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Proc Natl Acad Sci U S A
2018 May 29;11522:E5000-E5007. doi: 10.1073/pnas.1803415115.
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Helical rotation of the diaphanous-related formin mDia1 generates actin filaments resistant to cofilin.
Mizuno H
,
Tanaka K
,
Yamashiro S
,
Narita A
,
Watanabe N
.
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The complex interplay between actin regulatory proteins facilitates the formation of diverse cellular actin structures. Formin homology proteins (formins) play an essential role in the formation of actin stress fibers and yeast actin cables, to which the major actin depolymerizing factor cofilin barely associates. In vitro, F-actin decorated with cofilin exhibits a marked increase in the filament twist. On the other hand, a mammalian formin mDia1 rotates along the long-pitch actin helix during processive actin elongation (helical rotation). Helical rotation may impose torsional force on F-actin in the opposite direction of the cofilin-induced twisting. Here, we show that helical rotation of mDia1 converts F-actin resistant to cofilin both in vivo and in vitro. F-actin assembled by mDia1 without rotational freedom became more resistant to the severing and binding activities of cofilin than freely rotatable F-actin. Electron micrographic analysis revealed untwisting of the long-pitch helix of F-actin elongating from mDia1 on tethering of both mDia1 and the pointed end side of the filament. In cells, single molecules of mDia1ΔC63, an activated mutant containing N-terminal regulatory domains, showed tethering to cell structures more frequently than autoinhibited wild-type mDia1 and mDia1 devoid of N-terminal domains. Overexpression of mDia1ΔC63 induced the formation of F-actin, which has prolonged lifetime and accelerates dissociation of cofilin. Helical rotation of formins may thus serve as an F-actin stabilizing mechanism by which a barbed end-bound molecule can enhance the stability of a filament over a long range.
Fig. 1. Helical rotation of mDia1 attenuates filament severing by Xac2. (A–H) Representative time-lapse images (A–D) and kymographs (E–H) of mDia1(−) F-actin (A and E), mDia1(+) untrapped F-actin (B and F), mDia1(+) buckled F-actin (C and G), and mDia1(+) stuck F-actin (D and H). These filaments were nucleated for 3 min (white arrows in E–H) and then elongated (green arrows in E–H) in the presence of 0.1 μM DL488-actin and 3 μM profilin. The pointed end sides of buckled and stuck F-actin were trapped during elongation (green arrowheads in G and H). Filament severing was initiated by the addition of 50 nM Xac2 (red arrowheads in E–H), and filaments were severed (white arrowheads in A–C and E–G). In A–D, barbed ends are showed with white circles. Time is after the addition of 50 nM Xac2. (Scale bars: 5 μm.) (I) The severing frequency of four types of F-actin in the presence of 0.1 μM DL488-actin, 3 μM profiling, and 50 nM Xac2. (J) The severing frequency of F-actin induced by 50 nM Xac2 in the presence of 0.2 μM DL488-actin with 5 μM profilin (Left) and 0.5 μM DL488-actin with 5 μM profilin (Right).
Fig. 2. Helical rotation of mDia1 reduces binding of cofilin to F-actin. (A) Representative fluorescence images of four types of F-actin bound to cofilin-EGFP 1 min after the addition of 2 μM cofilin-EGFP at pH 6.8. Merged images show DyLight 550-labeled actin (DL550-actin; red) and cofilin-EGFP (green). Note the poor association of cofilin to stuck F-actin. (Scale bars: 5 μm.) (B) Fluorescence intensity of cofilin-EGFP along F-actin 1 min after the addition of 2 μM cofilin-EGFP. Gray lines show the fluorescence intensities of cofilin-EGFP bound to each F-actin [mDia1(−), n = 10; mDia1(+) untrapped, n = 10; mDia1(+) buckled, n = 10; mDia1(+) stuck, n = 13]. Black lines show the average fluorescence intensities of cofilin-EGFP bound to F-actin.
Fig. 3. The autoinhibition defective mDia1 mutant containing N-terminal domains attenuates actin disassembly in cells. (A) Representative images of EGFP-tagged mDia1 and its mutants expressed in XTC cells at a low density. Under this condition, individual EGFP-mDia1 molecules bound to cellular structures were observed as a discrete spot. Dotted lines indicate the area for the measurement in B. (Scale bars: 10 μm.) (B) The fraction of speckles of mDia1 and its mutants bound to cellular structures. mDia1 speckles were classified according to the criteria in our previous report (36). Processive is the fraction showing directional motion over five consecutive images (white), stationary is the fraction that stops the motion presumably trapped by rigid cell structures (black), random is the fraction showing slow random diffusing motion (red), and unclassified is the fraction that did not fall into three categories within the observation time (green). (C) The cumulative survival chance of DL550-actin SiMS incorporated into F-actin in nontransfected XTC cells and in cells overexpressing mDia1 and its mutants. Error bars show SD.
Fig. 4. The autoinhibition-defective mDia1 mutant containing N-terminal domains accelerates dissociation of cofilin from cellular F-actin. (A) Representative time-lapse images of EGFP-cofilin SiMS in XTC cells. Images indicated by rectangles are paneled. Red circles indicate cofilin-EGFP SiMS remaining from the first frame. (Scale bars: Left, 10 μm; Right, 5 μm.) (B) The fraction of cofilin-EGFP SiMS persisting from the initial image frame is plotted after correction for the photobleaching rate of cofilin-EGFP (T1/2 = 14.84 s). The decay rates between 1.0 and 0.2 were obtained by fitting the data with single exponentials (continuous lines).
Fig. 5. Helical rotation of mDia1 untwists the helical structure of F-actin when mDia1 and the pointed end side are immobilized. (A) Representative micrograph of a negatively stained F-actin elongated from an mDia1 aggregate in the presence of streptavidin. The black area on the left side of the image is an mDia1 aggregate. The rectangle indicates the area for digital processing in B. (Scale bar: 200 nm.) (B) Digital processing of electron micrographs. An extracted F-actin image was straightened (unbent) and then filtered in the Fourier space (1 and 2). The 1 image shows the filament after masking out the spatial frequencies >(30.0 nm)−1 in the Fourier space according to the method by Bremer et al. (57). The 2 image shows the filament filtered by a bandpass filter (SI Appendix, Fig. S8). In the 2 image, white lines indicate the position of cross-over. (Scale bar: 50 nm.) (C) Digital processing of electron micrographs of F-actin elongated from immobilized mDia1 aggregates in the absence of streptavidin. The procedure of digital processing is the same as B. (Scale bar: 50 nm.) (D) Box and whisker plot of cross-over length averaged from F-actin containing more than four consecutive cross-overs in the absence of streptavidin (−SA; n = 106) and the presence of streptavidin (+SA; n = 107). *Statistical significance (P < 0.01).
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