Cooperation between tropomyosin and α-actinin inhibits fimbrin association with actin filament networks in fission yeast
- The University of Chicago, United States
Figures
Figure 1 with 3 supplements
Fimbrin Fim1 and α-actinin Ain1 compete for association with the contractile ring.
(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). Scale bar, 5 μm. (B-D, top …
Figure 1—figure supplement 1
Contractile ring ABP localization following CK-666 treatment.
Fluorescent micrographs of fission yeast cells either immunostained (anti-Cdc8) or expressing the indicated fluorescently tagged contractile ring ABPs from their endogenous locus: formin Cdc12-GFP, …
Figure 1—figure supplement 2
Tropomyosin Cdc8 does not leave the contractile ring following CK-666 treatment.
Mean anti-Cdc8 contractile ring fluorescence normalized to total cell fluorescence. Error bars = s.e. Two-tailed t-test for data sets with unequal variance yielded p-value=0.9338. n ≥ 10 cells.
Figure 1—figure supplement 3
Fimbrin Fim1 displaces α-actinin Ain1 from the contractile ring following CK-666 treatment.
(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 …
Figure 2 with 2 supplements
Fimbrin Fim1 and α-actinin Ain1 compete for association with actin patches.
(A–B) 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 hr in a wild-type …
Figure 2—figure supplement 1
Cellular expression of GFP-tagged α-actinin Ain1 constructs.
Fluorescence intensity of GFP-tagged Ain1 constructs for cells in cytokinesis (with contractile ring) or interphase (no contractile ring). Error bars = s.e. A single factor ANOVA yielded p-values …
Figure 2—video 1
Overexpressed α-actinin Ain1-GFP localizes to actin patches in the absence of fimbrin, related to Figure 1.
Fission yeast cells expressing actin patch marker ArpC5-mCherry (right) with Ain1-GFP (left) at endogenous or overexpressed levels. (Top panels) Endogenous Ain1-GFP in a fim1-1Δ background. (Middle …
Figure 3
Fimbrin Fim1 and α-actinin Ain1 compete for F-actin binding F-actin in vitro.
(A–B) 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 …
Figure 4 with 1 supplement
Fimbrin Fim1 and α-actinin Ain1 competition at actin patches is driven by their residence time on F-actin.
(A,B, top) Fluorescence micrographs of fission yeast in an ain1-1Δ background overexpressing GFP-tagged wild-type Ain1 (A) or mutant Ain1(R216E) (B) from the 41Xnmt1 promoter. Scale bars, 5 μm. …
Figure 4—video 1
Overexpressed mutant α-actinin Ain1(R216E)-GFP, but not Ain1-GFP, localizes to actin patches in the presence of fimbrin Fim1, related to Figure 4.
(Top) Fission yeast cells with Ain1-GFP overexpressed under the 41xnmt promoter (left) and expressing Fim1-mCherry at the endogenous locus (right). (Bottom) Fission yeast cells with Ain1(R216E)-GFP …
Figure 5 with 1 supplement
α-actinin Ain1 does not displace tropomyosin Cdc8 from F-actin bundles in vitro.
(A-C, top) Two-color TIRFM of 1.5 μM Mg-ATP actin (15% Alexa 488-labeled) with 2.5 µM tropomyosin Cdc8 (TMR-labeled) and unlabeled 500 nM (A) wild-type α-actinin Ain1, (B) mutant Ain1(R216E), or (C) …
Figure 5—video 1
α-actinin Ain1 does not displace tropomyosin Cdc8 from F-actin bundles, related to Figure 5.
TIRFM of 1.5 μM actin (Alexa-488 labeled) (left column) with 2.5 μM tropomyosin Cdc8 (TMR- or Cy5-labeled) (middle column) and 500 nM Ain1 (top row), 500 nM Ain1(R216E) (middle row), or 500 nM Fim1 …
Figure 6 with 1 supplement
Tropomyosin Cdc8 enhances α-actinin Ain1-mediated F-actin bundling in vitro.
(A–B) Low-speed (10,000 x g) sedimentation assays of 5 µM Mg-ATP preassembled actin filaments, 500 nM Ain1, and increasing concentrations of Cdc8 (0–5 µM). (A) Coomassie blue-stained gel of pellets …
Figure 6—video 1
Tropomyosin Cdc8 enhances α-actinin Ain1-mediated bundling, related to Figure 6.
TIRFM of 1.5 μM actin (Alexa-488 labeled) with the indicated varying concentrations of unlabeled Ain1 and Cdc8. Scale bar, 5 μm. Time in s.
Figure 7
α-actinin Ain1 facilitates the association of tropomyosin Cdc8 with bundled F-actin in the presence of fimbrin Fim1 in vitro.
(A,B) Low-speed sedimentation comparing Cdc8 in the pellet with F-actin bundled by Fim1 (left), Ain1 (middle), or both (right). (A) Coomassie blue-stained gel of representative triplicate samples of …
Figure 8 with 1 supplement
Tropomyosin Cdc8 and α-actinin Ain1 cooperate to compete with fimbrin Fim1 for association with F-actin in vitro.
(A–D) Four-color TIRFM of 1.5 μM Mg-ATP actin (15% Alexa 488-labeled) with 50 nM fimbrin Fim1 (Alexa 405-labeled) and 2.5 μM tropomyosin Cdc8 (TMR-labeled) in the (A) absence or (B) presence of 500 …
Figure 8—video 1
Tropomyosin Cdc8 and α-actinin Ain1 cooperate to compete with fimbrin Fim1 for bundling F-actin, related to Figure 5.
Four-color TIRFM of 1.5 μM actin (Alexa-488 labeled), 2.5 μM tropomyosin Cdc8 (TMR-labeled) and 50 nM fimbrin Fim1 (Alexa 405-labeled) with (bottom) or without (top) 500 nM Ain1 (Cy5-labeled). Scale …
Tables
Table 1
Fission yeast strains used in this study.
https://doi.org/10.7554/eLife.47279.018| Strain name | Genotype | Reference |
|---|---|---|
| KV91 | h+, kanMX6::myo2p::gfp-myo2p+, ade6-M210, leu1-32, ura4-D18 | Wu et al., 2003 |
| KV343 | h?, cdc12-mGFP::KanR | This study |
| KV459 | h+, rlc1-tdTomato-natMX6 ade6-M210 leu1-32 ura4-D18 | This study |
| KV579 | h-, ain1-GFP-KanMX6, URA+ | Wu et al., 2001 |
| KV588 | h+, pAct1 Lifeact-GFP::Leu+; ade6-m216; leu1-32; ura4-D18 | Huang et al., 2012 |
| Laporte et al., 2011 KV707 | h-, leu1-32, his3-D1, ura4-D18, ade6-M216, Pnmt41-SpAin1-mGFP::Leu+ | This study |
| KV804 | h?, fim1-mCherry-natMX6, ain1-Δ1:: kanMX6, Pnmt41-SpAin1-mGFP::Leu+ | This study |
| KV818 | h + kanMX6-Prng2-mEGFP-rng2 ade6-M210 leu1-32 ura4-D18 | Laporte et al., 2011 |
| KV856 | h?, ain1-Δ1:: kanMX6, Pnmt41-SpAin1(R216E)-mGFP::Leu+, fim1-mCherry-natMX6, ade6, leu1-32, ura4-D18 | This study |
| KV861 | h?, ain1-GFP-kanMX6, sad1-tdTomato-natMX6, ade6-m21?, leu1-32, ura4-D18 | This study |
| KV878 | h+, fim1-mGFP-kanMX6, sad1-tdTomato-natMX6 | This study |
| KV908 | h? fim1-1Δ-kanMX6, ain1-GFP-kanMX6, sad1-tdTomato::natMX6 | This study |
| KV963 | h?, fim1-1Δ::kanMX6, Pnmt41-SpAin1-mGFP::Leu+ | This study |
| KV968 | h? Pnmt41-SpAin1-mGFP::Leu+, ARPC5-mCherry-natMX6 | This study |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.47279.020
