Figure 1.
ETV1 is a novel binding partner of Pkp3-catenin.
(A) Schematics of ETV1 and ETV5 protein structures. Red line indicates the binding region as suggested by sequenced prey clones identified from yeast-two hybrid screen. (B) Either HA-Pkp3 or HA-ARVCF was co-transfected with Myc-ETV1 in HEK 293T cells. Anti-Myc (ETV1) or –HA (Pkp3 or ARVCF) immune-precipitates were subjected to immuno-blotting with anti-Myc (ETV1) and –HA (Pkp3 or ARVCF) antibodies. Pkp3-catenin co-precipitates ETV1, but the structurally homologous ARVCF-catenin does not (negative/specificity control). ETV1 co-precipitates Pkp3, but does not co-precipitate ARVCF. (C) Untreated AB-1 wild type mouse embryonic stem cells were lysed and fractionated at 90% confluency and endogenous Pkp3 or ETV1 was immuno-precipitated from nuclear extracts, followed by immuno-blotting for Pkp3 and ETV1. Five percent inputs were acquired separately due to the low levels of endogenous proteins.
Figure 2.
Ectopic mitochondrial outer membrane (MOM) co-relocalization in HeLa cells of xPkp3 with xETV1, to map ETV1’s binding domains.
(A) Co-expression of Myc-xETV1 with MOM-targeted HA-xPkp3. Cells were co-immunostained for the ectopic co-relocalization of xETV1 (Myc-epitope, left panels), as well as for the MOM-targeted catenin (HA-epitope, middle panels). Ectopic co-relocalization of xETV1 with MOM-targeted xPkp3 was only observed when amino acids 257–335 of xETV1 were present. Representative images from a minimum of sixty cells expressing both constructs analyzed are shown. (B) List of Myc-tagged (MT) xETV1 constructs and their interaction status with MOM-targeted HA-xPkp3. A positive score (+) indicates that >60% of cells coexpressing the indicated xETV1 deletion construct showed co-relocalization of xETV1 with the MOM-targeted HA-xPkp3 construct. Our prior laboratory studies have shown that authentic partner proteins re-localize to the MOM (fusion protein), as visualized by their increased signal at the mitochondria [46]. (C) Quantification of the extent of signal overlap from MOM-targeted xPkp3 or MOM-targeted xARVCF with the indicated xETV1 mapping constructs. Minimally 30 cells from each replicate experiment (totaling over 100 cells from triplicate experiments) were quantitated for the extent of signal overlap.
Figure 3.
Ectopic mitochondrial outer membrane (MOM) co-relocalization in HeLa cells of xETV1 with xPkp3, to map Pkp3’s binding domains.
(A) Co-expression of HA-xPkp3 deletion constructs with MOM-targeted Myc-xETV1, or co-expression of Myc-xETV1 with MOM-targeted HA-xPkp3. Cells were co-immunostained for the ectopic co-relocalization of xETV1 (Myc-epitope, left panels), as well as for xPkp3 (HA-epitope, middle panels). Ectopic co-relocalization of xPkp3 with MOM-targeted xETV1 was observed when amino acids 1–325 of xPkp3 were present, while co-relocalization of xPkp3’s ARM domain was inconclusive due to its broad expression. Shown are representative images from a minimum of sixty cells that expressed both constructs and were analyzed. (B) List of HA-tagged (HA) xPkp3 constructs, and their interaction status with MOM-targeted Myc-xETV1 (or the inverse). A positive score (+) indicates that >60% of cells coexpressing the indicated xPkp3 deletion construct showed its co-relocalization with MOM-targeted Myc-xETV1. (C) Quantification of cells showing overlapping relocalization of MOM-targeted xPkp3 or xARVCF and xETV1 mapping constructs from Fig. 3A. Minimally 30 cells from each replicate experiment (totaling over 100 cells from triplicate experiments) were quantitated for their ability to co-relocalize.
Figure 4.
Rescue of neural crest establishment defects, induced upon xPkp3-catenin depletion, through ectopic expression of xETV1.
(A) The indicated morpholinos (20 ng) and mRNAs (0.5 ng) were co-injected into one dorsal blastomere at the four-cell stage with rhodamine dextran as a lineage tracer. After in situ hybridization for neural crest markers, Twist and FoxD3, embryos were imaged. The ectopic expression of ETV1-Myc largely rescued Twist and FoxD3 expression in embryos depleted for Pkp3-catenin (compare right-most images to middle images). (B) Quantification of embryos processed for Twist in situ hybrization, comparing the uninjected side to the injected side, indicated by an asterisk, for reduction in signal. (C) Quantification of embryos processed for FoxD3 in situ hybrization, comparing the uninjected side to the injected side for reduction in signal. P-values indicate statistical significance.
Figure 5.
Pkp3-catenin regulation of ETV1 reporters or target genes.
HEK 293T cells were transfected with expression plasmids for Myc-xETV1 or HA-xPkp3 in the indicated combinations. (A) Pkp3’s functional relationship with ETV1 was tested using an established luciferase reporter for Ets family member transcriptional activity harboring a fes gene promoter fragment in triplicate (Fes3xWT-Luc, [57]). Labelled p-values indicate changes that are statistically significant. (B) Pkp3’s functional relationship with ETV1 was tested using a second established ETV1 reporter construct harboring a native matrix metalloproteinase-1 (MMP1) promoter fragment (MMP1-Luc,[48]–[50]). (C) MMP1(−87/−88)-Luc is a negative-control construct containing a non-functional ETV1 consensus binding site. Labelled p-values indicate changes that are statistical significant. Performed in parallel to Figure 4B. (D) To assess Pkp3 and ETV1 activity in a cell-free (in vitro) context, we use the Fes3xWT-Luc reporter and in vitro translated the ETV1 and Pkp3 proteins (TnT Wheat Germ Extract). (E) The indicated morpholinos (40 ng) and mRNAs (0.5 ng) were injected into one-cell (cleavage) stage Xenopus embryos followed by RNA extraction at late neurula stages, DNAse treatment, reverse transcription, and real-time PCR to assess Pkp3- depletion effects upon ETV1 target-gene transcription. The co-injection of ETV1 mRNA with Pkp3 morpholino (MO) significantly rescued the impact of Pkp3-catenin depletion. The ETV1 gene targets assessed are components of the dopamine synthesis and transport pathways and include: DDC = dopa decarboxylase; TH = tyrosine hydroxylase; SLC = solute carrier family 18 member 2; and GCH = GTP cyclohydrolase I. All real-time PCR was repeated more than three times with samples tested in triplicate with similar outcomes. Error bars represent the standard deviation from three independent experiments. Differences between negative control (SC MO+β-gal) and Pkp3 knockdown (Pkp3 MO1+ β-gal) are all statistically significant based on the Student’s T-Test, demonstrating p-values <0.05. Differences between Pkp3 knockdown (Pkp3 MO1+ β-gal) and its rescue with ectopic ETV1 (Pkp3 MO1+ ETV1) are all statistically significant based on the Student’s T-Test, demonstrating p-values <0.05.