Multiple antagonist calcium-dependent mechanisms control CaM kinase-1 subcellular localization in a C. elegans thermal nociceptor
- Department of Biology, University of Fribourg, Switzerland
Figures
Figure 1 with 1 supplement
Multiple nuclear export sequence (NES) and nuclear localization signal (NLS) elements work in concert to regulate CaM kinase-1 (CMK-1) localization.
(A) Schematic of CMK-1 with positions of the tested NES and NLS candidates and their sequence. (B–F) Subcellular localization of CMK-1::mNeonGreen reporters expressed in FLP and scored after 90 min …
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Figure 1—source data 1
Nuclear/cytoplasmic CaM kinase-1 (CMK-1) expression ratio raw data for Figure 1.
- https://cdn.elifesciences.org/articles/85260/elife-85260-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
CaM kinase-1 (CMK-1) localization data distributions and ANOVA results.
Violin plots with superimposed datapoints depicting the distribution of nucleus/cytoplasmic ratios of the indicated CMK-1::mNG reporters and robust ANOVA results. Red bars: average. (A) Data …
Figure 2 with 1 supplement
CaM kinase-1 (CMK-1) T179 phosphorylation is required for CMK-1 nuclear entry downstream of intracellular calcium elevation.
(A) Schematic of calcium-dependent CMK-1 activation pathway involving CaM binding and CaM kinase kinase-1 (CKK-1) phosphorylation on T179. Illustration of the tested mutations and their effects. (B–D…
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Figure 2—source data 1
Nuclear/cytoplasmic CaM kinase-1 (CMK-1) expression ratio raw data for Figure 2.
- https://cdn.elifesciences.org/articles/85260/elife-85260-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
CaM kinase-1 (CMK-1) localization data distributions and ANOVA results.
Violin plots with superimposed datapoints depicting the distribution of nucleus/cytoplasmic ratios of the indicated CMK-1::mNG reporters and robust ANOVA results. Red bars: average. (A) Data …
Figure 3 with 1 supplement
CaM kinase-1 (CMK-1) T179 phosphorylation promotes both NLS71-78-dependent import and NES288-294-dependent export.
(A) Schematic of calcium-dependent CMK-1 activation pathways involving CaM binding and CaM kinase kinase-1 (CKK-1) phosphorylation on T179. Illustration of the tested mutations and their effects. (B–…
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Figure 3—source data 1
Nuclear/cytoplasmic CaM kinase-1 (CMK-1) expression ratio raw data for Figure 3.
- https://cdn.elifesciences.org/articles/85260/elife-85260-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
CaM kinase-1 (CMK-1) localization data distributions and ANOVA results.
Violin plots with superimposed datapoints depicting the distribution of nucleus/cytoplasmic ratios of the indicated CMK-1::mNG reporters and robust ANOVA results. Red bars: average. (A) Data …
Figure 4 with 1 supplement
CaM kinase kinase-1 (CKK-1) subcellular localization in FLP.
(A) Schematic of ckk-1 gene locus showing genomic coordinates and the two predicted isoforms produced via alternative transcription start. Coding exon (black boxes), untranslated regions (gray …
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Figure 4—source data 1
Nuclear/cytoplasmic CaM kinase-1 (CMK-1) and CaM kinase kinase-1 (CKK-1) expression ratio raw data for Figure 4.
- https://cdn.elifesciences.org/articles/85260/elife-85260-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
CaM kinase kinase-1 (CKK-1) localization data distributions and ANOVA results.
Violin plots with superimposed datapoints depicting the distribution of nucleus/cytoplasmic ratios of the indicated CMK-1::mNG and CKK-1::mNG reporters and robust ANOVA results. Red bars: average. …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (C. elegans) | DAG439 | Ippolito et al., 2021 | domSi439[mec-3p::cmk-1 (1–348)::mNG::unc-54 3’UTR] II | Expression of CMK-1(wt)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG703-704-705 | Ippolito et al., 2021 | domEx703-704-705[mec-3p::cmk-1(V292A/V294A)::mNG, unc-122p::RFP] | Expression of CMK-1(V292A/V294A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1032 | Ippolito et al., 2021 | domSi439[mec-3p::cmk-1::mNG::3xFlag::unc-54 3’UTR] II;[unc-68(dom13)] V | Expression of CMK-1(wt)::mNG in FLP in unc-68 mutant background |
| Genetic reagent (C. elegans) | DAG900-901-902 | Ippolito et al., 2021 | domEx900-901-902[mec-3p::cmk-1(K71A/R74Q/R77S/V292A/V294A)::mNG, unc-122p::RFP] | Expression of CMK-1(K71A/R74Q/R77S/V292A/V294A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG700-701-702 | Ippolito et al., 2021 | domEx700-701-702[mec-3p::cmk-1(W305S)::mNG, unc-122p::RFP] | Expression of CMK-1(W305S)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG706-707-708 | Ippolito et al., 2021 | domEx706-707-708[mec-3p::cmk-1(K71A/R74Q/R77S)::mNG, unc-122p::RFP] | Expression of CMK-1(K71A/R74Q/R77S)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG709-710-711-1134-1135-1136 | This study | domEx709-710-711-1134-1135-1136 [mec-3p::cmk-1(T179D)::mNeonGreen; unc-122p::RFP] | Expression of CMK-1(T179D)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG744-745-746-812-813 | This study | domEx744-745-746-812-813[mec-3p::cmk-1(T179A)::mNeonGreen; unc-122p::RFP] | Expression of CMK-1(T179A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG906-907-908 | This study | domEx906-907-908[mec-3p::cmk-1(K71A/R74Q/R77S/ T179D)::mNeonGreen, unc-122p::RFP] | Expression of CMK-1(K71A/R74Q/ R77S/T179D)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG909-910-911 | This study | domEx909-910-911[mec-3p::cmk-1(T179D/W305S)::mNeonGreen, unc-122p::RFP] | Expression of CMK-1(W305S/T179D)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG924-925-926 | This study | domEx924-925-926[mec-3p::cmk-1(T179A/V292A/V294A)::mNeonGreen, unc-122p::RFP] | Expression of CMK-1(T179A/ V292A/V294A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1015-1016-1017 | This study | domEx1015-1016-1017[mec-3p::cmk-1(T179A/W305S)::mNeonGreen, unc-122p::RFP] | Expression of CMK-1(W305S/T179A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1600-1601-1602 | This study | domEx1600-1601-1602[mec-3p::cmk-1(S325D)::mNeonGreen; unc-122p::RFP] | Expression of CMK-1(S325D)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1621-1622 | This study | domEx1621-1622[mec-3p::cmk-1(S325A)::mNeonGreen; unc-122p::RFP] | Expression of CMK-1(S325A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1727-1728-1729 | This study | domEx1727-1728-1729[mec-3p]::cmk-1(T179D/V292A/V294A)::mNeonGreen; unc-122p::RFP | Expression of CMK-1(T179D/V292A/V294A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1733-1734-1735 | This study | domEx1733-1734-1735[mec-3p::cmk-1(K71A/R74Q/R77S/V292A/V294A/L321A/L323A)::mNeonGreen; unc-122p::RFP] | Expression of CMK-1(K71A/R74Q/R77S/V292A/V294A/L321A/L323A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1739-1740-1741 | This study | domEx1739-1740-1741[mec-3p::cmk-1(K71A/R74Q/R77S/V292A/V294A/K307Q)::mNeonGreen; unc-122p::RFP] | Expression of CMK-1(K71A/R74Q/R77S/V292A/V294A/K307Q)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1748-1749-1750 | This study | domEx1748-1749-1750[mec-3p::QF]; [QUAS::ckk-1b::gfp]; [unc-122p::RFP] | Expression of CKK-1b::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1751-1752-1753 | This study | domEx1751-1752-1753[mec-3p::QF]; [QUAS::ckk-1a::gfp]; [unc-122p::RFP] | Expression of CKK-1a::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1793-1794-1795 | This study | domEx1793-1794-1795[mec-3p::cmk-1(V292A/V294A/S325A)::mNeonGreen; unc-122p::RFP] | Expression of CMK-1(V292A/V294A/S325A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1787-1788-1789 | This study | domEx1787-1788-1789[mec-3p::cmk-1(K71A;R74Q;R77S;V292A;V294A;S325A)::mNG]; [unc-122p::RFP] | Expression of CMK-1(K71A/R74Q/R77S/V292A/V294A/S325A)::mNG in FLP |
| Genetic reagent (C. elegans) | DAG1430-1431-1432 | This study | domEx1430-1431-1432[mec-3p::cmk-1(T179A)::mNeonGreen, unc-122p::RFP];[unc-68(dom13)]V | Expression of CMK-1(T179A)::mNG in FLP in unc-68 mutant background |
Additional files
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Supplementary file 1
Plasmid name, cloning, and primer information.
- https://cdn.elifesciences.org/articles/85260/elife-85260-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85260/elife-85260-mdarchecklist1-v1.pdf
