhabditis elegans has 302 neurons and is ideal for studying nervous system development. The cytoplasmic adaptor protein, MIG-10, is necessary for the long range anteroposterior migration during embryogenesis of the neurons CAN, ALM, and HSN. Mutations in the mig-10 gene result in incomplete migrations of all three neurons. MIG-10 is a homologue of the vertebrate proteins lamellipodin and RIAM-1, which are involved in directing actin polymerization during axon outgrowth and guidance. RIAM-1 is known to interact with proteins from the Ras GTPase family. The MIG-10 protein has a pleckstrin homology (PH) domain, a Ras-associating (RA) domain, and a proline-rich region. We used a yeast two-hybrid system to investigate which Ras family proteins MIG-10 interacts with. Three isoforms of MIG-10, MIG-10A, MIG-10B, and MIG-10C, as well as the RAPH domain alone, were used as baits. No evidence of interaction was observed for any of the baits used. These results do not reject our hypothesis as the constitutively active Ras clones may need to be used or there may not be a direct interaction between MIG-10 and the Ras family members. We are currently screening a C. elegans cDNA library for interactions with all three isoforms of MIG-10. In the future we plan to investigate how MIG-10 may be involved in the WAVE/SCAR actin nucleation pathway.
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Analysis of mig-10, a Gene Involved in Nervous System Development in Caenorhabditis elegansStovall, Elizabeth L. 30 April 2004 (has links)
The mig-10 gene in C. elegans is required for proper axon guidance and/or cell migration of certain neurons during development. In mig-10 (ct41) mutant worms, there is incomplete migration of the anterior lateral microtubule cells (ALMs), hermaphrodite specific neuron (HSN), left coelomocyte cells (ccL), and canal associated neuron (CAN) (Manser and Wood, 1990). The mig-10 (ct41) mutation also causes axon guidance defects in the IL2 neurons, and it enhances unc-6 defects in the axon guidance of the anterior ventral microtubule cell (AVM) (Rusiecki, 1999; C. Quinn, personal communication). mig-10's function in axon guidance and neuronal migration is unknown, but is believed to be involved in a signal transduction pathway that uses a G-protein, such as ras. The two mig-10 transcripts discussed in this thesis, mig-10 A and mig-10 B, encode proteins that are similar to Grb-7 and Grb-10 proteins, which are also believed to function in a signal transduction pathway (Manser et al., 1997). One of these similarities is the presence of a proline-rich region, which may be used to bind another protein (Manser et al., 1997). The MIG-10 A protein has an additional proline region, compared to MIG-10 B, which may indicate that the MIG-10 A and B proteins are utilized in different cells, or at different developmental stages. As a first step in learning where MIG-10 is expressed, mig-10 (ct41) mutant worms containing a wild-type mig-10 B::GFP fusion were constructed. Rescue of the mutant phenotype would indicate that the expression pattern of the transgene was similar to that of the endogenous gene. As this experiment did not allow for rescue, even after integration of the construct, a strain of worms containing a mig-10 promoter::GFP transgene was used. Preliminary observations of this strain indicated that mig-10 is expressed in neuronal tissue. The AIY neurons were observed in wild-type and mig-10 (ct41) worms to determine if they are affected by the mig-10 mutation as previously reported (O. Hobert, personal communication). As no difference was detected, the AIYs were not used in any further experiments. In order to determine which cells require functional MIG-10 protein for the proper development/migration of neurons to occur, mig-10 (ct41) worms containing mec-3 promoter::mig-10 A or B cDNA transgenes were constructed. The mec-3 promoter drives expression of the mig-10 cDNA in the ALM neurons and other touch cells early in the development of the embryo. If these transgenes rescued the ALM migration defect, then mig-10 would be acting cell autonomously in ALM. Partial rescue was obtained, which may be due to the need for both of the mig-10 transcripts to be expressed in the same cell; alternatively, one or both transcripts may need to be expressed in a cell nonautonomous fashion in addition to being expressed cell autonomously. Low production of the rescuing protein, or expression of the protein at a later developmental stage than is needed for rescue to occur, may also have been the cause of the partial rescue. Future work in this area includes putting mig-10 promoter::mig-10 A or B cDNA in mig-10 (ct41) background to investigate if the different transcripts rescue different aspects of the mig-10 phenotype. The mig-10 A and mig-10 B cDNA constructs could also be expressed in the same worm in an attempt to correct for partial rescue that may be due to the lack of both MIG-10 proteins.
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