SUMMARY Cells assemble and maintain functionally distinct actin cytoskeleton networks with various actin filament organizations and dynamics through the coordinated action of different sets of actin binding proteins. The biochemical and functional properties of diverse actin binding proteins, both alone and in combination, have been increasingly well studied. Conversely, how different sets of actin binding proteins properly sort to distinct actin filament networks in the first place is not nearly as well understood. Actin binding protein sorting is critical for the self-organization of diverse dynamic actin cytoskeleton networks within a common cytoplasm. Using in vitro reconstitution techniques including biomimetic assays and single molecule multi-color TIRF microscopy, we discovered that sorting of the prominent actin bundling proteins fascin and α-actinin to distinct networks is an intrinsic behavior, free of complicated cellular signaling cascades. When mixed, fascin and α-actinin mutually exclude each other by promoting their own recruitment and inhibiting recruitment of the other, resulting in the formation of distinct fascin- or α-actinin-bundled domains. Subdiffraction-resolution light microscopy and negative staining electron microscopy revealed that fascin domains are densely packed, while α-actinin domains consist of widely spaced parallel actin filaments. Importantly, other actin binding proteins such as fimbrin and espin show high specificity between these two bundle types within the same reaction. Here we directly observe that fascin and α-actinin intrinsically segregate to discrete bundled domains that are specifically recognized by other actin binding proteins.

Dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) leads to many cellular consequences, including perinuclear accumulation of free cholesterol due to impaired endosomal transport. The hypothesis being tested is that CF-related perinuclear cholesterol accumulation due to disrupted endocytic trafficking occurs as a result of reduced microtubule (MT) acetylation. Here, it is identified that acetylated-α-tubulin (Ac-tub) content is reduced by ∼40% compared with respective wild-type controls in both cultured CF cell models (IB3) and primary Cftr-/- mouse nasal epithelial tissue. Histone deacetylase 6 (HDAC6) has been shown to regulate MT acetylation, which provides reasonable grounds to test its impact on reduced Ac-tub content on CF cellular phenotypes. Inhibition of HDAC6, either through tubastatin treatment or HDAC6 knockdown in CF cells, increases Ac-tub content and results in redistributed free cholesterol and reduced stimulation of NF-κB activity. Mechanistically, endoplasmic reticulum stress, which is widely reported in CF and leads to aggresome formation, is identified as a regulator of MT acetylation. F508del CFTR correction with C18 in primary airway epithelial cells restores MT acetylation and cholesterol transport. A significant role for phosphatidyl inositol-3 kinase p110α is also identified as a regulator of MT acetylation.

Ena/VASP tetramers are processive actin elongation factors that localize to diverse F-actin networks composed of filaments bundled by different cross-linking proteins, such as filopodia (fascin), lamellipodia (fimbrin), and stress fibers (α-actinin). Previously, we found that Ena takes approximately threefold longer processive runs on trailing barbed ends of fascin-bundled F-actin. Here, we used single-molecule TIRFM (total internal reflection fluorescence microscopy) and developed a kinetic model to further dissect Ena/VASP’s processive mechanism on bundled filaments. We discovered that Ena’s enhanced processivity on trailing barbed ends is specific to fascin bundles, with no enhancement on fimbrin or α-actinin bundles. Notably, Ena/VASP’s processive run length increases with the number of both fascin-bundled filaments and Ena “arms,” revealing avidity facilitates enhanced processivity. Consistently, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Moreover, enhanced processivity on trailing barbed ends of fascin-bundled filaments is an evolutionarily conserved property of Ena/VASP homologues, including human VASP and Caenorhabditis elegans UNC-34. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and that avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory process whereby bundle size and bundling protein specificity control activities of a processive assembly factor.

How the bacterial virulence factors VopL/F from Vibrio catalyze actin nucleation is unclear. Using multicolor TIRF microscopy imaging, Burke et al. find that VopL and VopF stimulate actin assembly via identical pointed-end nucleation mechanisms.

Delivery of bacterial toxins to host cells is hindered by host protective barriers. This obstruction dictates a remarkable efficiency of toxins, a single copy of which may kill a host cell. Efficiency of actin-targeting toxins is further hampered by an overwhelming abundance of their target. The actin cross-linking domain (ACD) toxins of Vibrio species and related bacterial genera catalyze the formation of covalently cross-linked actin oligomers. Recently, we reported that the ACD toxicity can be amplified via a multivalent inhibitory association of actin oligomers with actin assembly factors formins, suggesting that the oligomers may act as secondary toxins. Importantly, many proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding Wiskott-Aldrich syndrome protein (WASP)-homology motifs 2 (WH2) organized in tandem and therefore may act as a multivalent platform for high-affinity interaction with the ACD-cross-linked actin oligomers. Using live-cell single-molecule speckle (SiMS) microscopy, total internal reflection fluorescence (TIRF) microscopy, and actin polymerization assays, we show that, in addition to formins, the oligomers bind with high affinity and potently inhibit several families of actin assembly factors: Ena/vasodilator-stimulated phosphorprotein (VASP); Spire; and the Arp2/3 complex, both in vitro and in live cells. As a result, ACD blocks the actin retrograde flow and membrane dynamics and disrupts association of Ena/VASP with adhesion complexes. This study defines ACD as a universal inhibitor of tandem-organized G-actin binding proteins that overcomes the abundance of actin by redirecting the toxicity cascade toward less abundant targets and thus leading to profound disorganization of the actin cytoskeleton and disruption of actin-dependent cellular functions.

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.

23Ena/VASP are tetrameric assembly factors that bind F-actin barbed ends continuously while increasing 24 their elongation rate within dynamic bundled networks such as filopodia. We used single-molecule 25 TIRFM and developed a kinetic model to dissect Ena/VASP's processive mechanism on bundled 26 filaments. Notably, Ena/VASP's processive run length increases with the number of both bundled 27 filaments and Ena arms, revealing avidity facilitates enhanced processivity. Moreover, Ena tetramers form 28 more filopodia than mutant dimer and trimers in Drosophila culture cells. Finally, enhanced processivity 29 on trailing barbed ends of bundled filaments is an evolutionarily conserved property of Ena/VASP 30 homologs and is specific to fascin-bundled filaments. These results demonstrate that Ena tetramers are 31 tailored for enhanced processivity on fascin bundles and avidity of multiple arms associating with multiple 32 filaments is critical for this process. Furthermore, we discovered a novel regulatory mechanism whereby 33 bundle size and bundling protein specificity control activities of a processive assembly factor. 34. CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/386961 doi: bioRxiv preprint first posted online Aug. Fascin bundles are composed of parallel filaments with narrow spacing, between 8-10 nm (Cant et al., 51 1994;Edwards and Bryan, 1995;Jansen et al., 2011; Yang et al., 2013). Ena/VASP continues to localize 52 to the tips of mature filopodia, where fascin-bundled filaments ultimately are the same length (Faix and 53 Rottner, 2006;Gupton and Gertler, 2007), presumably assuring uniform thickness of filopodia required 54 for protrusive force Winkelman et al., 2014). . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/386961 doi: bioRxiv preprint first posted online Aug. 7, 2018; 3 1999; Kuhnel et al., 2004). Between the EVH1 and EVH2 domains there is a poly-proline rich region that 66 binds profilin as well as SH3 domains (Ferron et al., 2007;Hansen and Mullins, 2010). 67In addition to the leading edge and tips of filopodia, Ena/VASP proteins also localize to focal 68 adhesions and stress fibers (Brindle et al., 1996;Reinhard et al., 1992), which are composed of filaments 69 crosslinked by CH domain superfamily crosslinkers, fimbrin/plastin and α-actinin. Fimbrin also localizes 70 to the lamellipodia and base of filopodia, and it bundles both parallel and antiparallel filaments with 71 narrow spacing (10-12 nm), similar to fascin (Hanein et al., 1998). In comparison, α-actinin bundles 72 filaments of mixed polarity with much wider spacing (30-36 nm) (Sjöblom et al., 2008). 73...

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