A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast.

Cell-cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure-the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion

Fission Yeast Sec3 and Exo70 Are Transported on Actin Cables and Localize the Exocyst Complex to Cell Poles

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk

Conditional Lethality, Division Defects, Membrane Involution, and Endocytosis in mre and mrd Shape Mutants of Escherichia coli▿ †

Maintenance of rod shape in Escherichia coli requires the shape proteins MreB, MreC, MreD, MrdA (PBP2), and MrdB (RodA). How loss of the Mre proteins affects E. coli viability has been unclear. We generated Mre and Mrd depletion strains under conditions that minimize selective pressure for undefined suppressors and found their phenotypes to be very similar. Cells lacking one or more of the five proteins were fully viable and propagated as small spheres under conditions of slow mass increase but formed large nondividing spheroids with noncanonical FtsZ assembly patterns at higher mass doubling rates. Extra FtsZ was sufficient to suppress lethality in each case, allowing cells to propagate as small spheres under any condition. The failure of each unsuppressed mutant to divide under nonpermissive conditions correlated with the presence of elaborate intracytoplasmic membrane-bound compartments, including vesicles/vacuoles and more-complex systems. Many, if not all, of these compartments formed by FtsZ-independent involution of the cytoplasmic membrane (CM) rather than de novo. Remarkably, while some of the compartments were still continuous with the CM and the periplasm, many were topologically separate, indicating they had been released into the cytoplasm by an endocytic-like membrane fission event. Notably, cells failed to adjust the rate of phospholipid synthesis to their new surface requirements upon depletion of MreBCD, providing a rationale for the “excess” membrane in the resulting spheroids. Both FtsZ and MinD readily assembled on intracytoplasmic membrane surfaces, and we propose that this contributes significantly to the lethal division block seen in all shape mutants under nonpermissive conditions

Schematic model suggesting the presence of the entire exocyst complex on exocytic vesicles.

<p>Exocytic vesicles labeled with the exocyst holocomplex may reach cell poles through one of two alternative routes: they are either transported along actin cables by myosin V Myo52 or reach cell poles by random walk. At the cell poles, Sec3 and Exo70 tether the exocyst complex and the vesicle by binding PIP₂ and Rho proteins. In absence of myosin V transport (<i>myo52</i>Δ), vesicles and the entire exocyst reach the pole by random walk. In absence of both Sec3 and Exo70, vesicles and the rest of the exocyst complex fail to be tethered at cell poles and form aggregates. Exo represents Sec5, Sec6, Sec8, Sec10, Sec15 and Exo84.</p

Strains used in this study.

<p>Strains used in this study.</p

Sec3, Exo70 and Sec5 are transported towards cell poles by myosin V Myo52.

<p><b>A-B.</b> Timelapse images of Sec3-, Sec5- and Exo70-GFP in wildtype (A) and <i>myo52</i>Δ cells (B). Arrowheads point to moving dots. A kymograph along the path of the indicated dot is shown on the right. Average rate of movement is shown on the right. Time is indicated on top in seconds. <b>C.</b> Timelapse images of Sec3-, Sec5- and Exo70-GFP in <i>myo52</i>Δ cells expressing a Tea2^N-CFP-Myo52^C chimera. GFP and CFP signals are shown. Arrowheads point to moving exocyst dots, which colocalize with the motor chimera. Note other non-moving signal also colocalize. A kymograph along the path of the indicated dot is shown on the right. Average rate of movement is shown on the right. Time is indicated on top in seconds. All images are single widefield images. Bar is 2 µm.</p

Synthetic lethality of <i>sec3</i> and <i>exo70</i>.

<p><b>A. </b><i>sec3</i>Δ, but not <i>sec6</i>Δ or <i>sec8</i>Δ, is viable in presence of 1 M sorbitol. Indicated diploids were sporulated and spores dissected on YE-sorbitol plates. Small colonies were <i>sec3</i>Δ, as they grew in presence of G418, while large colonies were wildtype as they failed to grow (not shown). <b>B.</b> 10-fold serial dilutions of indicated strains, showing <i>sec3</i>Δ grows in presence of 1 M sorbitol, but dies at 36°C. <b>C.</b> DIC images of wildtype and sec3Δ grown in YE-sorbitol at 25°C. Bar is 5 µm. <b>D.</b> 10-fold serial dilutions of indicated strains, showing <i>exo70</i>Δ <i>sec3-2</i> synthetic lethality. <b>E.</b> Tetrad dissection of <i>sec3</i>Δ<i>::kanMX/sec3+ exo70</i>Δ<i>::natMX/exo70+</i> diploid. Tetrad 1 shows a parental ditype, tetrads 2 to 4 are tetratypes, from which the double mutant spore is unviable, indicating synthetic lethality of <i>sec3</i>Δ and <i>exo70</i>Δ.</p

Sec3, like other exocyst subunits, localizes to cell poles in a Cdc42 and PIP₂-dependent, but largely actin-independent manner.

<p><b>A.</b> Maximum intensity projection of spinning disk confocal sections of Sec3-GFP in indicated genotypes. Bar is 5 µm. <b>B.</b> Timelapse of FRAP experiment of Sec3-GFP with or without 200 µM LatA. Indicated region was bleached. Time 0 was taken immediately post-bleach. Time is indicated in seconds. Single spinning disk confocal sections are shown. <b>C-D.</b> Average recovery of GFP-tagged exocyst subunits to bleached cell poles. Top: Untreated wildtype or <i>for3</i>Δ cells with indicated GFP-tagged subunits. Bottom: wildtype cells with indicated GFP-tagged subunits treated with 200 µM LatA or DMSO as control. The same data is shown in C and D. In C, all curves were normalized such that maximal recovery  =  1, to highlight similar recovery halftimes between 6 and 12 s for all curves. Note that <i>for 3</i>Δ and LatA curves globally show a tendency for slightly longer halftimes as compared to wildtype and untreated samples. In D, curves were normalized such that pre-bleach signal  =  1, to highlight the differences in mobile fractions. Note a tendency for reduced mobile fraction in <i>for3</i>Δ and LatA curves. For each condition, the curves show average of 16 or more bleached cell tips. <b>E-F.</b> Average recovery halftimes (E) and average mobile fraction ratio (F) for all strain analyzed in panels C-D. Error bars show the standard deviation of the mean. Significantly different values (unpaired Student’s t-test p<0.05) are indicated by asterisks.</p

Function and localization of <i>S. pombe</i> Sec3.

<p><b>A.</b> Scheme of Sec3 in <i>S. cere</i>visiae and <i>S. pombe</i>. Cryptic PH domains are shown in red, predicted coils in green. The C-terminal Sec5-binding region of <i>S. cerevisiae</i> Sec3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040248#pone.0040248-Guo1" target="_blank">[25]</a> is not conserved in <i>S. pombe</i>. <b>B.</b> Alignment of Sec3 PH domain. Critical residues predicted to contact phospholipids are indicated with red arrowheads and are conserved in <i>S. pombe</i>. <b>C.</b> Tetrad dissection of <i>sec3</i>Δ<i>::kanMX/sec3+</i> diploids on YE plate and terminal multi-septated phenotype of unviable spore. None of the viable spores grew on G418 plates (not shown). <b>D.</b> Maximum projection of spinning disk confocal sections of Sec3-GFP. Bar is 5 µm. <b>E.</b> Calcofluor-stained wildtype and <i>sec3-2</i> cells grown at 36°C for 6 h. Note that the multi-septated phenotype of <i>sec3-2</i> is rescued by plasmid-expression of <i>sec3+</i> (pREP41-sec3+), but not empty vector (pREP41). Bar is 5 µm. <b>F.</b> Secretion of acid phosphatase in wildtype and <i>sec3</i> mutants. Left: wildtype and <i>sec3</i>Δ mutants were pre-grown in EMM 1 M sorbitol at 25°C and kept at 25°C or shifted to 36°C at t = 0. Right: wildtype, <i>sec3-2</i> and <i>sec8-1</i> mutants were pre-grown at 25°C in EMM 1 M sorbitol and shifted to 36°C ± sorbitol at t = 0.</p

Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX, DedD, and RlpA) in Escherichia coli Cell Constriction ▿ †

Of the known essential division proteins in Escherichia coli, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an α-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain (EFtsN) of at most 35 residues that is centered about helix H2 in the periplasm. EFtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain (SFtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of SFtsN depended on the ability of cells to constrict and, thus, on the activity of EFtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional E. coli proteins of unknown function, DamX, DedD, and RlpA, as well as that of Bacillus subtilis CwlC, also accumulated sharply at constriction sites in E. coli, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process