Journal: Molecular Biology of the Cell

Article Title: The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

doi: 10.1091/mbc.E16-01-0010

Figure Lengend Snippet: SpAin1 bundles F-actin. (A) Domain organization of fission yeast α-actinin SpAin1 and human α-actinin 4 HsACTN4, with amino acid numbers indicated next to domain boundaries. Dashed lines indicate significant residue identity between specific spectrin repeats (SR). ABD, actin-binding domain. (B) Regions of Coomassie blue–stained gels showing purified proteins used in this study. (C and D) Low-speed (10,000 × g ) sedimentation of F-actin preassembled from 5 μM Mg-ATP actin, following incubation with a range of SpAin1 or HsACTN4 concentrations for 20 min at 25°C. (C) Coomassie blue–stained gel of pellets from a representative experiment with SpAin1. (D) Plot of the dependence of actin in the pellets on the concentration of SpAin1 or HsACTN4. Error bars, SE; n = 3. (E) Fluorescence micrographs of preassembled F-actin labeled with rhodamine–phalloidin following incubation with the indicated cross-linkers for 20 min. Scale bar: 5 μm.

Article Snippet: Insect cell expression constructs for full-length SpAin1 (pAcSG2-SpAin1-His) were prepared by PCR amplification from synthesized SpAin1 cDNA in a pUC57 plasmid (GenScript, Piscataway, NJ) with iProof DNA polymerase (Bio-Rad, Hercules, CA).

Techniques: Residue, Binding Assay, Staining, Purification, Sedimentation, Incubation, Concentration Assay, Fluorescence, Labeling

Journal: Molecular Biology of the Cell

Article Title: The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

doi: 10.1091/mbc.E16-01-0010

Figure Lengend Snippet: SpAin1 makes F-actin bundles of mixed polarity. (A–C) TIRFM observation of the addition of 0.75 μM Mg-ATP actin (33.3% Oregon Green-actin) to preassembled F-actin bundles (red boxes) (10.0% Oregon Green-actin) made by the indicated concentrations of human fascin HsFSCN1, fission yeast fimbrin SpFim1, SpAin1, or HsACTN4. (A) Time-lapse fluorescence micrographs. Scale bar: 5 μm. (B) Time-lapse fluorescence micrographs of representative parallel (top) and antiparallel (bottom) two-filament bundles made by SpAin1, used for quantification in C. Scale bar: 5 μm. Green and blue arrowheads mark F-actin barbed ends. (C) Percentage of two-filament bundles that are parallel and antiparallel.

Article Snippet: Insect cell expression constructs for full-length SpAin1 (pAcSG2-SpAin1-His) were prepared by PCR amplification from synthesized SpAin1 cDNA in a pUC57 plasmid (GenScript, Piscataway, NJ) with iProof DNA polymerase (Bio-Rad, Hercules, CA).

Techniques: Fluorescence

Journal: Molecular Biology of the Cell

Article Title: The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

doi: 10.1091/mbc.E16-01-0010

Figure Lengend Snippet: SpAin1 bundles F-actin less well than HsACTN4. (A) Merged two-color TIRFM time-lapse fluorescence micrographs showing the assembly of 2.5 μM Mg-ATP actin (33.3% Oregon Green-actin) in the presence of 300 nM SpAin1 (20% labeled with TMR). Blue and yellow arrowheads mark elongating barbed ends and SpAin1 decoration of a two-filament bundle. Scale bar: 5 μm. (B–E) Single-color TIRFM of the assembly of 2.5 μM Mg-ATP actin (33.3% Oregon Green-actin) in the absence and presence of the indicated concentrations of HsACTN4 or SpAin1. (B) Representative field of filaments 12 min after reactions were initiated. Fluorescence micrographs (left) and pseudocolored panels (right) with individual and bundled filaments traced with different colors: yellow, 1 filament; red, 2; green, 3; magenta, 4; cyan, ≥ 5. Scale bar: 5 μm. (C) Dependence of the number of filaments per bundle on the concentration of SpAin1 or HsACTN4. Error bars, SE; n = 4. (D) Distribution of Oregon Green-actin pixel intensity of filaments assembled in the absence and presence of SpAin1 or HsACTN4. (E) Polarity of two-filament bundles.

Article Snippet: Insect cell expression constructs for full-length SpAin1 (pAcSG2-SpAin1-His) were prepared by PCR amplification from synthesized SpAin1 cDNA in a pUC57 plasmid (GenScript, Piscataway, NJ) with iProof DNA polymerase (Bio-Rad, Hercules, CA).

Techniques: Fluorescence, Labeling, Concentration Assay

Journal: Molecular Biology of the Cell

Article Title: The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

doi: 10.1091/mbc.E16-01-0010

Figure Lengend Snippet: SpAin1 is a highly dynamic F-actin–bundling protein. (A–E) Two-color TIRFM of 2.5 μM Mg-ATP actin (33.3% Oregon Green-actin) assembled in the presence of 900 nM SpAin1, HsACTN4 or SpAin1(R216E) (1% TMR-labeled). (A) Merged fluorescence micrographs of α-actinin on two-filament bundles. Scale bar: 5 μm. (B) Time-lapse fluorescence micrographs of TMR-labeled α-actinin on corresponding bundles in A. Arrowheads mark speckles that remain for more (red) or less (green) than 1.5 s. (C) Kymographs of α-actinin on bundles in A over 20 s. Red boxes mark the 1.5 s of montages shown in B. Scale bars: 5 μm (horizontal); 5 s (vertical). (D) Histograms of the residence time of α-actinin speckles on two- and three-filament bundles. (E) Exponential fits of the fraction of α-actinin bound over time on two- and three-filament bundles, revealing the indicated residence times.

Article Snippet: Insect cell expression constructs for full-length SpAin1 (pAcSG2-SpAin1-His) were prepared by PCR amplification from synthesized SpAin1 cDNA in a pUC57 plasmid (GenScript, Piscataway, NJ) with iProof DNA polymerase (Bio-Rad, Hercules, CA).

Techniques: Labeling, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

doi: 10.1091/mbc.E16-01-0010

Figure Lengend Snippet: Mutant SpAin1(R216E) bundles F-actin better than WT SpAin1. (A) Domain organization of SpAin1 indicating the position of the R216E mutation in the second of two CH domains (top). ABD, actin-binding domain; CH, calponin homology domain. Alignment of the last α-helix of the CH2 domain of α-actinin from different species, with the 100% conserved residue SpAin1 R216 labeled in red with an asterisk (bottom) ( Borrego-Diaz et al. , 2006 ; Weins et al. , 2007 ). (B) Low-speed sedimentation of F-actin over a range of concentrations of SpAin1 or SpAin1(R216E). Error bars, SE; n = 3. (C and D) Single-color TIRFM of the assembly of 2.5 μM Mg-ATP actin (33.3% Oregon Green-actin) in the absence and presence of 300 nM SpAin1 or SpAin1(R216E). (C) Representative field of filaments 10 min after a reaction with SpAin1(R216E). Fluorescence micrograph (left) and pseudocolored panel (right) with individual and bundled filaments traced with different colors: yellow, 1 filament; red, 2; green, 3; magenta, 4; cyan, ≥ 5. Scale bar: 5 μm. (D) Average number of filaments per bundle. Error bars, SE; n = 4. (E) Quantification of bundle polarity of 300 nM WT SpAin1 or mutant SpAin1 (R216E). Error bars, SE; n = 2.

Article Snippet: Insect cell expression constructs for full-length SpAin1 (pAcSG2-SpAin1-His) were prepared by PCR amplification from synthesized SpAin1 cDNA in a pUC57 plasmid (GenScript, Piscataway, NJ) with iProof DNA polymerase (Bio-Rad, Hercules, CA).

Techniques: Mutagenesis, Binding Assay, Residue, Labeling, Sedimentation, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

doi: 10.1091/mbc.E16-01-0010

Figure Lengend Snippet: SpAin1(R216) causes severe cytokinetic defects in fission yeast. (A–D) WT cells, SpAin1 null ain1-Δ1 cells, and ain1-Δ1 cells expressing either WT SpAin1-GFP or mutant SpAin1(R216E)-GFP from low- ( 81nmt1 ) and medium- ( 41nmt1 ) strength promoters integrated at the leu locus. (A and B) General cytokinetic defects 24 h after nmt1 -promoter induction by removal of thiamine. (A) Fluorescence micrographs of cells stained with DAPI and calcofluor to visualize nuclei and septa. (B, left) Percentage of cells with 1 (white), 2 (light blue), or > 2 nuclei (dark blue). n ≥ 400 cells. (B, right) Percentage of normal (n, white) and abnormal (a, blue) septa. n ≥ 100 septa. (C) Z-projection fluorescence micrographs of the indicated cells expressing the contractile ring marker Rlc1-tdTomato (left), SpAin1-GFP or SpAin1(R216E)-GFP (middle), and merge (right). Scale bar: 5 μm. (D) SpAin1-GFP and SpAin1(R216E)-GFP whole cell fluorescence for the indicated strains. Error bars, SE; n ≥ 10 cells.

Article Snippet: Insect cell expression constructs for full-length SpAin1 (pAcSG2-SpAin1-His) were prepared by PCR amplification from synthesized SpAin1 cDNA in a pUC57 plasmid (GenScript, Piscataway, NJ) with iProof DNA polymerase (Bio-Rad, Hercules, CA).

Techniques: Expressing, Mutagenesis, Fluorescence, Staining, Marker

Journal: Molecular Biology of the Cell

Article Title: The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

doi: 10.1091/mbc.E16-01-0010

Figure Lengend Snippet: SpAin1(R216) causes defects in both contractile ring assembly and constriction. (A–F) Cytokinetic kinetics of WT cells, SpAin1 null ain1-Δ1 cells, and ain1-Δ1 cells expressing either WT SpAin1-GFP or mutant SpAin1(R216E)-GFP from low- ( 81nmt1 ) and medium- ( 41nmt1 ) strength promoters integrated at the leu locus. (A–D) Fluorescence micrograph montages of the time course of Rlc1-tdTomato–labeled contractile ring formation and constriction (right), corresponding to the boxed regions in DIC images (left). Cytokinetic stages are marked with red numbers as indicated in Supplemental Figure S7C: 1, Rlc1-tdTomato appearance (start); 2, ring assembly complete; 3, ring constriction begins; 4, ring constriction ends; 5, Rlc1-tdTomato disappears. Scale bars: 5 μm. (E) Average duration of ring assembly. Error bars, SD; n ≥ 10 cells. * p < 0.05. (F) Average duration of ring constriction. Error bars, SD; n ≥ 10 cells. * p < 0.05.

Article Snippet: Insect cell expression constructs for full-length SpAin1 (pAcSG2-SpAin1-His) were prepared by PCR amplification from synthesized SpAin1 cDNA in a pUC57 plasmid (GenScript, Piscataway, NJ) with iProof DNA polymerase (Bio-Rad, Hercules, CA).

Techniques: Expressing, Mutagenesis, Fluorescence, Labeling

Journal: Molecular Biology of the Cell

Article Title: The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

doi: 10.1091/mbc.E16-01-0010

Figure Lengend Snippet: The role of dynamic SpAin1 during contractile ring assembly. (A and B) Cartoon model for SpAin1-mediated contractile ring assembly and constriction. (A) Condensation of pre ring nodes (red and blue circles) into a contractile ring by the SCPR model in a wild-type cell ( Vavylonis et al. , 2008 ). Actin filaments assembled from nodes in random orientations are aligned into antiparallel bundles by SpAin1 (α-actinin) (i), which allows filaments to be efficiently captured by myosin-II motors on adjacent nodes (ii). The dynamic nature of SpAin1 allows myosin II to pull actin filaments without impediment, promoting proper ring constriction (iii). (B) Contractile ring formation in a cell expressing the less dynamic bundling mutant SpAin1(R216E). Actin filaments are elongated from nodes in random orientations (i), but get stalled as a result of excessive bundling, preventing actin filaments from reaching neighboring nodes (ii and iii). (C and D) Simulation of node capture at varying cross-linker affinities for F-actin (unitless K d −1 ). (C) Examples of node search and capture at low (0.3) cross-linker affinity (top) or high (3.0) cross-linker affinity (bottom). (D) Dependence of node capture rate ( k first-hit ( s −1 )) over a range of cross-linker affinities (τ x k on) at filament elongation angles of 15° (black circles), 25° (red squares), or 35° (blue triangles) from the axis connecting the two nodes.

Article Snippet: Insect cell expression constructs for full-length SpAin1 (pAcSG2-SpAin1-His) were prepared by PCR amplification from synthesized SpAin1 cDNA in a pUC57 plasmid (GenScript, Piscataway, NJ) with iProof DNA polymerase (Bio-Rad, Hercules, CA).

Techniques: Expressing, Mutagenesis