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MTM-6, a phosphoinositide phosphatase, is required to promote synapse formation in Caenorhabditis elegans.

Ericson VR, Spilker KA, Tugizova MS, Shen K - PLoS ONE (2014)

Bottom Line: We found that loss of function of the phosphoinositide phosphatase mtm-6 results in a reduction in the number of synaptic puncta.The reduction in synapses is partially the result of MTM-6 regulation of the secretion of the Wnt ligand EGL-20 from cells in the tail and partially the result of neuronal action.We conclude that regulation of secretion of different Wnt ligands can use different components.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Howard Hughes Medical Institute, Stanford University, Stanford, California, United States of America.

ABSTRACT
Forming the proper number of synapses is crucial for normal neuronal development. We found that loss of function of the phosphoinositide phosphatase mtm-6 results in a reduction in the number of synaptic puncta. The reduction in synapses is partially the result of MTM-6 regulation of the secretion of the Wnt ligand EGL-20 from cells in the tail and partially the result of neuronal action. MTM-6 shows relative specificity for EGL-20 over the other Wnt ligands. We suggest that the ability of MTM-6 to regulate EGL-20 secretion is a function of its expression pattern. We conclude that regulation of secretion of different Wnt ligands can use different components. Additionally, we present a novel neuronal function for MTM-6.

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Related in: MedlinePlus

Loss of mtm-6 causes a reduction in the number of DA9 presynaptic puncta.(A) Schematic of DA9. The dendrite of DA9 extends anterior from a cell body in the ventral side of the animal. The axon of DA9 extends towards the posterior before extending a commissure to the dorsal nerve cord, where the axon turns and continues towards the anterior of the animal. Synapses form with a stereotyped number and location on to muscle and Ventral D neurons. (B) Quantification of the number of puncta formed in wild-type and mtm-6 (ok330) animals using GFP::RAB-3 to mark presynapses and UNC-10::GFP as a marker for active zones (n>60, **** is p<0.0001). (C-F) mtm-6 (ok330) animals show a reduced number of dorsal synaptic puncta when compared to wild-type animals for the GFP::RAB-3 and UNC-10::GFP markers. (C) Wild-type; GFP::RAB-3. (D) Wild-type; UNC-10::GFP. (E) mtm-6(ok330); GFP::RAB-3. (F) mtm-6(ok330); UNC-10::GFP. The UNC-10::GFP puncta on the ventral side of the animal correspond to the VA12 motor neuron. The dashed boxes mark the regions that are enlarged for the insets above each window. Error bars are SD. Scale bar is 10 µm.
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pone-0114501-g001: Loss of mtm-6 causes a reduction in the number of DA9 presynaptic puncta.(A) Schematic of DA9. The dendrite of DA9 extends anterior from a cell body in the ventral side of the animal. The axon of DA9 extends towards the posterior before extending a commissure to the dorsal nerve cord, where the axon turns and continues towards the anterior of the animal. Synapses form with a stereotyped number and location on to muscle and Ventral D neurons. (B) Quantification of the number of puncta formed in wild-type and mtm-6 (ok330) animals using GFP::RAB-3 to mark presynapses and UNC-10::GFP as a marker for active zones (n>60, **** is p<0.0001). (C-F) mtm-6 (ok330) animals show a reduced number of dorsal synaptic puncta when compared to wild-type animals for the GFP::RAB-3 and UNC-10::GFP markers. (C) Wild-type; GFP::RAB-3. (D) Wild-type; UNC-10::GFP. (E) mtm-6(ok330); GFP::RAB-3. (F) mtm-6(ok330); UNC-10::GFP. The UNC-10::GFP puncta on the ventral side of the animal correspond to the VA12 motor neuron. The dashed boxes mark the regions that are enlarged for the insets above each window. Error bars are SD. Scale bar is 10 µm.

Mentions: The C. elegans motor-neuron DA9 has a stereotyped synapse localization and number. The axon of DA9 extends posteriorly from a ventral cell body and reaches the dorsal nerve cord through a commissure. Within the dorsal nerve cord, the axon extends anteriorly and forms a string of en passant synapses in a specific region along the axon that begins approximately 25 µm anterior to the dorsal commissural turn in adult animals (Fig. 1A). The DA9 synapses can be labeled by cell-specific expression of GFP fused with synaptic vesicle or active zone proteins [56]. During the search for genes required for proper development of the DA9 synapses, we found that a mutant for a myotubularin phosphatase, mtm-6(ok330), shows a reduction in synapse number (Fig. 1C-F). Using GFP::RAB-3 as a marker for synapses, we observed that in wild-type animals there are an average of 22.4 (+/−2.6) puncta within the dorsal cord. Both the number and the subcellular distribution pattern of the GFP::RAB-3 puncta closely agree with what was reported from serial electron microscopy reconstruction studies, suggesting that the RAB-3 marker represents true synapses. We found that the number of DA9 synapses is reduced to 14.4 (+/−2.8) puncta in mtm-6 mutant animals (Fig. 1B and Table S1, n>60, SD, p<0.0001). The reduction in synapse number is also present when synapses are visualized with an active zone marker: quantifying GFP::UNC-10, the C.elegan's RIM homolog, we observed that mtm-6 mutants have an average of 14.4 (+/−3.3) puncta in comparison to the 21.6 (+/−3.4) puncta found in wild-type (Fig. 1B and Table S1, n>60, SD, p<0.0001). The mtm-6(ok330) allele is a 1,235 bp deletion that is predicted to cause a frameshift towards the end of the phosphatase that affects all four isoforms of the gene. Due to the temperature sensitive nature of some mutants used in the paper and the temperature sensitivity of the observed phenotypes (as marked by GFP::RAB-3 or GFP::UNC-10) all animals were maintained at 20°C for at least two generations prior to quantification.


MTM-6, a phosphoinositide phosphatase, is required to promote synapse formation in Caenorhabditis elegans.

Ericson VR, Spilker KA, Tugizova MS, Shen K - PLoS ONE (2014)

Loss of mtm-6 causes a reduction in the number of DA9 presynaptic puncta.(A) Schematic of DA9. The dendrite of DA9 extends anterior from a cell body in the ventral side of the animal. The axon of DA9 extends towards the posterior before extending a commissure to the dorsal nerve cord, where the axon turns and continues towards the anterior of the animal. Synapses form with a stereotyped number and location on to muscle and Ventral D neurons. (B) Quantification of the number of puncta formed in wild-type and mtm-6 (ok330) animals using GFP::RAB-3 to mark presynapses and UNC-10::GFP as a marker for active zones (n>60, **** is p<0.0001). (C-F) mtm-6 (ok330) animals show a reduced number of dorsal synaptic puncta when compared to wild-type animals for the GFP::RAB-3 and UNC-10::GFP markers. (C) Wild-type; GFP::RAB-3. (D) Wild-type; UNC-10::GFP. (E) mtm-6(ok330); GFP::RAB-3. (F) mtm-6(ok330); UNC-10::GFP. The UNC-10::GFP puncta on the ventral side of the animal correspond to the VA12 motor neuron. The dashed boxes mark the regions that are enlarged for the insets above each window. Error bars are SD. Scale bar is 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4257696&req=5

pone-0114501-g001: Loss of mtm-6 causes a reduction in the number of DA9 presynaptic puncta.(A) Schematic of DA9. The dendrite of DA9 extends anterior from a cell body in the ventral side of the animal. The axon of DA9 extends towards the posterior before extending a commissure to the dorsal nerve cord, where the axon turns and continues towards the anterior of the animal. Synapses form with a stereotyped number and location on to muscle and Ventral D neurons. (B) Quantification of the number of puncta formed in wild-type and mtm-6 (ok330) animals using GFP::RAB-3 to mark presynapses and UNC-10::GFP as a marker for active zones (n>60, **** is p<0.0001). (C-F) mtm-6 (ok330) animals show a reduced number of dorsal synaptic puncta when compared to wild-type animals for the GFP::RAB-3 and UNC-10::GFP markers. (C) Wild-type; GFP::RAB-3. (D) Wild-type; UNC-10::GFP. (E) mtm-6(ok330); GFP::RAB-3. (F) mtm-6(ok330); UNC-10::GFP. The UNC-10::GFP puncta on the ventral side of the animal correspond to the VA12 motor neuron. The dashed boxes mark the regions that are enlarged for the insets above each window. Error bars are SD. Scale bar is 10 µm.
Mentions: The C. elegans motor-neuron DA9 has a stereotyped synapse localization and number. The axon of DA9 extends posteriorly from a ventral cell body and reaches the dorsal nerve cord through a commissure. Within the dorsal nerve cord, the axon extends anteriorly and forms a string of en passant synapses in a specific region along the axon that begins approximately 25 µm anterior to the dorsal commissural turn in adult animals (Fig. 1A). The DA9 synapses can be labeled by cell-specific expression of GFP fused with synaptic vesicle or active zone proteins [56]. During the search for genes required for proper development of the DA9 synapses, we found that a mutant for a myotubularin phosphatase, mtm-6(ok330), shows a reduction in synapse number (Fig. 1C-F). Using GFP::RAB-3 as a marker for synapses, we observed that in wild-type animals there are an average of 22.4 (+/−2.6) puncta within the dorsal cord. Both the number and the subcellular distribution pattern of the GFP::RAB-3 puncta closely agree with what was reported from serial electron microscopy reconstruction studies, suggesting that the RAB-3 marker represents true synapses. We found that the number of DA9 synapses is reduced to 14.4 (+/−2.8) puncta in mtm-6 mutant animals (Fig. 1B and Table S1, n>60, SD, p<0.0001). The reduction in synapse number is also present when synapses are visualized with an active zone marker: quantifying GFP::UNC-10, the C.elegan's RIM homolog, we observed that mtm-6 mutants have an average of 14.4 (+/−3.3) puncta in comparison to the 21.6 (+/−3.4) puncta found in wild-type (Fig. 1B and Table S1, n>60, SD, p<0.0001). The mtm-6(ok330) allele is a 1,235 bp deletion that is predicted to cause a frameshift towards the end of the phosphatase that affects all four isoforms of the gene. Due to the temperature sensitive nature of some mutants used in the paper and the temperature sensitivity of the observed phenotypes (as marked by GFP::RAB-3 or GFP::UNC-10) all animals were maintained at 20°C for at least two generations prior to quantification.

Bottom Line: We found that loss of function of the phosphoinositide phosphatase mtm-6 results in a reduction in the number of synaptic puncta.The reduction in synapses is partially the result of MTM-6 regulation of the secretion of the Wnt ligand EGL-20 from cells in the tail and partially the result of neuronal action.We conclude that regulation of secretion of different Wnt ligands can use different components.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Howard Hughes Medical Institute, Stanford University, Stanford, California, United States of America.

ABSTRACT
Forming the proper number of synapses is crucial for normal neuronal development. We found that loss of function of the phosphoinositide phosphatase mtm-6 results in a reduction in the number of synaptic puncta. The reduction in synapses is partially the result of MTM-6 regulation of the secretion of the Wnt ligand EGL-20 from cells in the tail and partially the result of neuronal action. MTM-6 shows relative specificity for EGL-20 over the other Wnt ligands. We suggest that the ability of MTM-6 to regulate EGL-20 secretion is a function of its expression pattern. We conclude that regulation of secretion of different Wnt ligands can use different components. Additionally, we present a novel neuronal function for MTM-6.

Show MeSH
Related in: MedlinePlus