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 multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction

Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. β-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3 d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction

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

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

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

Localization dependencies between Sbg1p and Bgs1p.

<p>(A) Maximum Z projection spinning disk confocal images of germinated cells of the indicated genotype from diploid <i>sbg1Δ/sbg1</i>^<i>+</i> mCherry-Atb2p GFP-Bgs1p (MBY9160). Green, GFP-Bgs1p. Red, mCherry-Atb2p. (B) Quantification for the experiment described in (a). (C) Determination of Bgs1p protein levels in wild type cells (GFP-Bgs1p MBY8865) and sporulated <i>sbg1Δ</i>cells from diploid <i>sbg1Δ/sbg1</i>^<i>+</i> GFP-Bgs1p (MBY11106) respectively. (D) Time-lapse maximum Z projection spinning disk confocal montage of the indicated strains: <i>hyg</i>^<i>r</i>-GFP-Sbg1p (MBY8967) and <i>cps1-191 hyg</i>^<i>r</i>-GFP-Sbg1p (MBY9097) after 3 hr at 34°C and 36°C. Images are inverted for Sbg1p fluorescence. (E) Quantification for the experiment described in (d). (F) Determination of Sbg1p protein levels in <i>cps1-191 hyg</i>^<i>r</i>-GFP-Sbg1p (MBY9097) cells at 24°C and 36°C after 4 hr. (G) Maximum Z projection spinning disk confocal images of <i>cps1-191</i> cells carrying multi-copy expression plasmid with mCherry at N-terminus of Sbg1p (<i>cps1-191</i>+pmCherry-Sbg1—MBY9285) at 24°C, 34°C and 36°C–shift up for 3 hr. Images are inverted for Sbg1p fluorescence. Arrowheads indicate presence of mCherry-Sbg1p signal at the division site while asterisk marks the absence of it. (n = 2, 50 cells). (H) Tetrad dissection analysis of a cross between <i>sbg1-3</i> and <i>cps1-191</i>. Boxes indicate double mutant <i>sbg1-3 cps1-191</i>. Scale bar 5μm.</p