DAPK interacts with Patronin and the microtubule cytoskeleton in epidermal development and wound repair

  1. Marian Chuang
  2. Tiffany I Hsiao
  3. Amy Tong
  4. Suhong Xu
  5. Andrew D Chisholm  Is a corresponding author
  1. University of California, San Diego, United States
8 figures, 8 videos, 4 tables and 1 additional file

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 …

https://doi.org/10.7554/eLife.15833.002
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 …

https://doi.org/10.7554/eLife.15833.003
Figure 2 with 1 supplement
ptrn-1 suppresses dapk-1(ju4) innate immune and wound repair phenotypes.

(Adapk-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 …

https://doi.org/10.7554/eLife.15833.005
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 …

https://doi.org/10.7554/eLife.15833.006
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 …

https://doi.org/10.7554/eLife.15833.007
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) …

https://doi.org/10.7554/eLife.15833.008
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 …

https://doi.org/10.7554/eLife.15833.009
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 …

https://doi.org/10.7554/eLife.15833.010
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 …

https://doi.org/10.7554/eLife.15833.015
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 …

https://doi.org/10.7554/eLife.15833.016
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)

https://doi.org/10.7554/eLife.15833.018
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 …

https://doi.org/10.7554/eLife.15833.019
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 …

https://doi.org/10.7554/eLife.15833.020
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-…

https://doi.org/10.7554/eLife.15833.021
Model for DAPK-1-mediated regulation of epidermal MTs via PTRN-1.
https://doi.org/10.7554/eLife.15833.028

Videos

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.

https://doi.org/10.7554/eLife.15833.022
Video 6
GFP::DAPK-1 dynamics in adult lateral epidermis.

Lateral seam is in center.

https://doi.org/10.7554/eLife.15833.023
Video 7
GFP::DAPK-1 dynamics in adult dorsoventral epidermis.

Dorsal midline is at bottom of movie.

https://doi.org/10.7554/eLife.15833.024
Video 8
Pdpy-7-GFP::RAB-5 dynamics.
https://doi.org/10.7554/eLife.15833.026

Tables

Table 1

Suppressors and enhancers of dapk-1 morphological defects.

https://doi.org/10.7554/eLife.15833.004
GeneAlleles and sequence changeMammalian orthologs
A. Suppressors (forward screen)
ptrn-1ju698CAMSAP/Patronin
*lt1
tm5597
dhc-1ju697Dynein heavy chain
*or195ts
dapk-1ju1143(intragenic)
ju1145
B. Suppressors (candidates)
unc-116e2310Kinesin-1
sma-1e30Beta-heavy spectrin
C. Enhancers
klp-7tm2143Kinesin-13
mei-1or642tsp60 katanin
spas-1tm683Spastin
cat-4tm773GTP cyclohydrolase I
F47G4.5ok2667p80 katanin
D. No interaction
ebp-1tm1357Plus-end binding protein
ebp-2gk756Plus-end binding protein
ccpp-1ok1821Cytosolic Carboxypeptidase
ccpp-6ok382Cytosolic Carboxypeptidase
mcrs-1tm3681Microspherule Protein 1
efa-6tm3124EFA6
ttll-5tm3360Tubulin tyrosine ligase-like
ttll-11tm4059Tubulin tyrosine ligase-like
ttll-12tm4957Tubulin tyrosine ligase-like
unc-70e524β-Spectrin
dylt-2gk762Dynein light chain
dnc-1or404tsp150 dynactin
nud-1ok552NDE1/NDEL1
nud-2ok949NDE1/NDEL1
unc-14e57kinesin-1 adaptor
tbg-1t1465γ-tubulin
ptl-1ok621tau
pinn-1tm2235Pin1
par-1zu310tsMARK
  1. 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.

https://doi.org/10.7554/eLife.15833.017
LocalizationFunction
Protein
Fragment
PunctaThick filamentsThin filamentsRestore MorInduce MorCo-loc with MTs
Full lengthxxxyesnoyes
CH---nonono
CCx--nonoyes
CKK--xyesyesyes
ΔCKKx--nonoyes
ΔCHxx-yesnoND
ΔCC--xyesnoND
ΔCHCC1?x-NDnoND
ΔCHCC1CC2--xNDslightlyND
ΔCC2?x*xNDnoND
ΔCC3x--NDnoND
  1. 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.

https://doi.org/10.7554/eLife.15833.025
Type of DynamicsTransgeneVelocity (mean ± SEM) μm s−1Directionality
LateralDorso-ventralLateralDorso-ventral
MT plus-end growthPcol-19-EBP-2::GFP0.30 ± 0.000.22 ± 0.007A→P
P→A
Lat→DV
DV→Lat
Early endosome transportPdpy-7-GFP::RAB-50.96 ± 0.0140.93 ± 0.014A→P
P→A
Lat→DV
DV→Lat
DAPK-1Pdpy-7-GFP::DAPK-11.33 ± 0.0280.96 ± 0.021A→P
P→A
Lat→DV
DAPK-1Pcol-19-GFP::DAPK-11.26 ± 0.020.90 ± 0.017A→P
P→A
Lat→DV
  1. 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.

https://doi.org/10.7554/eLife.15833.027
Protein FragmentPuncta?Motile?Rescues Mor*Induces MorEnhances MorLethal in dapk-1(ju4)
Full Lengthyesyesyesnonono
Δ Kinaseyesyesnonoyesyes
Δ Kinase + CaM Bind Domain (BD)yesyesnonoyesyes
Δ Cytoskeletal BDnononononono
Δ Death Domain + C terminusnononononono
Kinase onlysomenononoNDND
CaM BD onlynonononoNDND
Ankyrin Domain onlysomenonononono
Cytoskeletal BD onlysomenonononono
Mid + DD + Ctnononononono
S179L dapk-1(ju4)yesyesnoyesyesyes
K57A (kinase dead)yesyesnononono
  1. 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

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