Journal: bioRxiv

Article Title: Tropomyosin and α-actinin cooperation inhibits fimbrin association with actin filament networks in fission yeast

doi: 10.1101/169961

Figure Lengend Snippet: (A) Fluorescence micrographs of fission yeast cells expressing Lifeact-GFP following treatment with DMSO (control, top) or 200 μM Arp2/3 complex inhibitor CK-666 (bottom). (B-D, top panels) Fluorescence micrographs of fission yeast cells expressing Fim1-GFP (B) , Ain1-GFP (C) , or Ain1-GFP in a fim1 - 1Δ background (D) , following treatment with DMSO (left) or 200 μM CK-666 (right). Dotted lines outline cells. Scale bars, 5 μm. (B-D, bottom panels) Mean Fim1-GFP (B) or Ain1-GFP (C,D) fluorescence at the contractile ring normalized to whole cell fluorescence. Error bars=s.e. Two-tailed t-tests for data sets with unequal variance yielded p-values ∗ p=2.35×10 −5 , ∗∗ p=1.11×10 −5 , ∗∗∗ p=0.017. n≥13 cells total in each condition from two independent experiments. (E-F) Time-lapse fluorescent micrographs of fission yeast cells expressing ArpC5-mCherry (bottom) and overexpressing GFP-tagged α-actinin Ain1 from the 41xnmt promoter (top) for 20 hours in a wild-type (E) or fim1 - 1Δ background (F). Yellow box highlights Ain1-GFP localization at actin patches. Scale bars, 5 μm. Time in sec. (G) Percentage of cells in which Ain1-GFP is observed in actin patches. Error bars=s.e. Two-tailed t-tests for data sets with unequal variance yielded p-values + p=0.113, ++ p=0.002, +++ p=0.012. n=3 experimental replicates.

Article Snippet: Fimbrin Fim1 and tropomyosin AlaSer-Cdc8 (WT and I76C mutant) were expressed in BL21-Codon Plus (DE3)-RP (Agilent Technologies, Santa Clara, CA).

Techniques: Fluorescence, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Tropomyosin and α-actinin cooperation inhibits fimbrin association with actin filament networks in fission yeast

doi: 10.1101/169961

Figure Lengend Snippet: (A-C, top) Fluorescent micrographs of fission yeast cells expressing spindle pole body marker Sad1-tdTomato and Fim1-GFP (A) , Ain1-GFP (B) , or Ain1-GFP in a fim1 - 1Δ background (C), following treatment with DMSO (control, top) or 200 μM Arp2/3 complex inhibitor CK-666 (bottom). Scale bars, 5 μm. (A-C, bottom) Mean Fim1-GFP (A) or Ain1-GFP (B,C) contractile ring fluorescence normalized to whole cell fluorescence for cells in stage 1 (contractile ring formation), stage 2 (contractile ring dwell), or stage 3 (contractile ring constriction) of cytokinesis following treatment with DMSO (control) or 200 μM CK-666. Quantification of cells from stages 2 and 3 are also shown in . Dotted lines outline cells. Error bars=s.e. n≥6 cells for each condition.

Article Snippet: Fimbrin Fim1 and tropomyosin AlaSer-Cdc8 (WT and I76C mutant) were expressed in BL21-Codon Plus (DE3)-RP (Agilent Technologies, Santa Clara, CA).

Techniques: Expressing, Marker, Fluorescence

Journal: bioRxiv

Article Title: Tropomyosin and α-actinin cooperation inhibits fimbrin association with actin filament networks in fission yeast

doi: 10.1101/169961

Figure Lengend Snippet: (A-C) Two-color TIRFM of 1.5 μM Mg-ATP actin (15% Alexa 488-labeled) with 50 nM fimbrin Fim1 (Cy5-labeled) alone, or with either 1 μM wild-type α-actinin Ain1 or mutant Ain1(R216E). Scale bars, 5 μm. Dotted lines denote bundled regions. (B) Dot plots of the amount of Fim1-Cy5 fluorescence on the background (coverglass), single actin filaments, or two-filament F-actin bundles in either the absence (red circles) or presence of Ain1 (orange triangles) or Ain1(R216E) (yellow squares). Error bars=s.e. Two-tailed t-tests for data sets with unequal variance yielded p-values ∗ p=2.16×10 −4 , ∗∗ p=0.054, ∗∗∗ p=0.026, + p=1.13×10 −10 , ++ p=0.46, +++ p=2.39×10 −12 , and # p=3.90×10 −4 , ## p=0.18, ### p=1.97×10 −5 . Two independent experiments were performed for each condition. In total, n=20 background measurements, n≥54 single filament measurements, and n≥16 two-filament bundle measurements were taken for each condition. (C) Percentage of cells in which Ain1-GFP is observed in actin patches. Two-tailed t-test for data sets with unequal variance yielded ‡ p-value=0.0029. (D,E, top) Fluorescence micrographs of fission yeast in an ain1 - 1Δ background overexpressing GFP-tagged wild-type Ain1 (D) or mutant Ain1(R216E) (E) from the 41Xnmt1 promoter. (D,E, bottom) Timelapse (in sec.) of cell end taken from a single Z-plane.

Article Snippet: Fimbrin Fim1 and tropomyosin AlaSer-Cdc8 (WT and I76C mutant) were expressed in BL21-Codon Plus (DE3)-RP (Agilent Technologies, Santa Clara, CA).

Techniques: Labeling, Mutagenesis, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Tropomyosin and α-actinin cooperation inhibits fimbrin association with actin filament networks in fission yeast

doi: 10.1101/169961

Figure Lengend Snippet: (A,C,E) Two-color TIRFM of 1.5 μM Mg-ATP actin (15% Alexa 488-labeled) with 2.5 μM tropomyosin Cdc8 (TMR-labeled) and 500 nM (A) wild-type α-actinin Ain1, (B) mutant Ain1(R2l6E), or (C) fimbrin Fim1 (unlabeled). Scale bars, 1 μm. Dotted lines denote bundled regions. (B,D,F) Dot plots of the amount of Cdc8-TMR or Cdc8-Cy5 fluorescence on single filaments or two-filament bundles in the presence of Ain1 (B) , Ain1(R216E) (D), or Fim1 (F) Error bars=s.e. Two-tailed t-tests for data sets with unequal variance yielded p-values ∗ p=8.24×10 −18 , ∗∗ p=5.47×10 −12 , ∗∗∗ p=5.72×10 −11 .

Article Snippet: Fimbrin Fim1 and tropomyosin AlaSer-Cdc8 (WT and I76C mutant) were expressed in BL21-Codon Plus (DE3)-RP (Agilent Technologies, Santa Clara, CA).

Techniques: Labeling, Mutagenesis, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Tropomyosin and α-actinin cooperation inhibits fimbrin association with actin filament networks in fission yeast

doi: 10.1101/169961

Figure Lengend Snippet: (A-C) Three-color TIRFM of 1.5 μM Mg-ATP actin (15% Alexa 488-labeled) with 50 nM fimbrin Fim1 (Cy5-labeled) and 2.5 μM tropomyosin Cdc8 (TMR-labeled) in the (A) absence or (B) presence of 500 nM unlabeled α-actinin Ain1. (A-B, left) Representative TIRF field 540 seconds following reaction initiation. (A-B, right) Kymographs of actin, Fim1, and Cdc8 during bundle formation. Dotted lines denote bundled regions. Scale bars, 2.5 μm. Time bar, 30 seconds. (C-D) Dot plots of the amount of Cdc8-TMR (C) or Fim1-Cy5 fluorescence (D) on two-filament bundles in experiments with Cdc8 and Fim1, or Cdc8, Fim1 and Ain1. Purple and red lines denotes mean. Error bars=s.e. Two-tailed t-tests for data sets with unequal variance yielded p-values ∗ p=0.00716, ∗∗ p=1.15×10 −15 , ∗∗∗ p=2.68×10 −30 , ∗∗∗∗ p=1.14×10 −14 . n≥30 measurements from two independent experiments. (E) Model of the involvement of ABP competition in ABP sorting in the fission yeast cell. In endocytic actin patches, fimbrin Fim1 and cofilin Adf1 enhance each other’s activities, resulting in the displacement of tropomyosin Cdc8 from the F-actin network ( Christensen et al ., 2017 ). In the contractile ring, α-actinin Ain1 and tropomyosin Cdc8 work together to prevent fimbrin Fim1 association with the F-actin network.

Article Snippet: Fimbrin Fim1 and tropomyosin AlaSer-Cdc8 (WT and I76C mutant) were expressed in BL21-Codon Plus (DE3)-RP (Agilent Technologies, Santa Clara, CA).

Techniques: Labeling, Fluorescence, Two Tailed Test