Rho1 Directs Formin-Mediated Actin Ring Assembly during Budding Yeast Cytokinesis

AbstractIn eukaryotic cells, dynamic rearrangement of the actin cytoskeleton is critical for cell division. In the yeast Saccharomyces cerevisiae, three main structures constitute the actin cytoskeleton: cortical actin patches, cytoplasmic actin cables, and the actin-based cytokinetic ring [1–4]. The conserved Arp2/3 complex and a WASP-family protein mediate actin patch formation, whereas the yeast formins (Bni1 and Bnr1) promote assembly of actin cables [5–9]. However, the mechanism of actin ring formation is currently unclear. Here, we show that actin filaments are required for cytokinesis in S. cerevisiae, and that the actin ring is a highly dynamic structure that undergoes constant turnover. Assembly of the actin ring requires the formin-like proteins and profilin, but is not Arp2/3-mediated. Furthermore, the formin-dependent actin ring assembly pathway is regulated by the Rho-type GTPase Rho1 but not Cdc42. Finally, we show that the formins are not required for localization of Cyk1/Iqg1, an IQGAP-like protein previously shown to be required for actin ring formation, suggesting that formin-like proteins and Cyk1 act synergistically but independently in assembly of the actin ring

High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors

The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors

Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation

International audienceUsing a high-throughput screening (HTS)-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well-defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O 2 • –), hydrogen peroxide (H 2 O 2), and peroxynitrite (ONOO –), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO – formation in activated macrophages. New diagnostic marker product for ONOO – is reported. We conclude that the newly developed HTS/ROS assays could also be used to identify potential inhibitors of ONOO – formed from Nox2-derived O 2 • – and nitric oxide synthase (NOS)-derived nitric oxide

Stability of the mCherry Fluorescence Signal.

<p>(A) Stability of the total cellular fluorescence after transfer to 4°C. Cells producing spTorA-mCherry-SsrA under Tat⁺ or Tat⁺⁺ conditions were incubated at 25°C for 8 h, and then transferred to 4°C to inhibit growth. The total mCherry cellular fluorescence was monitored periodically (<i>n</i> = 3). (B) Stability of the total cellular fluorescence after glucose addition. The total cellular fluorescence was quantified as in (A), except that spTorA-mCherry-SsrA production was repressed by addition of 0.5% glucose after 8 h of growth. Growth was continued at 25°C (<i>n</i> = 3). These data demonstrate that the intensity of the mCherry fluorescence signal can be maintained at a constant value for long time periods, thereby providing consistency when reading many HTS plates.</p

Summary of the HTS's and Hit Analysis by Follow-up Screens.

<p>Surviving and eliminated compounds from the local (<i>left</i>) and BIPDeC (<i>right</i>) HTS's are denoted by <i>black</i> and <i>red</i> numbers, respectively. The number of eliminated compounds at each step is shown to the right of the arrows and the number of surviving compounds are shown on the left in bold. Active (123) and inconclusive (1263*) compounds after the primary BIPDeC HTS were evaluated for chemical intractability and then retested in dose response and cell growth assays.</p

Optimization of the Total mCherry Fluorescence Signal and Validation of Substrate Design Properties.

<p>(A) Effect of spTorA-mCherry-SsrA induction levels on the total mCherry cellular fluorescence in wild type (Tat⁺; MC4100(DE3)) and TatABC overproduced (Tat⁺⁺) backgrounds. The production of spTorA-mCherry-SsrA was induced for 8 h at 25°C with the indicated concentrations of arabinose and the total mCherry cellular fluorescence was determined (<i>n</i> = 3; EX = 587 nm, EM = 610 nm). TatABC was induced with 1 mM IPTG. (B) Cell fractionation. Cytoplasmic (C) and periplasmic (P) fractions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149659#pone.0149659.ref060" target="_blank">60</a>] of the indicated strains were analyzed after 8 h induction by SDS-PAGE and immunoblotting using anti-mCherry antibodies. For the top three gels, the cells overproduced spTorA-mCherry-SsrA. For the bottom gel, the SsrA tag on spTorA-mCherry-SsrA was replaced with a 6xHis-tag (yielding spTorA-mCherry-H6). Precursor (p) and mature (m) proteins are indicated. The * and ** identify what appears to be C-terminally truncated products. (C) Fluorescence microscopy of the strains in (B). The mCherry proteins were induced at 25°C for 15 h with 2 mM arabinose. Cells were grown in fresh media for an additional 5 h with no arabinose before imaging. More than 95% of cells showed a clear and dominating periplasmic localization of mCherry in the Tat⁺⁺ strain versus ~70% for the Tat⁺ strain. No periplasmic mCherry was observed in the ΔTat strain, confirming that the spTorA-mCherry-SsrA protein was indeed targeted to and transported by the Tat system. Replacing the SsrA tag with a 6xHis-tag resulted in an increase in the amount of cytoplasmic mCherry (compare with Fig 2B), confirming that the SsrA tag promotes cytoplasmic degradation of the spTorA-mCherry-SsrA protein. Visually, about 80% of cells showed noticeable cytoplasmic localization of spTorA-mCherry-H6. Bar = 1.3 μm. (D) Effect of TatABC pre-induction on the total cellular fluorescence of mCherry. TatABC was induced 3 h prior to or simultaneously with spTorA-mCherry-SsrA (100 mM arabinose; <i>n</i> = 3). (E) Effect of induction time on the total cellular fluorescence of mCherry. The spTorA-mCherry-SsrA protein was produced in the Tat⁺⁺ background and the total mCherry cellular fluorescence was obtained as in (A) (<i>n</i> = 3).</p

<i>IC</i>_<i>50</i>'s for Hits Surviving the <i>In Vitro</i> Tat Transport Assays.

<p><i>IC</i>_<i>50</i>'s for Hits Surviving the <i>In Vitro</i> Tat Transport Assays.</p