Colleen T. Skau

Identification and Characterization of a Small Molecule Inhibitor of Formin-Mediated Actin Assembly

Formins stimulate actin filament assembly for fundamental cellular processes including division, adhesion, establishing polarity, and motility. A formin inhibitor would be useful because most cells express multiple formins whose functions are not known and because metastatic tumor formation depends on the deregulation of formin-dependent processes. We identified a general small molecule inhibitor of formin homology 2 domains (SMIFH2) by screening compounds for the ability to prevent formin-mediated actin assembly in vitro. SMIFH2 targets formins from evolutionarily diverse organisms including yeast, nematode worm, and mice, with a half-maximal inhibitor concentration of approximately 5 to 15 microM. SMIFH2 prevents both formin nucleation and processive barbed end elongation and decreases formins affinity for the barbed end. Furthermore, low micromolar concentrations of SMIFH2 disrupt formin-dependent, but not Arp2/3 complex-dependent, actin cytoskeletal structures in fission yeast and mammalian NIH 3T3 fibroblasts.

Actin Filament Bundling by Fimbrin Is Important for Endocytosis, Cytokinesis, and Polarization in Fission Yeast

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament “gatekeeper” and as a filament cross-linker.

The Cytokinesis Formins from the Nematode Worm and Fission Yeast Differentially Mediate Actin Filament Assembly

Formins drive actin filament assembly for diverse cellular processes including motility, establishing polarity, and cell division. To investigate the mechanism of contractile ring assembly in animal cells, we directly compared the actin assembly properties of formins required for cytokinesis in the nematode worm early embryo (CYK-1) and fission yeast (Cdc12p). Like Cdc12p and most other formins, CYK-1 nucleates actin filament assembly and remains processively associated with the elongating barbed end while facilitating the addition of profilin-actin above the theoretical diffusion-limited rate. However, specific properties differ significantly between Cdc12p and CYK-1. Cdc12p efficiently nucleates filaments that in the presence of profilin elongate at approximately the same rate as control filaments without formin (∼10.0 subunits/s). CYK-1 is an inefficient nucleator but allows filaments to elongate profilin-actin 6-fold faster than Cdc12p (∼60 subunits/s). Both Cdc12p and CYK-1 bind to pre-assembled actin filaments with low nanomolar affinity, but CYK-1 dissociates 2 orders of magnitude more quickly. However, CYK-1 rapidly re-associates with free barbed ends. Cdc12p allows barbed ends to elongate in the presence of excess capping protein, whereas capping protein inhibits CYK-1-mediated actin assembly. Therefore, these evolutionarily diverse formins can drive contractile ring assembly by a generally similar mechanism, but cells with unique dimensions and physical parameters might require proteins with carefully tuned actin assembly properties.

Minimal requirements for actin filament disassembly revealed by structural analysis of malaria parasite actin-depolymerizing factor 1

Malaria parasite cell motility is a process that is dependent on the dynamic turnover of parasite-derived actin filaments. Despite its central role, actins polymerization state is controlled by a set of identifiable regulators that is markedly reduced compared with those of other eukaryotic cells. In Plasmodium falciparum, the most virulent species that affects humans, this minimal repertoire includes two members of the actin-depolymerizing factor/cofilin (AC) family of proteins, P. falciparum actin-depolymerizing factor 1 (PfADF1) and P. falciparum actin-depolymerizing factor 2. This essential class of actin regulator is involved in the control of filament dynamics at multiple levels, from monomer binding through to filament depolymerization and severing. Previous biochemical analyses have suggested that PfADF1 sequesters monomeric actin but, unlike most eukaryotic counterparts, has limited potential to bind or depolymerize filaments. The molecular basis for these unusual properties and implications for parasite cell motility have not been established. Here we present the crystal structure of an apicomplexan AC protein, PfADF1. We show that PfADF1 lacks critical residues previously implicated as essential for AC-mediated actin filament binding and disassembly, having a substantially reduced filament-binding loop and C-terminal α4 helix. Despite this divergence in structure, we demonstrate that PfADF1 is capable of efficient actin filament severing. Furthermore, this severing occurs despite PfADF1’s low binding affinity for filaments. Comparative structural analysis along with biochemical and microscopy evidence establishes that severing is reliant on the availability of an exposed basic residue in the filament-binding loop, a conserved minimal requirement that defines AC-mediated filament disassembly across eukaryotic cells.

Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly

Significance The ability of cells to interact with and remodel their extracellular environment is a critical process in developmental morphogenesis, wound healing, and cancer. How the physical and chemical responses of fibroblasts to the extracellular matrix are integrated across the cell remains a major open question. Previous data has shown a major role for the actin cytoskeleton in coordinating deposition and organization of the extracellular matrix by fibroblasts. Our study examines the role of inverted formin 2 (INF2), a protein known to create new actin filaments, in mediating cellular response to extracellular conditions and control of extracellular matrix remodeling. We find that INF2 is responsible for generating specific actin structures and specialized integrin-based fibrillar adhesions that are required for remodeling of the fibronectin extracellular matrix by fibroblasts. Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF–FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM.

Rationally simplified bistramide analog reversibly targets actin polymerization and inhibits cancer progression in vitro and in vivo.

We describe structure-based design and chemical synthesis of a simplified analog of bistramide A, which potently and reversibly binds monomeric actin with a K(d) of 9.0 nM, depolymerizes filamentous actin in vitro and in A549 (nonsmall cell lung cancer) cells, inhibits growth of cancer cell lines in vitro at submicromolar concentrations, and significantly suppresses proliferation of A549 cells in a nude mice tumor xenograft model in terms of both tumor growth delay and average tumor volume. This study provides a conceptual framework for the design and development of new antiproliferative compounds that target cytoskeletal organization of cancer cells in vivo by a combination of reversible G-actin binding and effective F-actin severing.

Retraction Notice to: FMN2 Makes Perinuclear Actin to Protect Nuclei during Confined Migration and Promote Metastasis

Our study reported that the formin-family actin nucleator FMN2 has a critical role in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage, facilitating cell survival during confined cell migration associated with cancer metastasis. Shortly following publication, a lab with whom we had shared reagents noticed that cell lines that were supposed to be stably expressing GFP-FMN2 were not. We subsequently found that a western blot in the paper had been inappropriately manipulated and that multiple cell lines were not as reported. When we constructed and validated new cell lines and reagents, our attempts to reproduce critical results in the paper were unsuccessful. Based on an assessment by the NIH, analysis by the Department of Health and Human Services Office of Research Integrity (ORI), and Dr. Skau’s admission, the ORI found that the first author Colleen Skau engaged in research misconduct by fabrication and falsification of results reported in Figures 2, 3, 5, 6, 7, S2, S4, S5, S6, and S7, including reporting data that did not originate from experimental observations, selectively including and omitting data points, selectively omitting images and conditions from analyses, falsifying the quantitation of data including statistical analyses, and falsifying a western blot. We are therefore retracting the paper, and we apologize for the inconvenience we have caused.

Chlamydomonas reinhardtii formin and profilin are optimized for acute rapid actin filament assembly

Chlamydomonas reinhardtii is a unicellular green alga that appears less dependent upon a conventional actin cytoskeleton than other eukaryotes, in part due to overlapping functions of a second non-conventional actin. One network that contains exclusively conventional F-actin is the fertilization tubule, a mating structure at the apical cell surface in gametes. Therefore, Chlamydomonas is an excellent system to investigate how actin polymerization is regulated in space and time. Chlamydomonas expresses a profilin (CrPRF), and a formin (CrFor1) that we have characterized for the first time. We found that unlike typical profilins, CrPRF prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed end elongation at equimolar concentrations to actin. However, CrFor1 is able to stimulate rapid actin filament assembly of CrPRF-bound actin. CrPRF further favors CrFor1-mediated actin assembly by potently inhibiting Arp2/3 complex-mediated actin assembly. The small molecule formin inhibitor SMIFH2 prevents fertilization tubule formation in gametes, suggesting that mating is a primary function of CrFor1. Together, these findings indicate that CrFor1 and CrPRF cooperate to selectively and rapidly assemble F-actin at the right time and place.