DAPK interacts with Patronin and the microtubule cytoskeleton in epidermal development and wound repair
- University of California, San Diego, United States
Figures
Figure 1 with 1 supplement
dapk-1 epidermal phenotypes are modified by mutations in cytoskeletal regulating genes.
(A) Anterior epidermal morphology of wild type adult and aberrant morphology (Mor phenotype, red arrowhead) of dapk-1(ju4) animals; suppression by ptrn-1(ju698) and dhc-1(ju697). DIC images; scale …
Figure 1—figure supplement 1
Partial suppression of dapk-1 phenotypes by loss of function in cytoskeletal regulators
(A) Dissection microscope images of WT and Mor animals. (B) Schematic of DAPK-1 protein showing domains and locations of previous and newly identified alleles. (C) Suppression of dapk-1(ju4) Mor …
Figure 2 with 1 supplement
ptrn-1 suppresses dapk-1(ju4) innate immune and wound repair phenotypes.
(A) dapk-1(ju4) animals display an elevated expression of the antimicrobial peptide nlp-29 (Pnlp-29-GFP, frIs7); this is suppressed by ptrn-1(0), whereas dhc-1(or195) and other mutations do not …
Figure 2—figure supplement 1
Most dapk-1 modifiers do not affect the dapk-1(ju4) constitutively active innate immune response.
(A) Loss of function in unc-116, sma-1, or klp-7 does not drastically modify the elevated expression of the Pnlp-29-GFP transcriptional reporter in dapk-1(ju4) mutants. (B) dhc-1(or195) is epistatic …
Figure 3 with 1 supplement
dapk-1 epidermal morphology defects are mimicked or enhanced by MT stabilization and suppressed by MT depolymerization.
(A) MT stabilization by paclitaxel treatment induces dapk-1-like morphological defects in the wild type and in ptrn-1(0) mutants, and enhances the morphological defects of dapk-1(gk219). Colchicine …
Figure 3—figure supplement 1
Effects of MT-altering drugs on epidermal morphology and innate immune responses in mutant backgrounds.
(A) MT modulation of epidermal morphology in dapk-1 mutants. Colchicine suppresses the epidermal morphology defects of dapk-1(gk219) cat-4 in a dose-dependent manner. dhc-1(or195) and dapk-1(gk219) …
Figure 4 with 1 supplement
dapk-1 defects in epidermal MT architecture are suppressed by ptrn-1.
(A) ptrn-1(0) and dapk-1(ju4) display distinct effects on epidermal MT organization; MTs visualized in young adults (Pcol-19-GFP::TBB-2, juSi239). Left column, lateral views of the head; right …
Figure 4—figure supplement 1
Epidermal MTs in wild type and dapk-1 mutants.
(A) Cartoon of C. elegans post-embryonic epidermis (hyp7 syncytium and lateral seam cells) showing the organization of MTs in the lateral and dorsoventral (muscle-adjacent) compartments. A large …
Figure 5 with 1 supplement
The CKK domain of PTRN-1 is required and sufficient to cause dapk-1-like defects in epidermal morphology.
(A) PTRN-1 domain organization. (B) Quantitation of epidermal defects in animals expressing different fragments of GFP::PTRN-1. N>100. Fisher’s exact test; ***p<0.001. (C) Representative DIC images …
Figure 5—figure supplement 1
Structure-function analyses of PTRN-1.
(A) Localization of GFP-tagged fragments of the PTRN-1 coiled coil (CC) domain, in the head (left) and anterior lateral epidermis (right). Scale bars, 10 µm. (B) Quantitation of epidermal defects of …
Figure 6 with 1 supplement
dapk-1(ju4) mutants display aberrant PTRN-1 localization.
(A) Localization of PTRN-1::GFP expressed from single-copy insertion in L4 stage anterior epidermis (left) and midbody lateral epidermis (right); transgene Pdpy-7-PTRN-1::GFP, ltSi541, in ptrn-1(0) …
Figure 6—figure supplement 1
PTRN-1 localization is sensitive to MT polymerization and dapk-1.
(A) PTRN-1 is not overexpressed in dapk-1(ju4) animals; western blot using anti-CePTRN-1. Control, anti-actin. (B,C) The number of PTRN-1 filaments in the head and lateral epidermis is sensitive to …
Figure 7 with 1 supplement
DAPK-1 undergoes MT-dependent transport in the epidermis.
(A,B) Representative images of GFP::DAPK-1 puncta in the larval epidermis (Pdpy-7-GFP::DAPK-1, juEx1774), showing lateral and dorsoventral regions, respectively. Dotted arrows indicate line scans …
Figure 7—figure supplement 1
Structure function analysis of DAPK-1.
(A) Representative images of GFP-tagged DAPK-1 protein fragments and their localization in the lateral epidermis in the head. (B) Velocity of DAPK-1 puncta, with or without colchicine treatment (cat-…
Figure 8
Model for DAPK-1-mediated regulation of epidermal MTs via PTRN-1.
https://doi.org/10.7554/eLife.15833.028Videos
Video 1
EBP-GFP dynamics in adult lateral epidermis (Pcol-19).
https://doi.org/10.7554/eLife.15833.011
Video 2
EBP-GFP dynamics in adult dorsoventral epidermis.
https://doi.org/10.7554/eLife.15833.012
Video 3
EBP-GFP dynamics in adult lateral epidermis, dapk-1(ju4) background.
https://doi.org/10.7554/eLife.15833.013
Video 4
EBP-GFP dynamics in adult dorsoventral epidermis, dapk-1(ju4) background.
https://doi.org/10.7554/eLife.15833.014
Video 5
GFP::DAPK-1 dynamics in larval epidermis.
Lateral seam cells are in the center of the movie.
Video 6
GFP::DAPK-1 dynamics in adult lateral epidermis.
Lateral seam is in center.
Video 7
GFP::DAPK-1 dynamics in adult dorsoventral epidermis.
Dorsal midline is at bottom of movie.
Video 8
Pdpy-7-GFP::RAB-5 dynamics.
https://doi.org/10.7554/eLife.15833.026Tables
Table 1
Suppressors and enhancers of dapk-1 morphological defects.
| Gene | Alleles and sequence change | Mammalian orthologs |
|---|---|---|
| A. Suppressors (forward screen) | ||
| ptrn-1 | ju698 | CAMSAP/Patronin |
| *lt1 | ||
| tm5597 | ||
| dhc-1 | ju697 | Dynein heavy chain |
| *or195ts | ||
| dapk-1 | ju1143 | (intragenic) |
| ju1145 | ||
| B. Suppressors (candidates) | ||
| unc-116 | e2310 | Kinesin-1 |
| sma-1 | e30 | Beta-heavy spectrin |
| C. Enhancers | ||
| klp-7 | tm2143 | Kinesin-13 |
| mei-1 | or642ts | p60 katanin |
| spas-1 | tm683 | Spastin |
| cat-4 | tm773 | GTP cyclohydrolase I |
| F47G4.5 | ok2667 | p80 katanin |
| D. No interaction | ||
| ebp-1 | tm1357 | Plus-end binding protein |
| ebp-2 | gk756 | Plus-end binding protein |
| ccpp-1 | ok1821 | Cytosolic Carboxypeptidase |
| ccpp-6 | ok382 | Cytosolic Carboxypeptidase |
| mcrs-1 | tm3681 | Microspherule Protein 1 |
| efa-6 | tm3124 | EFA6 |
| ttll-5 | tm3360 | Tubulin tyrosine ligase-like |
| ttll-11 | tm4059 | Tubulin tyrosine ligase-like |
| ttll-12 | tm4957 | Tubulin tyrosine ligase-like |
| unc-70 | e524 | β-Spectrin |
| dylt-2 | gk762 | Dynein light chain |
| dnc-1 | or404ts | p150 dynactin |
| nud-1 | ok552 | NDE1/NDEL1 |
| nud-2 | ok949 | NDE1/NDEL1 |
| unc-14 | e57 | kinesin-1 adaptor |
| tbg-1 | t1465 | γ-tubulin |
| ptl-1 | ok621 | tau |
| pinn-1 | tm2235 | Pin1 |
| par-1 | zu310ts | MARK |
-
Suppressors indicated * were tested for suppression of dapk-1(ju4) and dapk-1(gk219). Enhancers were tested with dapk-1(gk219). Genes in section D were mostly tested for interaction with dapk-1(ju4). dapk-1 tbg-1 double mutants were extremely sick, and a stable strain could not be obtained; n > 100 animals scored per genotype.
Table 2
PTRN-1 Structure-function analysis.
| Localization | Function | |||||
|---|---|---|---|---|---|---|
| Protein Fragment | Puncta | Thick filaments | Thin filaments | Restore Mor | Induce Mor | Co-loc with MTs |
| Full length | x | x | x | yes | no | yes |
| CH | - | - | - | no | no | no |
| CC | x | - | - | no | no | yes |
| CKK | - | - | x | yes | yes | yes |
| ΔCKK | x | - | - | no | no | yes |
| ΔCH | x | x | - | yes | no | ND |
| ΔCC | - | - | x | yes | no | ND |
| ΔCHCC1 | ? | x | - | ND | no | ND |
| ΔCHCC1CC2 | - | - | x | ND | slightly | ND |
| ΔCC2 | ? | x* | x | ND | no | ND |
| ΔCC3 | x | - | - | ND | no | ND |
-
Notes: *: Thick filaments present, but fewer compared to PTRN-1 full length or ΔCH. ?: unclear. ND: Not Determined.
Table 3
Dynamics parameters in the epidermis.
| Type of Dynamics | Transgene | Velocity (mean ± SEM) μm s−1 | Directionality | ||
|---|---|---|---|---|---|
| Lateral | Dorso-ventral | Lateral | Dorso-ventral | ||
| MT plus-end growth | Pcol-19-EBP-2::GFP | 0.30 ± 0.00 | 0.22 ± 0.007 | A→P P→A | Lat→DV DV→Lat |
| Early endosome transport | Pdpy-7-GFP::RAB-5 | 0.96 ± 0.014 | 0.93 ± 0.014 | A→P P→A | Lat→DV DV→Lat |
| DAPK-1 | Pdpy-7-GFP::DAPK-1 | 1.33 ± 0.028 | 0.96 ± 0.021 | A→P P→A | Lat→DV |
| DAPK-1 | Pcol-19-GFP::DAPK-1 | 1.26 ± 0.02 | 0.90 ± 0.017 | A→P P→A | Lat→DV |
-
Notes: A→P: Anterior to Posterior. P→A: vice versa. Lat→DV: Lateral to dorsoventral epidermis. DV→Lat: vice versa.
Table 4
DAPK-1 structure-function analysis.
| Protein Fragment | Puncta? | Motile? | Rescues Mor* | Induces Mor† | Enhances Mor‡ | Lethal in dapk-1(ju4) |
|---|---|---|---|---|---|---|
| Full Length | yes | yes | yes | no | no | no |
| Δ Kinase | yes | yes | no | no | yes | yes |
| Δ Kinase + CaM Bind Domain (BD) | yes | yes | no | no | yes | yes |
| Δ Cytoskeletal BD | no | no | no | no | no | no |
| Δ Death Domain + C terminus | no | no | no | no | no | no |
| Kinase only | some | no | no | no | ND | ND |
| CaM BD only | no | no | no | no | ND | ND |
| Ankyrin Domain only | some | no | no | no | no | no |
| Cytoskeletal BD only | some | no | no | no | no | no |
| Mid + DD + Ct | no | no | no | no | no | no |
| S179L dapk-1(ju4) | yes | yes | no | yes | yes | yes |
| K57A (kinase dead) | yes | yes | no | no | no | no |
-
Notes: *: In dapk-1(ju4). †: In WT background. ‡: In dapk-1(ju469).
Additional files
-
Supplementary file 1
Newly generatedstrains and plasmids.
(A) List of new strains and genotypes. (B) List of new plasmids.
- https://doi.org/10.7554/eLife.15833.029
