Research ArticleCell Migration

A large-scale screen reveals genes that mediate electrotaxis in Dictyostelium discoideum

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Science Signaling  26 May 2015:
Vol. 8, Issue 378, pp. ra50
DOI: 10.1126/scisignal.aab0562
  • Fig. 1 High-throughput screen determined electrotaxis phenotypes.

    (A) Compiled electrotaxis phenotypes of 563 strains from the mutant collection, arranged according to directedness value. The red rectangle on the left end shows severely defective strains, and that on the right shows the hyperresponsive mutants. WT, wild type. (B) Mutant cells were categorized into four groups according to the directedness value cos θ. Normal electrotaxis group: strains with cos θ between 0.5 and 0.9, accounting for 68% of the collection screened; low electrotaxis group: those with cos θ between 0.3 and 0.5, accounting for 24% of the collection; defective electrotaxis group: those with cos θ less than 0.3, accounting for 5% of the collection; hyperresponsive group: those with cos θ higher than 0.9, accounting for 3% of the collection. (C and D) Examples of defective and hyperresponsive strains. Labels of the x axis indicate strain codes. Results shown are from experiments with an electric field (EF) of 12 V/cm. Data are means ± SEM from at least 50 cells per strain from three independent experiments. *P < 0.05, **P < 0.01, Student’s t test, compared with AX2 WT cells in an electric field of the same strength. (E) Representative trajectories of typical strains from WT, defective, and hyperresponsive groups. Plots show migration paths of multiple cells with the start position of each cell centered at point 0,0. Field strength and polarity are as shown.

  • Fig. 2 Quantitative analyses of electrotactic responses in selective defective mutants.

    (A) Directedness values in selected severely defective strains. (B) Defective strains showed decreased migration persistence. (C and D) Migration speeds in the strains with significantly decreased directedness values. Data are means ± SEM from at least 50 cells per strain from three independent experiments in an electric field of 12 V/cm. Labels of the x axis indicate strain codes. *P < 0.05, **P < 0.01, Student’s t test, compared with AX2 WT cells in an electric field of the same strength.

  • Fig. 3 PiaA is essential for electrotaxis in D. discoideum.

    Null mutation of piaA significantly reduced electrotaxis, which was rescued by reexpression of piaA. (A) piaA cells displayed an aggregation defect, which was rescued by reexpression of piaA. WT, piaA, Flag-piaA/piaA (piaA/piaA), and pJK1/piaA cells were developed on nonnutrient agar plates and photographed at 48 hours. (B) Trajectories of cells in an electric field of 12 V/cm. The square is 200 μm × 200 μm. Polarity is as shown. (C) Directedness values of the four strains in an electric field. (D) PiaA cells showed a significant defect in electrotaxis at all voltages tested. Flag-piaA/piaA cells showed normal electrotaxis at all voltages. See also movies S1 to S4. Data are means ± SEM from at least 50 cells per genotype from three independent experiments. *P < 0.05, **P < 0.01, Student’s t test, compared with AX2 WT cells.

  • Fig. 4 The TORC2/PKB pathway in electrotaxis.

    (A and B) Pharmacological inhibition of TORC2-impaired electrotaxis, as shown by compiled cell migration trajectories (AX2) (A) and migration directedness values (B). Cells were treated with TORC2 inhibitor pp242 for 30 min before being exposed to an electric field of 12 V/cm in the continuous presence of the inhibitor. Data are means ± SEM from at least 50 cells per treatment from three independent experiments. *P < 0.05 when compared with no drug in an electric field. (C to F) Knockouts of components of TORC2 signaling pathway significantly affected electrotaxis, but to different extents on migration directedness and speed. Knockouts of components of TORC2 signaling pathway (piaA, rasC, gefA, rip3, and pkbR1) significantly reduced the directedness values (C and D) and differentially affected trajectory speed and displacement speed (E and F). Cell migration trajectories are presented with the start point of each cell set at the origin. WT cells (AX2) migrated directionally toward the cathode (to the left). Data are means ± SEM from 50 cells per treatment or per genotype in an electric field of 12 V/cm from three independent experiments. *P < 0.05, **P < 0.01, Student’s t test, compared with AX2 WT cells in an electric field of the same strength.

  • Fig. 5 Contribution of the TORC2/PKB, PI3K, and PLA2 pathways to electrotaxis.

    (A) Roles of three chemotaxis pathways—TORC2/PKB, PIP3, and PLA2—in electrotaxis. PiaA and PI3K played more critical roles. Knockout or inhibition of three pathways that function in chemotaxis—TORC2/PKB, PIP3, and PLA2—eliminated electrotaxis. (B to D) Effect of knockout of pla2 (pla2), double knockout of pla2 and piaA (pla2/piaA), or double knockout of pla2 and piaA and PI3K inhibition with LY294002 (LY) on cell migration directedness (B), trajectory speed (C), and displacement speed (D). (E) Cell migration trajectories are presented with the start point of each cell set at the origin. WT cells (AX2) migrated directionally toward the cathode (to the left). Electrotaxis was significantly impaired in pi3k/ cells (null mutation of pi3k1 and pi3k2). (F) The PI3K knockout strain showed significantly decreased directedness values and decreased track speed and displacement speed. Migration trajectories of cells in an EF with the cathode on the left. The square is 200 μm × 200 μm. Polarity is as shown. Data are means ± SEM from 50 cells per genotype or per treatment in an electric field of 12 V/cm from three independent experiments. *P < 0.05, **P < 0.01, Student’s t test, compared with AX2 WT cells.

  • Fig. 6 Diagram depicting hypothetical mechanisms of electrotaxis against chemotaxis.

    The major signaling network (TORC2 and PI3K) appears to be shared between electrotaxis and chemotaxis. Some receptors, for example, the cAMP receptor cAR1 and downstream G protein Gβ, are essential for chemotaxis, but not for electrotaxis. This suggests that there are some chemotaxis- and electrotaxis-specific pathways. Electric fields activate the major shared signaling network and an as yet to be identified specific pathway that converge on the cytoskeletal network, which is altered during both chemotaxis and electrotaxis (green box). Changes in the cytoskeleton network result in cell motility and directed migration.

  • Table 1 Strains that were severely defective in electrotaxis.

    Data are means ± SEM from at least 50 cells from three independent experiments.

    StrainsElectrotaxis indexMorphological
    defect
    Chromosome:
    insertion site
    GenesGene product
    SN-129(−)0.08 ± 0.02Streamer5:2430559mybI; cmfBmyb domain–containing protein;
    putative CMF receptor CMFR1
    SN-576(−)0.16 ± 0.02Mound3:1355756cldA;RGS13Clu domain–containing protein A;
    regulator of G protein signaling 13
    SN-183(−)0.17 ± 0.02Agg-2:8027355piaACytosolic regulator of ACA;
    alternative protein name is Pianissimo
    SN-494(−)0.19 ± 0.03Fuzzy6:771404qtrt1Queuine tRNA-ribosyltransferase
    SN-517(−)0.20 ± 0.03Mound1:4688833DDB_G0270350_psPseudogene
    SN-028(−)0.21 ± 0.05Mound6:1494014amdAAMP deaminase
    SN-447(−)0.21 ± 0.04Mound1:2285000nat10Putative N-acetyltransferase
    SN-613(−)0.22 ± 0.05Mound6:1494014amdAAMP deaminase
    SN-677(−)0.22 ± 0.04Mound1:2285000nat10Putative N-acetyltransferase
    SN-123(−)0.22 ± 0.04Mound6:2664855DDB_G0293234N-acetyltransferase, noncatalytic subunit
    SN-P27(−)0.23 ± 0.05Agg-5:2430475mybI; cmfBSame as above
    SN-139(−)0.24 ± 0.05Agg-2:8027355piaASame as above
    SN-150(−)0.24 ± 0.03Agg-2:8027355piaASame as above
    SN-333(−)0.25 ± 0.02Agg-5:1156568DDB_G0288175UV radiation resistance–associated gene protein (automated)
    SN-598(−)0.20 ± 0.04Agg-5:2430475mybI; cmfBSame as above
    SN-584(−)0.20 ± 0.06Stramer3:388181DDB_G0278163TM2 domain–containing protein
    SN-106a(−)0.21 ± 0.04Agg-piaASame as above
    SN-040(−)0.21 ± 0.07Agg-3:389227tgrA_ps8Pseudogene
    SN-xxx(−)0.23 ± 0.03Agg-3:388181DDB_G0278163TM2 domain–containing protein
    SN-165(−)0.24 ± 0.07Agg-2:8013514DDB_G0277539WEE family protein kinase DDB_G0277539
    SN-661(−)0.25 ± 0.03Culmination3:1355756cldA;DDB_G0278897Clu domain–containing protein A;
    regulator of G protein signaling 13
    SN-242(−)0.26 ± 0.05Agg-2:8027893piaASame as above
    SN-252(−)0.27 ± 0.04Mound1:4688833DDB_G0270350_psPseudogene
    SN-223(−)0.28 ± 0.05Agg-2:8027355piaASame as above
    SN-290(−)0.28 ± 0.04Streamer5:2430475mybI; cmfBSame as above
    SN-432(−)0.29 ± 0.06Mound5:2430475mybI; cmfBSame as above
    SN-403(−)0.29 ± 0.05Fuzzy5:2430475mybI; cmfBSame as above
    SN-170(−)0.29 ± 0.04Agg-6:2879930abcA3ABC transporter A family protein

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/378/ra50/DC1

    Fig. S1. The collection of mutant strains with morphological defects used in our screen.

    Fig. S2. High-throughput screening strategy to determine electrotaxis phenotypes.

    Fig. S3. Design and fabrication of the barcoded microplates.

    Fig. S4. Cells on barcoded microplates at different positions in an electrotaxis chamber showed consistent electrotaxis responses.

    Fig. S5. Dictyostelium cells displayed consistent migration phenotypes on both barcoded microplates and tissue culture dishes.

    Fig. S6. Recapitulation of the defective electrotaxis phenotype in the mutant strains by knockout cells.

    Fig. S7. Electrotaxis of vegetative cells.

    Fig. S8. Mutated strains of Dictyostelium cells displayed consistent migration phenotypes on barcoded microplates and tissue culture dishes.

    Table S1. Defective strains identified from the screen.

    Table S2. Hyperresponsive strains identified.

    Table S3. Knockouts confirmed the genes that underlie the 12 defective strains.

    Movie S1. Wild-type AX2 cell not in an electric field.

    Movie S2. Wild-type AX2 cell in an electric field of 12 V/cm.

    Movie S3. PiaA in an electric field of 12 V/cm.

    Movie S4. Reexpression of piaA in piaA cells restored electrotaxis.

  • Supplementary Materials for:

    A large-scale screen reveals genes that mediate electrotaxis in Dictyostelium discoideum

    Runchi Gao, Siwei Zhao, Xupin Jiang, Yaohui Sun, Sanjun Zhao, Jing Gao, Jane Borleis, Stacey Willard, Ming Tang, Huaqing Cai, Yoichiro Kamimura, Yuesheng Huang, Jianxin Jiang, Zunxi Huang, Alex Mogilner, Tingrui Pan, Peter N. Devreotes, Min Zhao*

    *Corresponding author. E-mail: minzhao{at}ucdavis.edu

    This PDF file includes:

    • Fig. S1. The collection of mutant strains with morphological defects used in our screen.
    • Fig. S2. High-throughput screening strategy to determine electrotaxis phenotypes.
    • Fig. S3. Design and fabrication of the barcoded microplates.
    • Fig. S4. Cells on barcoded microplates at different positions in an electrotaxis chamber showed consistent electrotaxis responses.
    • Fig. S5. Dictyostelium cells displayed consistent migration phenotypes on both barcoded microplates and tissue culture dishes.
    • Fig. S6. Recapitulation of the defective electrotaxis phenotype in the mutant strains by knockout cells.
    • Fig. S7. Electrotaxis of vegetative cells.
    • Fig. S8. Mutated strains of Dictyostelium cells displayed consistent migration phenotypes on barcoded microplates and tissue culture dishes.
    • Table S1. Defective strains identified from the screen.
    • Table S2. Hyperresponsive strains identified.
    • Table S3. Knockouts confirmed the genes that underlie the 12 defective strains.
    • Legends for movies S1 to S4

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 817 KB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Wild-type AX2 cell not in an electric field.
    • Movie S2 (.avi format). Wild-type AX2 cell in an electric field of 12 V/cm.
    • Movie S3 (.avi format). PiaA in an electric field of 12 V/cm.
    • Movie S4 (.avi format). Reexpression of piaA in piaA cells restored electrotaxis.

    [Download Movies S1 to S4]


    Citation: R. Gao, S. Zhao, X. Jiang, Y. Sun, S. Zhao, J. Gao, J. Borleis, S. Willard, M. Tang, H. Cai, Y. Kamimura, Y. Huang, J. Jiang, Z. Huang, A. Mogilner, T. Pan, P. N. Devreotes, M. Zhao, A large-scale screen reveals genes that mediate electrotaxis in Dictyostelium discoideum. Sci. Signal. 8, ra50 (2015).

    © 2015 American Association for the Advancement of Science

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