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      Cooperation between tropomyosin and α-actinin inhibits fimbrin association with actin filament networks in fission yeast

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          Abstract

          We previously discovered that competition between fission yeast actin binding proteins (ABPs) for binding F-actin facilitates their sorting to different cellular networks. Specifically, competition between endocytic actin patch ABPs fimbrin Fim1 and cofilin Adf1 enhances their activities, and prevents tropomyosin Cdc8’s association with actin patches. However, these interactions do not explain how Fim1 is prevented from associating strongly with other F-actin networks such as the contractile ring. Here, we identified α-actinin Ain1, a contractile ring ABP, as another Fim1 competitor. Fim1 competes with Ain1 for association with F-actin, which is dependent upon their F-actin residence time. While Fim1 outcompetes both Ain1 and Cdc8 individually, Cdc8 enhances the F-actin bundling activity of Ain1, allowing Ain1 to generate F-actin bundles that Cdc8 can bind in the presence of Fim1. Therefore, the combination of contractile ring ABPs Ain1 and Cdc8 is capable of inhibiting Fim1’s association with F-actin networks.

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          Most cited references36

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          Characterization of two classes of small molecule inhibitors of Arp2/3 complex

          Polymerization of actin filaments directed by the Arp2/3 complex supports many types of cellular movements1. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones owing to the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-636 binds between Arp2 and Arp3 where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying Arp2/3 complex in living cells.
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            Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide, phalloidin, and muscle regulatory proteins.

            Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy. The persistence length (Lp) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine-action. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 micron, in agreement with recent observations. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same Lp of 9 +/- 0.5 microns, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion. In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with Lp = 13.5 microns. Hence, newly formed F-ADP-Pi-actin filaments are more rigid than "old" F-ADP-actin filaments, a fact which has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (Lp = 20 +/- 1 micron) in the off state (-Ca2+), and flexible (Lp = 12 microns) in the on state (+Ca2+), consistent with the steric blocking model. In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (Lp = 20 +/- 1 micron). In conclusion, this method allows accurate measurement of small (< or = 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.
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              Homeostatic actin cytoskeleton networks are regulated by assembly factor competition for monomers.

              Controlling the quantity and size of organelles through competition for a limited supply of components is quickly emerging as an important cellular regulatory mechanism. Cells assemble diverse actin filament (F-actin) networks for fundamental processes including division, motility, and polarization. F-actin polymerization is tightly regulated by activation of assembly factors such as the Arp2/3 complex and formins at specific times and places. We directly tested an additional hypothesis that diverse F-actin networks are in homeostasis, whereby competition for actin monomers (G-actin) is critical for regulating F-actin network size. Here we show that inhibition of Arp2/3 complex in the fission yeast Schizosaccharomyces pombe not only depletes Arp2/3-complex-mediated endocytic actin patches, but also induces a dramatic excess of formin-assembled F-actin. Conversely, disruption of formin increases the density of Arp2/3-complex-mediated patches. Furthermore, modification of actin levels significantly perturbs the fission yeast actin cytoskeleton. Increasing actin favors Arp2/3-complex-mediated actin assembly, whereas decreasing actin favors formin-mediated contractile rings. Therefore, the specific actin concentration in a cell is critical, and competition for G-actin helps regulate the proper amount of F-actin assembly for diverse processes.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Role: Senior Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                10 June 2019
                2019
                : 8
                : e47279
                Affiliations
                [1 ]deptDepartment of Molecular Genetics and Cell Biology The University of Chicago ChicagoUnited States
                [2 ]deptDepartment of Biochemistry and Molecular Biology The University of Chicago ChicagoUnited States
                University of Warwick United Kingdom
                Utrecht University Netherlands
                University of Warwick United Kingdom
                Author notes
                [†]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0003-0323-6169
                https://orcid.org/0000-0002-5747-0949
                Article
                47279
                10.7554/eLife.47279
                6557641
                31180322
                99abd6f5-3646-4516-9289-6f817d3d3052
                © 2019, Christensen et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 30 March 2019
                : 29 May 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: Graduate Research Fellowship DGE-1144082
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: T32 GM0071832
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: Graduate Research Fellowship DGE-1746045
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: IMSD R25GM109439
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R01 GM079265
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000048, American Cancer Society;
                Award ID: RSG-11-126-01-CSM
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Cell Biology
                Custom metadata
                The contractile ring actin binding proteins tropomyosin Cdc8 and a-actinin Ain1 synergize to effectively compete with the endocytic actin patch protein fimbrin Fim1 for associating with F-actin networks.

                Life sciences
                endocytosis,actin patch,cytokinesis,contractile ring,ain1,cdc8,s. pombe
                Life sciences
                endocytosis, actin patch, cytokinesis, contractile ring, ain1, cdc8, s. pombe

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