Caenorhabditis elegans SEL-5/AAK1 regulates cell migration and cell outgrowth independently of its kinase activity
- Department of Cell Biology, Faculty of Science, Charles University, Czech Republic
- Institute of Molecular Genetics, Czech Academy of Sciences, Czech Republic
Figures
Figure 1 with 2 supplements
Loss of sel-5 potentiates QL migration defect in retromer and Wnt pathway mutants.
(A) QL neuroblast lineage and a cartoon indicating the position of terminally differentiated neurons (depicted with empty circles). (A has been adapted from Figure 1 of Rella et al., 2016.) The …
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Figure 1—source data 1
Source gel for Figure 1.
- https://cdn.elifesciences.org/articles/91054/elife-91054-fig1-data1-v2.zip
Figure 1—figure supplement 1
Additional examples of RNAi against sel-5 in wild type and vps-29 background.
Results are shown as % of WT and QL.d animals. Fisher’s exact test was performed to assess the difference between the samples. Bonferroni correction for multiple testing was applied, n > 55 per …
Figure 1—figure supplement 2
Schematic structure of the sel-5 genomic locus.
The exons of sel-5 are represented by white rectangles and are numbered. Deletions found in the ok149 and ok363 alleles are shown.
Figure 2 with 1 supplement
SEL-5 is expressed in multiple tissues and is required cell non-autonomously for QL.d migration.
(A) Transgenic rescue of the QL.d migration defect. sel-5 was expressed under the control of various promoters from an extrachromosomal array in sel-5 vps-29; muIs32 background and the effect of …
Figure 2—figure supplement 1
Transgenic rescue of the QL.d migration defect.
Several independent transgenic strains for each rescue construct were tested in sel-5 vps-29; muIs32 background and the effect of such expression on QL.d migration was quantified. Comparison was …
Figure 3 with 1 supplement
SEL-5 alters the phosphorylation status of DPY-23 but does not affect MIG-14 levels.
(A) The level of DPY-23 phosphorylation at position T160 is reduced in sel-5 mutant animals. Phosphorylation was detected by western blot analysis in lysates from a population of L4/young adults of …
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Figure 3—source data 1
Source blots for Figure 3.
- https://cdn.elifesciences.org/articles/91054/elife-91054-fig3-data1-v2.zip
Figure 3—figure supplement 1
MIG-14 does not re-localize to the plasma membrane in sel-5 or sel-5 vps-29 mutants.
L2/L3 animals expressing MIG-14::GFP from the huSi2 transgene together with mCherry::PH driven by egl-20 promoter were imaged using confocal microscope. The posterior part of the body with …
Figure 4
EGL-20 gradient formation.
(A) Endogenously tagged EGL-20::GFP was visualized in various backgrounds. Images were acquired with spinning disc microscopy using L2/L3 animals and represent maximum projections of seven …
Figure 5 with 2 supplements
SEL-5 kinase activity and DPY-23 phosphorylation are not required for QL.d migration.
(A) Transgenic rescue of the QL.d migration defect with kinase-inactive SEL-5. D178A or K75A SEL-5 mutant protein was expressed from egl-20 promoter in sel-5 vps-29; muIs32 mutant background from an …
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Figure 5—source data 1
Source blots for Figure 5.
- https://cdn.elifesciences.org/articles/91054/elife-91054-fig5-data1-v2.zip
Figure 5—figure supplement 1
Sequence alignment of the kinase domain of SEL-5 and human AAK1.
Residues mutated in C. elegans rescue constructs are highlighted in grey. The black rectangle marks the conserved HRD motif.
Figure 5—figure supplement 2
Transgenic rescue of QL.d migration defect by kinase-inactive SEL-5.
Additional independent transgenic strains for each rescue construct were tested in sel-5 vps-29; muIs32 background and the effect of such expression on QL.d migration was quantified. Comparison was …
Figure 6
sel-5 cooperates with the retromer complex to regulate the length of excretory cell canals.
(A) Posterior canals of the excretory cell are significantly shortened in sel-5 vps-29 mutants. The excretory cell was visualized by Ppgp-12::gfp expression from a mamEx11 transgene. The scale bar …
Figure 7
Wnt pathway components determine the length of the posterior excretory canal.
(A) Posterior canals of the excretory cell overgrow into the tip of the tail in lin-17 mutants. The excretory cell was visualized by Ppgp-12::gfp expression from the sIs10089 transgene. Boxed areas …
Figure 8
Model summarizing the inputs acting in the excretory cell outgrowth.
(A) Three possible outcomes of the canal outgrowth depending on the genetic background. (B) Model summarizing the role of Wnt pathway components, sel-5, and the retromer in the outgrowth of the …
Tables
Table 1
Phenotypes of sel-5 and vps-29 single and double mutants.
| Genotype | ALM (%)* | PLM (%)† | CAN (%) ‡ | Dye filling (%) § | Fecundity (n) ¶ |
|---|---|---|---|---|---|
| Wild type | 0.0 | 0.0 | 0.6 | 0.5 | 280 ± 41 |
| sel-5(ok149) | 0.0 | 0.0 | 8.9 | 4.7 | 236 ± 24 |
| sel-5(ok363) | 0.0 | 0,0 | 10.9 | 1.0 | 249 ± 37 |
| vps-29(tm1320) | 0.0 | 0.0 | 2.9 | 4.3 | 221 ± 28 |
| sel-5(ok149) vps-29 | 29.9 | 9.1 | 17.7 | 17.4 | 132 ± 40 |
| sel-5(ok363) vps-29 | 7.2 | 2.8 | 16.3 | 7.1 | 159 ± 23 |
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*
% of animals with axons of one or both ALM reversed/bipolar, n > 60.
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†
% of animals with axons of one or both PLM neurons reversed, n > 60.
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‡
% of CAN neurons located anteriorly of V3 seam cell, n > 40.
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§
% of animals with one or both phasmid sensilla not dyed with DiI, n > 40.
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¶
Average number of progeny from five hermaphrodites,± s.d.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Caenorhabditis elegans) | Wild type | Caenorhabditis Genetics Center | N2 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | lin-17(n671) I | CGC, Brenner, 1974 | MT1306 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | lin-44(n1792) I | CGC, Herman and Horvitz, 1994 | MT5383 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | mig-1(e1787) I | CGC, Brenner, 1974 | CB3303 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | pry-1(mu38) I; him-5(e1490) V | CGC, Maloof et al., 1999 | CF491 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | cwn-1(ok546) II | CGC | RB763 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | dsh-1(ok1445) II | CGC | RB1328 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | mig-14(mu71) II | CGC, Harris et al., 1996 | CF367 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | mig-5(cp385[mNG-GLO^AID::mig-5]) II | CGC, Heppert et al., 2018 | LP728 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | vps-35(hu68) II | CGC, Coudreuse et al., 2006 | KN555 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | sel-5(ok149) III | CGC | GS2381 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | sel-5(ok363) III | CGC | RB638 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | egl-20(n585) IV | CGC, Harris et al., 1996 | MT1215 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | cwn-2(ok895) IV | CGC | VC636 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | cfz-2(ok1201) V | CGC | RB1162 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | muIs32[Pmec-7::gfp; lin-15(+)] II | CGC, Ch’ng et al., 2003 | CF702 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | huSi2[Pmig-14::mig-14::gfp] II | Lorenowicz et al., 2014 | KN1312 | Korswagen lab |
| Strain, strain background (C. elegans) | dpy-5(e907) X; sIs10089 III | CGC, McKay et al., 2003 | BC10210 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | lin-15AB(n765) kyIs4 [Pceh-23::unc-76::gfp+lin-15(+)] X | CGC, Zallen et al., 1999 | CX2565 | Strain can be obtained from CGC |
| Strain, strain background (C. elegans) | fcho-1(ox477) II; dpy-23(mew25) X | Gunther Hollopeter | GUN27 | Hollopeter lab |
| Strain, strain background (C. elegans) | vps-29(tm1320)III; muIs32 [Pmec-7::gfp] II | Yang et al., 2008 | Korswagen lab | |
| Antibody | Anti-α-tubulin antibody (mouse monoclonal) | Sigma-Aldrich | RRID:AB_477593 | WB (1:10,000) |
| Antibody | Anti-GFP antibody (mouse monoclonal) | Roche | RRID:AB_390913 | WB (1:4000) |
| Antibody | Anti-phosphoAP2M1 (rabbit monoclonal) | Abcam | RRID:AB_10866362 | WB (1:2500) |
| Antibody | Goat anti-mouse HRP-conjugated antibody (goat polyclonal) | Jackson ImmunoResearch Laboratories | RRID:AB_2307392 | WB (1:10,000) |
| Antibody | Goat anti-rabbit HRP-conjugated antibody (goat polyclonal) | Jackson ImmunoResearch Laboratories | RRID:AB_2337938 | WB (1:10,000) |
| Recombinant DNA reagent | pDD282 (plasmid) | Dickinson et al., 2013 | RRID:Addgene_66823 | |
| Recombinant DNA reagent | pDD162 (plasmid) | Dickinson et al., 2013 | RRID:Addgene_47549 | |
| Recombinant DNA reagent | pCFJ104 (plasmid) | Frøkjaer-Jensen et al., 2008 | RRID:Addgene_19328 | |
| Recombinant DNA reagent | pPD95.81 (plasmid) | Addgene (Andrew Fire) | RRID:Addgene_1497 | |
| Software, algorithm | Fiji image processing package | Schindelin et al., 2012 | ||
| Software, algorithm | Real Statistics Resource Pack, release 7.6 | Zaiontz, 2021, https://www.real-statistics.com | ||
| Software, algorithm | MATLAB | https://www.mathworks.com/ |
Additional files
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Supplementary file 1
List of all C. elegans strains generated.
- https://cdn.elifesciences.org/articles/91054/elife-91054-supp1-v2.xlsx
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Supplementary file 2
List of primers used to construct plasmid vectors.
- https://cdn.elifesciences.org/articles/91054/elife-91054-supp2-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/91054/elife-91054-mdarchecklist1-v2.docx
