Nanoscale architecture of the Schizosaccharomyces pombe contractile ring.

Yeast methods and strain construction

S. pombe strains ( Supplementary file 1 ) were grown in yeast extract (YE) media at 25°C, with the exception of pnmt1-Acyl-mMaple3 ( Figure 1—figure supplement 1B ) and nmt1-Acyl-GFP ( Figure 5A ) which were grown in Edinburgh minimal medium (EMM) lacking thiamine to induce expression ( Basi et al., 1993 ). S. pombe transformations were performed with a lithium acetate method ( Keeney and Boeke, 1994 ). mMaple3 ( Wang et al., 2014 ) was cloned into a pFA6a vector at AscI and PacI sites. C-terminal fluorophore endogenous fusion strains were created by transforming a pFA6a integration cassette amplified with gene specific primers for insertion at the 3’ end of open reading frames ( Bähler et al., 1998 ). N-terminal mMaple3 or GFP fusion proteins were created with two methods. For nonessential genes (Cyk3, Imp2, Mid1, Pom1, Pxl1, Rga7, and Spa2), constructs containing (1) a 500 bp 5’ flank, (2) mMaple3 or GFP with a GGGGSGGGGSG C-terminal linker, (3) the coding sequence, and (4) a 500 bp 3’ flank were assembled into a pSK or pIRT2 vector ( Gibson et al., 2009 ). These cassettes were amplified from their 5’ and 3’ flanks, transformed into ura4 + deletion strains of the targeted gene, and selected for ura4 + loss and integration by resistance to 5-FOA. For essential genes (Cdc12, Cdc15, Myo2, Rng2, and Rgf3), constructs containing (1) a 500 bp 5’ flank, (2) mMaple3 or GFP with a GGGGSGGGGSG C-terminal flexible linker, (3) the coding sequence, (4) a Kan R cassette from pFA6a, and (5) a 500 bp 3’ flank were assembled into a pSK vector. These cassettes were digested before and after their 5’ and 3’ flanks and transformed into wildtype S. pombe . G418 resistant transformants were screened for correct integration by PCR. pAct-LifeAct-mMaple3 was constructed to be identical to the previously described pAct-LifeAct-GFP ( Huang et al., 2012 ). A 1 kb promotor from act1 , the LifeAct peptide (MGVADLIKKFESISKE) ( Riedl et al., 2008 ), a short GGPGG linker, and mMaple3 were assembled into a pJK148 plasmid which was subsequently digested by NruI and integrated into the leu1-32 locus ( Keeney and Boeke, 1994 ). Acyl-mMaple3 was constructed with the N-terminal acyl sequence from Gpa1 (MGCMSSKYADTSGGEV) and mMaple3 in a pREP1 vector ( Maundrell, 1993 ). All strains were confirmed by PCR and sequencing.

Sample preparation for fPALM imaging

To enrich for cells undergoing cytokinesis for imaging, newly separated short cells containing mMaple3-tagged proteins were isolated from a 7–30% lactose gradient, grown for 80 min in YE, labeled with 0.5 mM mCling-ATTO647N ( Revelo et al., 2014 ) (Synaptic Systems, Goettingen, Germany) for 10 min, and fixed. The resulting fixed sample was enriched for cells containing fully formed but unconstricted contractile rings. Cells were fixed with 3.7% paraformaldehyde directly in media for 20 min and subsequently washed 6X with PBS. Staining of actin with phalloidin was performed on fixed cells with 3.3 µM Phalloidin-Alexa488 in PBS + 0.1% NP-40 for 30 min, followed by 3 washes with PBS. Fixed and washed cells were resuspended in a small volume of PBS containing 1:100 diluted 80 nm gold particles (Microspheres-Nanospheres, Cold Spring, NY) previously sonicated for 1 hr to disperse clusters. The cell and gold particle suspension was mounted on a 3% agarose pad to prevent cell drifting during imaging. fPALM imaging fPALM imaging ( Figures 1 – 4 and 6 ) was performed on a Nikon dSTORM 4.0 system which included a Nikon Eclipse Ti microscope, 405, 488, 561, and 647 nm solid state lasers, a Hamamatsu ORCA-Flash4.0 camera, and a 100X CFI HP Apochromat TIRF 1.49NA objective with a 1.5X tubelens (resulting in 110 nm pixels). Imaging was performed at a 0° laser angle (‘straight through’) focused in Z at the center of cells for Figures 1 – 4 and the top of cells for Figure 6 . Z drift was minimized using a Nikon Perfect Focus system. The mMaple3 channel was imaged with simultaneous 0.5% 405 nm activation and 7.5% 561 nm excitation lasers, filtered through a polychroic mirror (ZT405/488/561/647rpc, Chroma, Bellows Falls, VT) and emission filter (ET585/65 m, Chroma), and captured with 30 ms exposures over 10–20 k frames. The ATTO647N channel was imaged with simultaneous 0.2% 405 nm activation and 2% 647 nm excitation lasers, filtered through a polychroic mirror (ZT405/488/561/647rpc, Chroma) and emission filter (ET705/75 m, Chroma), and captured with 10 ms exposures over 15 k frames. The Alexa488 channel for Phalloidin-Alexa488 was imaged with simultaneous 0.2% 405 nm activation and 2% 488 nm excitation lasers, filtered through a polychroic mirror (ZT405/488/561/647rpc, Chroma) and emission filter (ET525/50 m, Chroma), and captured with 10 ms exposures over 15 k frames. Laser powers and exposure times were optimized for single photoactivated localizations per ring in each frame. fPALM analysis fPALM images were analyzed using the ImageJ plugin ThunderSTORM ( Ovesný et al., 2014 ). Images were pre-filtered with a wavelet B-spline filter and molecules were approximately localized with the 8-neighborhood local maximum method. Molecules were sub-pixelly localized using the elliptical Gaussian weighted least squares method to identify precise lateral and Z positions. A Z-calibration was performed with 0.1 µm Tetraspek beads (ThermoFisher) for accurate Z positioning. Axial drift, though minimal with this microscope setup, was corrected in post-processing by tracking fiducial gold bead markers as well as cross-correlation analysis. fPALM images are visualized with localizations as normalized Gaussians, where each peak’s standard deviation (FWHM) equals its localization uncertainty ( Betzig et al., 2006 ). Processed and aligned 2-color images were restricted to a 500 nm plane through the center of the cells. Fully formed, unconstricted rings were identified visually through the following criteria: (1) a lack of precursor nodes, (2) a fully formed contiguous ring perpendicular to the long cell axis, and (3) a lack of membrane and septum ingression detected in the mCling channel. These criteria place all analyzed rings in a short window of 15–20 min between ring formation and constriction ( Wu et al., 2003 ). The edge of the plasma membrane was identified in an unbiased manner using an automated threshold method to determine where the signal from the mCling-ATTO647N channel drops to 5% of its maximum plasma membrane intensity ( Figure 1—figure supplement 1B ). The distance of each individual mMaple3 particle in the contractile ring to this edge was calculated. These data were fit with a Gaussian curve and the distance from the membrane (d center ) and a vertical width parameter (σ vert or FWHM) were determined using R ( Figure 1E ), as performed previously in a study of focal adhesion proteins ( Kanchanawong et al., 2010 ). A horizontal width parameter (σ width ) was also calculated to describe the FWHM of ring localizations parallel to the plasma membrane. d center , σ vert , and σ width values from multiple rings (see Figure 1—figure supplement 1 and Figure 2—figure supplement 1 for n values and other statistics) were plotted using ggplot2 in R. The contractile ring horizontal width dimension reported in the text (182 ± 26 nm) represents an average of all components’ σ width . The contractile ring maximum extension from the plasma membrane figure reported in the text (293 ± 64 nm) represents LifeAct-mMaple3’s (the most distal component) d center + ½(σ vert ), approximating the ring’s extension into the cytoplasm. Analysis of local homogeneity in Figure 6 was performed with downconverted 8-bit images using the ‘Texture Analysis’ ImageJ plugin ( https://imagej.nih.gov/ij/plugins/texture.html ). This plugin computes a grey-level co-occurrence matrix for all the pixels in the ROI of a contractile ring p(i, j) , and calculates multiple of Haralick’s textural features ( Haralick et al., 1973 ) from this matrix. We utilized the Inverse Difference Moment (IDM), a measure of local homogeneity: ∑ i = 1 N g ∑ j = 1 N g 1 1 + i - j 2 p ( i , j ) where N g is the maximum grey value and p(i, j) is the grey level co-occurrence matrix. This analysis was performed specifically in the direction of the ring circumferential axis.

FRET imaging

FRET imaging ( Figure 5 ) was performed on a Perkin Elmer Ultraview Vox spinning disk system equipped with a Zeiss Axio Observer microscope, 488 and 561 nm solid state lasers with a PhotoKinesis bleaching module, a Yokogawa CSU-X1 spinning disk, a 63X C-Apochromat objective, and a Hamamatsu ImageEM C9100-13 EMCCD camera. An acceptor photobleaching method was employed for FRET imaging in live cells. The mCherry-Cdc15 or Cdc15-mCherry fluorophores were bleached throughout the cells with 100% 561 nm laser power for 20 cycles. 10 frames of single Z slices in the donor GFP channel were acquired pre- and post-bleach. FRET percentages were calculated by first correcting for background and photobleaching over the 10 pre- and post-bleach frames, and subsequently calculating the percentage increase in GFP donor fluorescence at contractile ring ROIs of a consistent size. Statistical tests in Figure 5A–B and Figure 5—figure supplement 1B and S5B are ANOVA tests versus Rlc1-GFP a negative control which does not FRET with either mCherry-Cdc15 or Cdc15-mCherry, with uncorrected p values reported in Figure 5—figure supplement 1A–B . Statistical tests in Figure 5—figure supplement 1C were Fisher’s exact tests between the mCherry-Cdc15 and Cdc15-mCherry conditions.

Yeast methods and strain construction

S. pombe strains ( Supplementary file 1 ) were grown in yeast extract (YE) media at 25°C, with the exception of pnmt1-Acyl-mMaple3 ( Figure 1—figure supplement 1B ) and nmt1-Acyl-GFP ( Figure 5A ) which were grown in Edinburgh minimal medium (EMM) lacking thiamine to induce expression ( Basi et al., 1993 ). S. pombe transformations were performed with a lithium acetate method ( Keeney and Boeke, 1994 ). mMaple3 ( Wang et al., 2014 ) was cloned into a pFA6a vector at AscI and PacI sites. C-terminal fluorophore endogenous fusion strains were created by transforming a pFA6a integration cassette amplified with gene specific primers for insertion at the 3’ end of open reading frames ( Bähler et al., 1998 ). N-terminal mMaple3 or GFP fusion proteins were created with two methods. For nonessential genes (Cyk3, Imp2, Mid1, Pom1, Pxl1, Rga7, and Spa2), constructs containing (1) a 500 bp 5’ flank, (2) mMaple3 or GFP with a GGGGSGGGGSG C-terminal linker, (3) the coding sequence, and (4) a 500 bp 3’ flank were assembled into a pSK or pIRT2 vector ( Gibson et al., 2009 ). These cassettes were amplified from their 5’ and 3’ flanks, transformed into ura4 + deletion strains of the targeted gene, and selected for ura4 + loss and integration by resistance to 5-FOA. For essential genes (Cdc12, Cdc15, Myo2, Rng2, and Rgf3), constructs containing (1) a 500 bp 5’ flank, (2) mMaple3 or GFP with a GGGGSGGGGSG C-terminal flexible linker, (3) the coding sequence, (4) a Kan R cassette from pFA6a, and (5) a 500 bp 3’ flank were assembled into a pSK vector. These cassettes were digested before and after their 5’ and 3’ flanks and transformed into wildtype S. pombe . G418 resistant transformants were screened for correct integration by PCR. pAct-LifeAct-mMaple3 was constructed to be identical to the previously described pAct-LifeAct-GFP ( Huang et al., 2012 ). A 1 kb promotor from act1 , the LifeAct peptide (MGVADLIKKFESISKE) ( Riedl et al., 2008 ), a short GGPGG linker, and mMaple3 were assembled into a pJK148 plasmid which was subsequently digested by NruI and integrated into the leu1-32 locus ( Keeney and Boeke, 1994 ). Acyl-mMaple3 was constructed with the N-terminal acyl sequence from Gpa1 (MGCMSSKYADTSGGEV) and mMaple3 in a pREP1 vector ( Maundrell, 1993 ). All strains were confirmed by PCR and sequencing.

Additional files 10.7554/eLife.28865.017 Supplementary file 1. S. pombe strains used in this study. 10.7554/eLife.28865.018 Transparent reporting form