A new membrane protein Sbg1 links the contractile ring apparatus and septum synthesis machinery in fission yeast

Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the β-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1+ (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the β-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast

A New Membrane Protein Sbg1 Links the Contractile Ring Apparatus and Septum Synthesis Machinery in Fission Yeast

[EN]Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the β-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1+ (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the β-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast. © 2016 Sethi et al

Productivity Studies of Ultra High Density (UHD) Stitching Process

Abstract—This paper focuses on Ultra High Density (UHD) stitching process of connectors, where the company relies on 100 % manual inspection in UHD connectors. Based on historical data on production of UHD connectors, typically labour cost involved in inspection and testing is around 15 % of the total manufacturing cost. This poses both challenges and opportunities for improvement in the bottleneck process, which is the inspection process. The female connectors quantity produced is on the average of 2000 parts/shift. It takes around 47 sec of visual inspection per connector and around 2.89 sec per piece for go-gauge inspection. Considering these factors, the objectives of the study was to understand the whole process in its entirety and then use this knowledge to re-organize the stages staring from the automatic stitching process. By using process improvement tools of time study, methods and process mapping, a process re-orientation and re-organization has been carried out. This resulted in not only reduction of time of inspection and testing of the connectors, but also the piece inspection process reduced from an average total time of 112.59 sec to 86.15 sec. Overall improvement was also increased from 56 % to 79.5%. Index Terms—Process Mapping, Process Re-engineering, Ultra High Density. I

Temperature sensitive mutant <i>sbg1-3</i> deposits aberrant septa.

<p>(A) Multiple sequence alignment of <i>sbg1</i>, <i>sbg1-3</i> and related proteins from other filamentous fungi. Mutation in <i>sbg1-3</i> was identified at N147 as indicated in the image, just before the TM domain (residues 150–172). (B) Calcofluor white (CW) images of the medial plane of fixed cells from the indicated strains: wt (MBY192) and <i>sbg1-3</i> (MBY9359) after 6 hr at 36°C. Insets from other fields to show paired and dead cells. Coloured asterisks mark different phenotypes. (C) Quantification for experiment described in (b). (n = 3, ≥508 cells). (D) Calcofluor white (CW) images of the medial plane of fixed cells from the indicated strains: wt+pEmpty (MBY8558), <i>sbg1-3</i>+pEmpty (MBY9372), <i>sbg1-3</i>+pSbg1 (MBY9370) and <i>sbg1-3+</i>pBgs1 (MBY9366). Cells were grown overnight at 24°C in minimal media lacking leucine, shifted to YES for 2 hr and then shifted to 36°C for 6 hr. Coloured asterisks mark different phenotypes. (E) Quantification of experiment described in (d). (n = 2, ≥507 cells). (F) Aniline blue and DAPI images of medial plane of cells from the indicated strains: wt (MBY192), <i>imp2Δ</i> (MBY977), <i>sbg1-3</i> (MBY9359) and <i>imp2Δ sbg1-3</i> (MBY9358), fixed after shift to 36°C for 6 hr. Insets from another field to show tetranucleate cells. Coloured asterisks mark different phenotypes. (G) Quantification for experiment described in (f). (n = 3, ≥500 cells). Scale bar 5μm. Error bars indicate S.D.</p

Characterization of Sbg1p.

<p>(A) Bgs1p physically interacts with Sbg1p. Solubilized membrane proteins from the indicated strains: wt (MBY192), <i>hyg</i>^<i>r</i>-GFP-Sbg1p (MBY8967) and HA-Bgs1p <i>hyg</i>^<i>r</i>-GFP-Sbg1p (MBY9241) were immunoprecipitated (IP) with anti-GFP antibodies. Solubilized membrane proteins (input, top) and IP (bottom) were transferred to the same membrane and blotted with monoclonal anti-HA antibodies. (B) Time-lapse maximum Z projection spinning disk confocal montage of the indicated strain <i>hyg</i>^<i>r</i>-GFP-Sbg1p mCherry-Atb2p (MBY8977). Green, <i>hyg</i>^<i>r</i>-GFP-Sbg1p. Red, mCherry-Atb2p. 0min marks spindle pole body duplication as judged by a short microtubule spindle. (C) Time-lapse maximum Z projection spinning disk confocal montage of the indicated strain <i>hyg</i>^<i>r</i>-GFP-Sbg1p Rlc1p-tdTomato Pcp1p-mCherry (MBY9389). Green, <i>hyg</i>^<i>r</i>-GFP-Sbg1p. Red, Rlc1p-tdTomato Pcp1p-mCherry. 0min marks spindle pole body duplication as judged by Pcp1p signal. (D) Time-lapse maximum Z projection spinning disk confocal montage of the indicated strain <i>hyg</i>^<i>r</i>-GFP-Sbg1p tdTomato-Bgs1p (MBY9006). Green, <i>hyg</i>^<i>r</i>-GFP-Sbg1p. Red, tdTomato-Bgs1p. 0min marks initiation of nuclear division as judged by Sbg1p signal at nuclear periphery. Scale bar 5μm.</p

Multi-copy expression of <i>sbg1</i>^<i>+</i> improves cell wall structures of <i>cps1-191</i>.

<p>(A) Illustration depicting a longitudinal section of a septated wild type cell with both primary septum (PS) and secondary septum (SS). (B) Transmission electron microscopy images of septated cells of the indicated strains, I: wt+pEmpty (MBY8558), II: <i>cps1-191+</i>pEmpty (MBY8944) and III: <i>cps1-191+</i>pSbg1 (MBY8946) after 16 hr at 34°C. The bottom panel displays the division septum at a higher magnification. (C) Quantification of septated cells observed from the transmission electron microscopy images. (n≥14cells). Scale bar 1μm.</p

DaXi-high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy.

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 μm axially over an imaging volume of 3,000 × 800 × 300 μm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development - up to nine embryos at a time

Sbg1p interacts with other ring proteins.

<p>(A) Summary of yeast two hybrid interaction of truncated Sbg1TMΔp with the indicated proteins. (B) Tetrad dissection analysis of a cross between <i>sbg1-3</i> and <i>rga7Δ</i>. Boxes indicate double mutant <i>sbg1-3 rga7Δ</i>. (C) Maximum Z projection spinning disk confocal montage of the indicated strain (<i>sbg1-3</i> Rga7p-GFP Rlc1p-tdTomato Pcp1p-mCherry—MBY9485) after 6 hr at 36°C. Green, Rga7p-GFP. Red, Rlc1p-tdTomato Pcp1p-mCherry. Calcofluor White (CW) images were acquired as single medial plane images and are inverted for fluorescence. 0min indicates time of spindle body duplication. Scale bar 5μm.</p

Multi-copy expression of <i>sbg1</i>^<i>+</i> rescues <i>cps1-191</i>.

<p>(A) Time-lapse maximum Z projection spinning disk confocal montage of an actomyosin ring in wt Rlc1p-GFP Pcp1p-GFP (MBY 5732) after 2 hr at 36°C. No ring sliding was observed (n = 2, 8cells). 0min indicates time of spindle pole body duplication. Green, Rlc1p-GFP Pcp1p-GFP. (B) Time-lapse maximum Z projection spinning disk confocal montage of a sliding actomyosin ring in <i>cps1-191</i> Rlc1p-GFP Pcp1p-GFP (MBY 5730) after 2 hr at 36°C. Average no of cells with sliding rings: 20.8%±5.9% (n = 2, ≥30cells). 0min indicates time of spindle pole body duplication. Green, Rlc1p-GFP Pcp1p-GFP. (C) Spot assay comparing the viability of the strains of indicated genotypes. Cultures of the strains: wt+pEmpty (MBY8558), <i>cps1-191+</i>pEmpty (MBY8944), <i>cps1-191+</i>pSbg1 (MBY8946) and <i>cps1-191+</i>pBgs1 <i>(</i>MBY8947) were grown overnight at 24°C, were serially diluted in two-fold steps and spotted on minimal media agar plates without leucine and incubated at various growth temperatures. (D) Time-lapse maximum Z projection spinning disk confocal montages of actomyosin ring in the indicated strains: wt Rlc1p-GFP Pcp1p-GFP+pEmpty (MBY9493), wt Rlc1p-GFP Pcp1p-GFP+pSbg1 (MBY9508), <i>cps1-191</i> Rlc1p-GFP Pcp1p-GFP+pSbg1 (MBY9456) and <i>cps1-191</i> Rlc1p-GFP Pcp1p-GFP+pEmpty (MBY9454) after 3.5 hr at 34°C. 0min indicates time of spindle pole body duplication. Green, Rlc1p-GFP Pcp1p-GFP. (E) Graph shows the time taken (in minutes) for the ring to constrict in the indicated strains in (d). p-value<0.0001 indicated by **** (two-tailed T-test). (F) Maximum Z projection spinning disk confocal images of indicated strains: GFP-Cps1-191p+pEmpty_his3 (MBY9188) and GFP-Cps1-191p+pSbg1_his3 (MBY9193) after 6 hr at 34°C. Cells were fixed and stained with DAPI to identify binucleate cells. (G) Quantification for the presence or absence of medial GFP-Cps1-191p localization at 34°C for experiment in (f). (n = 3, ≥340cells). Scale bar 5μm. Error bars indicate S.D.</p