Open Access Highly Accessed Research article

Identification and characterization of a set of conserved and new regulators of cytoskeletal organization, cell morphology and migration

Siau Wei Bai15, Maria Teresa Herrera-Abreu1, Jennifer L Rohn2, Victor Racine35, Virginia Tajadura1, Narendra Suryavanshi1, Stephanie Bechtel4, Stefan Wiemann4, Buzz Baum2 and Anne J Ridley1*

Author Affiliations

1 Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK

2 MRC Laboratory of Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK

3 Institute of Molecular and Cellular Biology, 61 Biopolis Drive, Proteos, 138673, Singapore

4 Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, Heidelberg, Germany

5 SWB, Institut Pasteur, 25-28 rue du Docteur Roux, 75015 PARIS; VR, Fluofarma, 2 rue Robert Escarpit, 33600 Pessac, France

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BMC Biology 2011, 9:54  doi:10.1186/1741-7007-9-54

Published: 11 August 2011

Additional files

Additional file 1:

Table S1. Selection of PMMs. Data on gene names, alternative names, domains and interaction partners are taken from NCBI Gene information and/or GeneCards http://www.genecards.org webcite; x, no information available.

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Additional file 2:

Figure S1. Effects of PMM depletion on cell morphology and cytoskeletal organization. (A) PC3 cells were transfected with the indicated siRNA pools for each PMM in 384-well plates. (B) PC3 cells plated on Matrigel were transfected with the indicated siRNA pools. Cells were fixed after 72 h, then stained for F-actin (red), α-tubulin (green) and nuclei (DAPI, blue). Images in A were acquired on an automated Nikon microscope; images in B were acquired by confocal microscopy. Scale bar, 100 μm (A); 10 μm (B).

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Additional file 3:

Figure S2. Examples of actin and shape phenotypes in PMM-depleted cells. Description: Images show examples of cytoskeletal and shape phenotypes used to classify PMMs into groups in Table 2. PC3 cells were transfected with siRNA pools targeting ARC (stress fibres), FMNL1 (cortical actin), PDZK8 (actin patches), ZMYM4 (very elongated), FAM40B (elongated), or FMNL2 (processes). Images shown are taken from the images in Figure S1, and show the F-actin or microtubule channels separately as indicated. Arrows indicate examples of stress fibres, cortical actin, actin patches or processes.

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Additional file 4:

Table S2. Phenotypes induced by individual siRNAs. Description: Each of four different siRNA oligos targeting the indicated genes (listed in Table S2) was transfected into PC3 cells or HeLa cells. The number of oligos that gave the same morphological phenotype as the pool of four oligos (Figure 1 and Table S2 for PC3, Figure 6 for HeLa cells) is shown. NP, no phenotype; x, not tested (weak phenotype with pool).

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Additional file 5:

Movie 1. Control siRNA. PC3 cells were transfected with siRNA pools for each PMM in 96-well plates. After 72 h, cells were scratch wounded and then monitored by time-lapse microscopy. Phase contrast cell images were acquired every 30 minutes for 16 hours.

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Additional file 6:

Movie 2. ZRANB1 siRNA. PC3 cells were transfected with siRNA pools for each PMM in 96-well plates. After 72 h, cells were scratch wounded and then monitored by time-lapse microscopy. Phase contrast cell images were acquired every 30 minutes for 16 hours.

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Additional file 7:

Movie 3. ARC siRNA. PC3 cells were transfected with siRNA pools for each PMM in 96-well plates. After 72 h, cells were scratch wounded and then monitored by time-lapse microscopy. Phase contrast cell images were acquired every 30 minutes for 16 hours.

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Additional file 8:

Movie 4.

    FMNL3
siRNA. PC3 cells were transfected with siRNA pools for each PMM in 96-well plates. After 72 h, cells were scratch wounded and then monitored by time-lapse microscopy. Phase contrast cell images were acquired every 30 minutes for 16 hours.

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Additional file 9:

Movie 5. FNBP3 siRNA. PC3 cells were transfected with siRNA pools for each PMM in 96-well plates. After 72 h, cells were scratch wounded and then monitored by time-lapse microscopy. Phase contrast cell images were acquired every 30 minutes for 16 hours.

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Additional file 10:

Movie 6. LIMD1 siRNA. PC3 cells were transfected with siRNA pools for each PMM in 96-well plates. After 72 h, cells were scratch wounded and then monitored by time-lapse microscopy. Phase contrast cell images were acquired every 30 minutes for 16 hours.

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Additional file 11:

Movie 7. FAM40B siRNA. PC3 cells were transfected with siRNA pools for each PMM in 96-well plates. After 72 h, cells were scratch wounded and then monitored by time-lapse microscopy. Phase contrast cell images were acquired every 30 minutes for 16 hours.

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Additional file 12:

Movie 8. FAM40A siRNA. PC3 cells were transfected with siRNA pools for each PMM in 96-well plates. After 72 h, cells were scratch wounded and then monitored by time-lapse microscopy. Phase contrast cell images were acquired every 30 minutes for 16 hours.

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Additional file 13:

Figure S3. Migration speeds of PMM-depleted PC3 cells. PC3 cells transfected with siRNAs for each of the indicated PMMs were imaged by time-lapse analysis for 14 h. Cells were tracked (Figure 3) and speeds determined. A total of 70 to 99 cells were tracked from three movies for each PMM. Values are means +/- s.e.m.; *** P ≤ 0.001, compared to control siRNA-transfected cells (unpaired Student's t-test).

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Additional file 14:

Table S3. siRNAs used for experiments. The names of the 26 human PMM genes and actin regulatory genes, and 4 siRNA sequences (sense strand) that were used as a pool for knockdown of each gene are shown. Only one siRNA was used for RhoA.

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