1. Volume 25, Issue 9, Pages 1135-1145 (May 2015)
Anillin Regulates Neuronal Migration and Neurite Growth by Linking RhoG to the Actin Cytoskeleton 
Dong Tian, Min Diao, Yuxiang Jiang, Lingfei Sun, Yan Zhang, Zhucheng Chen, Shanjin Huang, Guangshuo Ou 
Current Biology 
Volume 25, Issue 9, Pages (May 2015)
DOI: /j.cub
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2. Figure 1
Dynamic Localization of Anillin during C. elegans Q Neuroblast Development and the Generation of Conditional Anillin Knockouts
(A) Schematic of GFP-tagged Anillin localization in Q neuroblast lineages. QL and QR neuroblasts divide three times and generate three neurons (QL: PQR, PVM, and SDQL; QR: AQR, AVM, and SDQR) and two apoptotic cells (X).
(B) Fluorescence images of GFP-tagged Anillin (green) and an mCherry-tagged plasma membrane and histone (red) in the QR lineage. Left, inverted images of GFP-Anillin; right, merged images. See Figure S1 for the QL lineage.
(C) Quantification of the GFP or mCherry fluorescence intensity ratio between the cell cortex and the nucleus in the Q cell lineages. The error bars indicate the SD. n = 10–20. ∗∗∗Significant difference at p < by Student’s t test.
(D) Fluorescence time-lapse images of GFP-tagged Anillin during the dendritic outgrowth of the QR.ap cell. Yellow arrow, the direction of neurite growth; white arrow heads, the enrichment of GFP::ANI-1 in the growth cone; asterisks; cell body labeled with mCherry-tagged plasma membrane and histones.
(E) Schematic of ani-1 gene model and sgRNA sequences (sg1 and sg2 target the first and second exon, respectively). MBD, myosin-binding domain; ABD, actin-binding domain; AHD, Anillin homology domain; PH, Pleckstrin homology domain.
(F) Representative gels of the T7EI assay for ani-1 PCR products amplified from the genomic DNA of worms expressing Phsp::Cas9 and PU6::ani-1-sg1 (left) or PU6::ani-1-sg2 (right) with or without heat-shock treatment.
(G and H) Quantification of embryo survival rates (G) and the loss of A/PQR neuronal phenotypes (H) in WT and ani-1 conditional knockouts. n = 50–100 from three generations.
Scale bars in (B) and (D), 5 μm. See also Figure S1 and Table S1, S2, and S3.
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3. Figure 2
Defects in Q Neuroblast Asymmetric Division, Migration, and Neurite Growth in Anillin Conditional Knockouts
(A and B) Fluorescence time-lapse images of GFP-tagged F-actin and mCherry-tagged plasma membrane during QR.a (yellow arrows) and QR.p (white arrows) cytokinesis in WT (A) or ani-1 conditional knockouts (B). Dotted white lines show the periphery of dividing QR.a cells.
(C) Schematics (top) and fluorescence inverted images (bottom) of the A/PQR position in WT and ani-1 conditional knockouts. A/PQR neurons were visualized using Pgcy-32::mCherry. The image is inverted so that high mCherry fluorescence intensity is black. The cell identities are denoted adjacent to the cells. Dotted blue lines show the periphery of C. elegans. The red arrow indicates the C. elegans anus.
(D) Neurites from AVM, PVM, and PQR neurons in WT (top) and ani-1 conditional mutants (bottom). The pink or blue arrows indicate the dorsal-ventral or anterior-posterior growth of the neurites in WT animals, and the red arrows show the abnormal neurites.
(E and F) Quantification of defects in Q cell migration (E) and neurite growth (F) in ani-1 conditional knockouts. (n = 60–100). Statistical significance: ∗∗∗p < based on Student’s t test.
Asterisk, cell body. Scale bars in (A), (B), and (D), 5 μm; scale bars in (C), 50 μm. See also Figure S2 and Tables S2 and S3.
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4. Figure 3
Actin Filaments at the Leading Edge during Q Cell Migration and Neurite Growth in WT and Anillin Conditional Knockouts
(A–D) Fluorescence time-lapse images of GFP-tagged F-actin and mCherry-tagged plasma membrane during QR.ap cell migration (A and B) and neurite growth (C and D) in WT (A) and (C) and ani-1 conditional mutants expressing Pegl-17::Cas9; PU6::ani-1-sg1 (B) and (D). For QR.ap migration, the magnified views of QR.ap are shown in the lower panels of (A) and (B). Yellow arrows indicate the normal directions of cell migration or neurite growth, and white arrowheads show the neurites with branched leading edges or defective guidance in ani-1 conditional knockouts. Scale bar, 5 μm.
(E) Quantification of F-actin fluorescence intensity at the leading edge during cell migration (left) or in the growth cone during neurite growth (right) in WT and ani-1 conditional knockouts.
(F) Fluorescence intensity ratio between GFP-F-actin and mCherry-membrane (the internal control). Statistical analyses of the reduction in F-actin at the leading edge or the growth cone in ani-1 conditional knockouts: ∗∗∗p < based on Student’s t test.
See also Figure S3 and Tables S2 and S3.
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5. Figure 4
Anillin Co-localizes with F-actin at the Leading Edge and Stabilizes Actin Filaments In Vitro
(A) Fluorescence images of GFP- or mCherry-tagged F-actin, myosin, and Anillin in Q cells. Dotted lines show cell peripheries. Anterior, left. Scale bar, 5 μm.
(B) Line scans of fluorescence intensities across the leading edge. The peak intensities of Anillin and F-actin overlap, whereas the peak intensities (dotted lines) of myosin and Anillin or F-actin do not.
(C) Quantification of the distance between Anillin, myosin, and F-actin at the leading edge (n = 10).
(D) Schematics of the full-length protein or the MBD-ABD or ABD domains of Anillin. Domain information is described in the Figure 1E legend. SDS-PAGE gels of the GST-tagged Anillin protein or its domains are located in Figures S4A–S4C.
(E) The low-speed co-sedimentation assay measured the bundling activities of ANI-1 and its domains. ANI-1 and its MBD-ABD domain, but not the ABD domain, bundles actin filaments. The reaction mixture contained 3 μM actin only, actin with 0.2 μM ANI-1, actin with 0.5 μM plastin 3 as the positive control (left), or actin with 2 μM MBD-ABD (right top) or with 2 μM ABD (right bottom). The mixtures were incubated and then sedimented at 13,600 × g. S, supernatant; P, pellet.
(F) Fluorescence micrographs of rhodamine-phalloidin staining of the actin filaments in the reactions described in (E). Scale bar, 20 μm.
(G) Anillin inhibits dilution-mediated actin depolymerization. The preassembled actin filaments (5 μM, 100% Pyrene labeled) were incubated with various concentrations of ANI-1 or the MBD-ABD or ABD of ANI-1 for 5 min at room temperature and then diluted 25-fold into buffer G. Actin depolymerization was monitored by tracing the changes in Pyrene fluorescence.
See also Figure S4 and Tables S2 and S3.
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6. Figure 5 Anillin Antagonizes Cofilin-Mediated Severing Activity
(A) ANI-1 inhibits the severing action of Cofilin/UNC-60 in vitro. The dynamics of single-actin filaments were visualized by time-lapse TIRF microscopy in reactions containing actin only; actin with 500 nM UNC-60; or actin with 500 nM UNC-60 and 200 nM ANI-1, 1.5 μM MBD-ABD of ANI-1, or 2 μM ABD of ANI-1. Red arrows show F-actin breaking events. See also Movie S6. Scale bar, 5 μm.
(B) Quantification of (A). Values in the upper and lower panels represent the F-actin severing frequency or actin subunit off rates, respectively. Data represent mean ± SE. n = 3. ∗∗Significant difference at p < 0.01 by Student’s t test.
(C) Quantification of Q cell migration defects in UNC-60(S3A), ani-1 conditional knockout, and ani-1 overexpression animals (n = 80–120). ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05 based on χ2 test.
(D) Quantification of neurite growth defects in ani-1 overexpression and UNC-60(S3A) animals (ANI-1::GFP, 50 ng/μl; n = 50–65). ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05 based on χ2 test.
See also Tables S2 and S3.
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7. Figure 6 MIG-2 Recruits Anillin to the Leading Edge
(A and B) Inverted fluorescence images (top) or merged images (bottom) of GFP-tagged Anillin in QR.ap cells in WT (A) and mig-2(mu28)-null mutants (B) during migration. The Q cell membrane and histones are marked with mCherry. Anterior, left. Scale bar, 5 μm.
(C and D) Fluorescence images (C) and quantification (D) of GFP-tagged Anillin and mCherry-tagged plasma membrane (M) and histone (H) in WT, mig-2(mu28)-null, and mig-2(rh17) constitutively active mutants. Traces in (D) are line-scan intensity plots (in a.u.) of the GFP::ANI-1 (green) and Myri-mCherry (red) signals around the periphery of QR.a. The trace starts from the posterior of QR.a and moves counterclockwise along the cell periphery to the anterior and back to the posterior.
(E) Quantification of the fluorescence intensity ratio of GFP::ANI-1 and mCherry-membrane between the anterior and posterior of the QR.ap cell in WT and mig-2 mutants (n = 10).
(F) Quantification of fluorescence intensity of GFP::ANI-1 and mCherry-membrane across the leading edge of the QR.ap cell in WT and mig-2 mutants.
(G) Distance between GFP::ANI-1 and plasma membrane at the leading edge of WT and mig-2 mutants (n = 10).
See also Figure S5 and Tables S2 and S3.
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8. Figure 7 Active MIG-2 Binds to Anillin, and a Proposed Model
(A) Interaction between MIG-2 and ANI-1 in the yeast two-hybrid system. Yeast transformants that express both Gal4 DNA-binding domain-MIG-2 WT or Q65L (constitutively active; CA) or T21N (dominant negative; DN) mutant fusions and the Gal4 transcription activation domain (AD)-ANI-1 C-terminal (759–1159) truncation fusion were streaked on synthetic complete medium lacking Trp and Leu (left) or lacking Trp, Leu, His, and Ade (SC-4, right). Growth on SC-4 showed the interaction between the two tested proteins.
(B) Interaction between MIG-2-CA and ANI-1 in a GST fusion protein pull-down assay. The binding reactions contained GST-tagged ANI-1 C-terminal (759–1159) protein or the control GST protein (top two rows) and His-tagged WT MIG-2 or MIG-2-CA. Shown are detections of His-MIG-2 and His-MIG-2-CA by western blot.
(C) A proposed model of the roles of Anillin in transducing the MIG-2 signal to stabilize the actin cytoskeleton at the leading edge during Q cell migration and neurite growth.
See also Tables S2 and S3.
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