1. Volume 39, Issue 2, Pages 224-238 (October 2016)
Functional Coordination of WAVE and WASP in C. elegans Neuroblast Migration 
Zhiwen Zhu, Yongping Chai, Yuxiang Jiang, Wenjing Li, Huifang Hu, Wei Li, Jia-Wei Wu, Zhi-Xin Wang, Shanjin Huang, Guangshuo Ou 
Developmental Cell 
Volume 39, Issue 2, Pages (October 2016)
DOI: /j.devcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
MIG-13 and Arp2/3 Regulate the C. elegans Q Neuroblast Migration
(A) Schematic overview of Q cell migration. The QR descendant (QR.x), AQR, migrates anteriorly, and the QL descendant (QL.x), PQR, migrates posteriorly. Apoptotic Q.aa cells are indicated by a black cross.
(B) MIG-13 protein domains. The amino acid changes or deletion in mig-13 mutant alleles are indicated.
(C) A color-coded heatmap scoring the distribution of AQR position in L4 animals of various genotypes as indicated. The full length between URX and PLM cells is divided into ten blocks, and the percentage of AQR that stopped within each block is given. The color (green) darkness of the blocks symbolizes the range of percentage values. n > 50 for all genotypes. Statistical significance compared with the control (■) with a matching color code is based on Student's t test, ∗p < N.S., not significant.
(D and E) Fluorescence time-lapse images of F-actin and plasma membrane and histone during AQR migration in WT (D) or mig-13(cas15) mutant (E) animals. Merged images, left; inverted fluorescence images of AQR morphology, right; white arrows, migration direction; white arrowhead, mispolarized leading edge; asterisks, nuclei; double-headed arrows, migration distance.
(F) Quantification of the F-actin fluorescence intensity ratio of the leading edge to the rear part of AQR in WT (n = 19) or mig-13 mutant (n = 15) animals. The corresponding mCherry-membrane intensity ratio was used as an internal control. Data are presented as mean ± SEM.
(G) Box plots show the distributions of the AQR migration speed in WT (n = 17), mig-13 mutant (n = 15), and arx-2-sg mutant (n = 14) animals. The anterior migration is referred to as “positive.”
(H) Quantification of the mean counts of filopodia-like structures. Bar segments represent the percentage of filopodia-like structures within the length range as indicated. The same imaging datasets as in (G) were used for analysis.
(I) Inverted fluorescence images of AQR morphology in arx-2-sg mutants. Arrows, leading edges; asterisks, nuclei.
(J–L) Fluorescence images (J) and quantification (K and L) of ARX-2 (GFP knockin). (J) Upper, merged image; middle, plasma membrane and histone; lower, ARX-2. Arrows, leading edges; asterisks, ARX-2::GFP from an adjacent apoptotic cell. Box plots show the quantification of ARX-2::GFP enrichment (K) or distribution (L) at the leading edge of AQR (n = 10–15 animals). The quantification method is described in Figure S1 and Experimental Procedures.
Statistical significances in (F)–(H), (K), and (L) are based on Student's t test, ∗p < 0.05, ∗∗∗p < Scale bars, 5 μm. See also Figure S1; Movies S1, S2, and S3.
Developmental Cell  , DOI: ( /j.devcel )
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3. Figure 2 Identification of MIG-13 and ABL-1 Interaction
(A) Suppressor screen strategy (see text). AQR and PQR were marked by the Pgcy-32::gfp reporter.
(B) A color-coded heatmap scoring the PQR position. The quantification scheme is the same as for Figure 1C. n > 50 for all genotypes. Statistical significance compared with the control (■) with a matching color code is based on Student's t test, ∗∗∗p <
(C) C. elegans SOEM-1 and ABL-1 protein domains. The amino acid changes or deletion in soem-1 and abl-1 mutant alleles are indicated.
(D) Interaction between SOEM-1 and ABL-1 in the yeast two-hybrid assay. Yeast transformants that express both the Gal4 DNA-binding domain-SOEM-1 WT or H140Q mutant fusions and the Gal4 transcription activation domain (AD)-ABL-1(1–1,214) fusion were streaked on synthetic complete medium lacking Trp and Leu (SC-2) or lacking Trp, Leu, His, and Ade (SC-4). Growth on SC-4 showed the interaction.
(E) Co-immunoprecipitation (IP) of MIG-13 and ABL-1 SH3-SH2 domain. Cell lysates were immunoprecipitated and subjected to western blot (IB) analysis with the indicated antibodies.
(F) Fluorescence images of AQR expressing GFP-tagged ABL-1 (green, lower) with mCherry-tagged plasma membrane and histone (red, middle; merged, upper). Scale bar, 5 μm.
(G) Quantification of the GFP or mCherry fluorescence intensity ratio between the leading edge and the rear cortex of AQR in WT (n = 30) and mig-13 mutant (n = 40) animals. Data are presented as mean ± SEM. Statistical significance is based on Student's t test, ∗∗∗p <
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 3 Identification of MIG-13 and SEM-5 Interaction
(A) A color-coded heatmap scoring the position of AQR. n > 50 for all genotypes. Statistical significance compared with the control (■) with a matching color code is based on Student's t test, ∗∗∗p <
(B) Interaction of MIG-13 with SEM-5, CED-2, or NCK-1 in a GST-tagged protein pull-down assay.
(C) Representative still images and quantification of SEM-5 (GFP knockin) in AQR. Traces in the lower panel are line-scan intensity plots (a.u.) of the SEM-5::GFP and Myri-mCherry signals along the leading edge. The trajectory and direction of the scanning line are shown in the upper and bottom panels. Asterisks show the positions of the leading edge.
(D) Quantification of AQR that contains SEM-5::GFP signal at the leading edge.
(E–G) Fluorescence time-lapse images of F-actin, plasma membrane, and histone during AQR migration in abl-1(ok171) (E), sem-5-sg (F), or abl-1(ok171);sem-5-sg double (G) mutant animals. Asterisks denote nuclei.
(H–J) Quantification of migration speed (box plots, H) F-actin GFP fluorescence intensity ratio (the leading edge to cytosol, I; data are presented as mean ± SEM), and the number of filopodia-like structures (J) of AQR.
n = 10–20. Statistical significances compared with WT (∗) or between underlined pairs (#) are based on Student's t test: ∗∗p < 0.01, ∗∗∗p < 0.001, #p < 0.05, and ###p < Scale bars, 5 μm.
See also Figure S3; Movies S4, S5, and S6.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 4 WAVE and WASP Regulate Q Cell Migration
(A) A color-coded heatmap scoring the position of AQR. n > 50 for all genotypes. Statistical significance compared with the control (■) with a matching color code is based on Student's t test, ∗∗∗p <
(B–D) Fluorescence time-lapse images of F-actin, plasma membrane, and histone during AQR migration in wsp-1(gm324) (B), wve-1-sg (C), or wsp-1(gm324);wve-1-sg double (D) mutant animals. Asterisks denote nuclei.
(E–G) Quantification of AQR migration speed (box plots, E), F-actin GFP fluorescence intensity ratio (the leading edge to the cytosol, F; data are presented as mean ± SEM), and the number of filopodia-like structures (G). n = 10–20.
Scale bar, 5 μm. Statistical significances compared with WT (∗) or between underlined pairs (#) are based on Student’s t test, ∗∗p < 0.01, ∗∗∗p < 0.001, #p < 0.05, ##p < 0.01.
See also Movies S7, S8, and S9.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 5 SEM-5 and MIG-2 Synergistically Activate WASP
(A) Interaction between WSP-1 and SEM-5 in a pull-down assay.
(B) Co-immunoprecipitation of GFP-tagged WSP-1 and 3×FLAG-tagged MIG-2CA.
(C) SDS-PAGE analysis of recombinant GST-WSP-1, GST-WSP-1-VCA domain, His-SEM-5, and His-MIG-2CA. Red asterisks indicate the lower bands, which are the degradation products validated by mass spectrometry.
(D) Assembly of branched actin networks monitored by TIRF microscopy. Actin monomers (1.2 μM; 50% Oregon green labeled) were polymerized for 2 min before addition of 15 nM Arp2/3 complex, 40 nM WSP-1, 1 μM SEM-5, 1 μM MIG-2CA, and 1.2 μM actin monomers. Red asterisks show the actin branching sites, and green arrows show multiple actin branches from one site. Scale bar, 5 μm.
(E) Branching frequency is calculated from TIRF movies. Branch events are calculated from eight fields (two independent experiments). Data are presented as mean ± SEM. Dunnett's multiple comparison test was used after one-way ANOVA to generate the p values. The statistical significances are: compared with “+WSP-1,” ∗p < 0.05, ∗∗∗p < 0.001; comparison between the underlined pairs, #p <0.05, ###p <
See also Figure S4 and Movie S10.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6 Distribution of Arp2/3, WAVE, and WASP at the Leading Edge
(A–C) Top: representative images of GFP-tagged ARX-2 (A), WVE-1 (B), and WSP-1 (C) in knockin animals. Left, GFP knockin fluorescence (green); middle, mCherry-tagged plasma membrane and histone (red, plot region is labeled with a dashed line); right, merged images. Bottom: the GFP fluorescence distribution plot along the leading edge of the representative image shown above. The GFP fluorescence intensity along the leading edge was normalized to 1.5 times that in the adjacent hyp7 cell. The area above the baseline of 1.0 is highlighted in green. Individual GFP::WSP-1 puncta are numbered in (C). Scale bar, 2.5 μm.
(D) The leading-edge distribution of ARX-2::GFP. Left, merged images; middle and right, the leading areas of mCherry-tagged plasma membrane and histone (red) and ARX-2::GFP knockin (green). Furthest right, the normalized GFP fluorescence distribution plot, as described in (A) to (C). Scale bar, 5 μm.
(E and F) Box plots show the distribution of ARX-2::GFP fluorescence intensity ratio between the leading edge and the cytosol of AQR (E) and the percentage of leading-edge periphery with enriched ARX-2::GFP (F). Enrichment is defined if the GFP intensity in AQR is 1.5 times greater than that in hyp7. n = 50. The statistical significances are: compared with WT, ∗∗∗p < 0.001; compared with wsp-1(gm324), ##p <0.01 and ###p <
(G) Triple fluorescence images of AQR. The corresponding normalized fluorescence knockin intensity distribution along the leading edge is shown on the right. Individual puncta of TagRFP::WVE-1 or GFP::WSP-1 are labeled and numbered in representative images and the corresponding plots. The asterisk indicates an overlapping cluster of TagRFP::WVE-1 and GFP::WSP-1. Hash indicates an autofluorescent particle. Scale bar, 2.5 μm.
(H) Quantification of the number of puncta of WVE-1 and WSP-1 at the leading edge. The black portion in each bar indicates the overlapped WVE-1 and WSP-1.
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 7 WAVE and WASP Coordinate at the Leading Edge
(A) Fluorescence images of GFP::WVE-1 knockin in WT (upper) or wsp-1(gm324) mutant (lower) animals. Right, the corresponding fluorescence line-scanning plot. The method to generate the plot is described in Figure 6D.
(B) Fluorescence images and line-scanning plot of GFP::WSP-1 KI.
(C and D) Box plots show the data distribution of the GFP::WVE-1 enrichment (C) and distribution (D) at the leading edge of AQR in WT and wsp-1 mutant animals. n = 50.
(E and F) Box plots show the data distribution of the GFP::WSP-1 signal enrichment (E) and distribution (F). n = 50.
(G) A proposed model of two parallel pathways transducing MIG-13 signal to the Arp2/3 complex.
Statistical significances are ∗p < 0.05, ∗∗∗p < based on Student's t test. Scale bar, 5 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions