(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 …
(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 …
(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 …
(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 …
(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 …
(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) …
(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 …
(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 …
(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 …
(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 …
(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) …
(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 …
(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 …
(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-…
Lateral seam cells are in the center of the movie.
Lateral seam is in center.
Dorsal midline is at bottom of movie.
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.
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.
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.
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).
Newly generatedstrains and plasmids.
(A) List of new strains and genotypes. (B) List of new plasmids.