Journal: Journal of Cell Science

Article Title: An acentrosomal aster with atypical microtubule polarity recruits cytokinesis signals to its center in Xenopus egg extracts

doi: 10.1242/jcs.263766

Figure Lengend Snippet: Formation of the noncanonical aster requires the microtubule motor MKLP2 and Aurora kinase B activity. (A) Confocal images of microtubule organization in control extracts and extracts with 100 µM MKLP2 inhibitor paprotrain. n =4. Each image is a maximum-intensity projection of nine confocal planes spanning 24 μm of depth. (B) Confocal images of microtubule dynamics in control extracts (top row) and extracts with 40 µM Aurora kinase B inhibitor barasertib (bottom row). n =6. Each image is a maximum-intensity projection of nine confocal planes spanning 16 µm of depth. For both A and B, imaging started at an arbitrary time point when asters had just begun to form in the untreated extracts. (C) Widefield epifluorescence images of microtubule organization in control extracts and extracts with 100 µM kinesin Eg5 inhibitor STLC. n =10. (D) Confocal images of microtubule organization in control extracts and extracts with 2 µM dynein inhibitor GST–p150-CC1. n =6. (E) Widefield epifluorescence images of microtubule and ER organization in control extracts and extracts with 0.68 µM GST–p150-CC1. n =2.

Article Snippet: The chicken DCTN1 p150Glued AA 217–548 (CC1) was from the plasmid pVEX-CC1 (Addgene plasmid 74170; http://n2t.net/addgene:74170 ; RRID:Addgene_74170; deposited by Trina Schroer).

Techniques: Activity Assay, Control, Imaging

Journal: Journal of Cell Science

Article Title: An acentrosomal aster with atypical microtubule polarity recruits cytokinesis signals to its center in Xenopus egg extracts

doi: 10.1242/jcs.263766

Figure Lengend Snippet: Noncanonical asters can merge. (A) Confocal time-lapse montage of microtubule and EB1–GFP dynamics in egg extracts, showing that the centers of two noncanonical asters merged with each another spontaneously, and that the EB1–GFP-enriched regions at the centers also merged. Each image is a maximum-intensity projection of four confocal planes spanning 6 µm of depth. Imaging started at an arbitrary time point after the asters had formed but had not merged. The plot below each image is the fluorescence intensity profile along a 1.65 µm thick, 22.8 µm long line segment (yellow dashed rectangle) that starts at the bottom left and ends at the top right. For each point on the curve in the plot, the horizontal coordinate is the distance from the start of the line segment, and the vertical coordinate is the average fluorescence intensity of the pixels across the width of the line segment at that distance (a.u., arbitrary units). The black arrows indicate intensity peaks for microtubule (second row) and EB1–GFP (fourth row) fluorescence at the aster centers. n =9. (B) Confocal images of microtubules in control and GST–p150-CC1-treated extracts, showing that noncanonical asters still merged when dynein was inhibited by 2 µM GST–p150-CC1. n =2.

Article Snippet: The chicken DCTN1 p150Glued AA 217–548 (CC1) was from the plasmid pVEX-CC1 (Addgene plasmid 74170; http://n2t.net/addgene:74170 ; RRID:Addgene_74170; deposited by Trina Schroer).

Techniques: Imaging, Fluorescence, Control

Journal: Science advances

Article Title: Afadin mediates cadherin-catenin complex clustering on F-actin linked to cooperative binding and filament curvature.

doi: 10.1126/sciadv.adu0989

Figure Lengend Snippet: Fig. 1. Vinculin and afadin synergistically enhance the cadherin-catenin complex’s F-actin binding. (A) Domain organization of E-cadherin, β-catenin, αE-catenin, vinculin, and afadin. Domains used in this study are colored and sequence boundaries are indicated. ABD, actin-binding domain; CC, coiled coil; FAB, F-actin binding. (B to C) Co-sedimentation assays showing the binding of indicated proteins and protein complexes to F-actin. S, supernatant; P, pellet. (D) Quantification of the fraction of indi- cated proteins or protein complexes that co-sedimented with F-actin in the pellet. Measurements were corrected by subtracting the amount of protein which sedimented in the absence of F-actin. Data are presented as means ± SD of three independent experiments. Conditions were compared using two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test: Not significant (n.s.), P ≥ 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Dotted lines indicate stitching interfaces between gels.

Article Snippet: We thank B. Weinberg for providing the cdnA of mouse e- cadherin (plasmid no. 18804) through Addgene, as well as valeri vasioukhin for providing the cdnAs of mouse β- catenin (plasmid no. 20140) and αe- catenin (plasmid no. 20139).

Techniques: Binding Assay, Sequencing, Sedimentation, Comparison

Journal: Science advances

Article Title: Afadin mediates cadherin-catenin complex clustering on F-actin linked to cooperative binding and filament curvature.

doi: 10.1126/sciadv.adu0989

Figure Lengend Snippet: Fig. 2. Cryo-EM structure of pentamer bound F-actin, visualizing αE-catenin ABD stabilization by afadin-CC. (A) Slice from cryo–electron tomogram highlighting pentameric complexes flexibly protruding from F-actin. Red and blue arrows indicate bare and pentamer bound F-actin, respectively. Scale bar, 50 nm. (B) Cryo-EM den- sity map (3.1-Å resolution), which resolves two αE-catenin ABDs and one afadin-CC bound to F-actin. BE, barbed end; PE, pointed end. (C) Ribbon representation of the associated atomic model.

Article Snippet: We thank B. Weinberg for providing the cdnA of mouse e- cadherin (plasmid no. 18804) through Addgene, as well as valeri vasioukhin for providing the cdnAs of mouse β- catenin (plasmid no. 20140) and αe- catenin (plasmid no. 20139).

Techniques: Cryo-EM Sample Prep

Journal: Science advances

Article Title: Afadin mediates cadherin-catenin complex clustering on F-actin linked to cooperative binding and filament curvature.

doi: 10.1126/sciadv.adu0989

Figure Lengend Snippet: Fig. 3. Structural remodeling of the αE-catenin ABD evoked by afadin-CC. (A to C) Structures of the αE-catenin ABD in three different conformational states. αE- catenin and F-actin are depicted in cartoon and surface representation, respectively. Pre-bound, crystal structure of αE-catenin (PDB: 4IGG, chain B); post-bound, cryo-EM structure of αE-catenin ABD alone bound to F-actin (PDB: 6UPV); afadin-stabilized, this study. The refolded CTE-C is highlighted in magenta. (D) Superposition of the αE- catenin ABD in the pre-bound and post-bound states. (E) Superposition of the αE-catenin ABD in the post-bound and afadin-stabilized states. (F) Detail view [boxed in (E)] highlighting αE-catenin ABD H1 and CTE structural rearrangements evoked by afadin-CC engagement.

Article Snippet: We thank B. Weinberg for providing the cdnA of mouse e- cadherin (plasmid no. 18804) through Addgene, as well as valeri vasioukhin for providing the cdnAs of mouse β- catenin (plasmid no. 20140) and αe- catenin (plasmid no. 20139).

Techniques: Cryo-EM Sample Prep

Journal: Science advances

Article Title: Afadin mediates cadherin-catenin complex clustering on F-actin linked to cooperative binding and filament curvature.

doi: 10.1126/sciadv.adu0989

Figure Lengend Snippet: Fig. 4. Afadin-CC engagement mediates enhanced binding of the αE-catenin ABD to F-actin. (A) Overview of the tripartite interface. BE, barbed end; PE, pointed end. (B) Contacts between actin subunit i, αE-catenin ABD2, and afadin-CC’s N-terminal coil. (C) Contacts at the interface between the displaced H1 and H2 of αE-catenin ABD1 and afadin-CC. (D) Interaction network formed by afadin-CC’s C-terminal coil, ABD1, and actin subunit i. (E) Contacts between the CTE of αE-catenin ABD1, actin subunit i, and H4 of αE-catenin ABD2. (F and G) Co-sedimentation assays analyzing the effects of afadin-CC fragments on F-actin binding by the tetrameric complex (F) and the isolated αE-catenin ABD (G). (H and I) Co-sedimentation assays analyzing the contributions of specific interfaces to the tetramer’s F-actin binding in the absence (H) and presence (I) of afadin-CC. (J) Quantification of (F) to (I). Data are presented as means ± SD of three independent experiments. Conditions were compared by two-way ANOVA with Tukey’s multiple comparison test: n.s., P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Dotted lines indicate stitching interfaces between gels.

Article Snippet: We thank B. Weinberg for providing the cdnA of mouse e- cadherin (plasmid no. 18804) through Addgene, as well as valeri vasioukhin for providing the cdnAs of mouse β- catenin (plasmid no. 20140) and αe- catenin (plasmid no. 20139).

Techniques: Binding Assay, Sedimentation, Isolation, Comparison

Journal: ACS Synthetic Biology

Article Title: Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in E. coli

doi: 10.1021/acssynbio.4c00564

Figure Lengend Snippet: Schematic overview of modular cloning and the contributions of this study. To facilitate Golden Gate Assembly with low- and medium copy plasmids, a kit containing 20 level one and 8 level two destination vectors was constructed (yellow background), carrying either p15A or pBR322-derived origins of replication. These destination vectors can be used for defined assembly of expression constructs (green background). The effect of the origin of replication, promoter, and RBS on GFP expression was characterized in this work to allow effective deployment of this kit, which has been made available on Addgene. The addition of these new low- and medium copy number destination vectors can be used to assemble libraries of expression constructs (blue background). Using growth-coupled selection highly diverse libraries can be effectively and simply screened for the most growth-advantageous expression solution.

Article Snippet: The destination vectors described in this study are publicly available on Addgene (catalog #217582–217609).

Techniques: Cloning, Construct, Derivative Assay, Expressing, Selection

Journal: ACS Synthetic Biology

Article Title: Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in E. coli

doi: 10.1021/acssynbio.4c00564

Figure Lengend Snippet: Burden assessment of MoClo-compatible destination vectors with different origins of replication. E. coli BW25113 carrying various constitutive GFP expression constructs in p15A, pBR322, or pUC19 backbones were grown in LB (A, C) or M9 minimal medium with 1% glucose (B, D) and assessed for their growth rates (A, B) and maximum achieved OD 600 (C, D). Data points represent averages of biological triplicate measurements, and error bars represent their standard deviations. The visualized data is also provided in table format in Supporting Table S4 , including a distinction of the exact promoter, RBS, and backbone combination used per result.

Article Snippet: The destination vectors described in this study are publicly available on Addgene (catalog #217582–217609).

Techniques: Expressing, Construct

Journal: ACS Synthetic Biology

Article Title: Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in E. coli

doi: 10.1021/acssynbio.4c00564

Figure Lengend Snippet: Growth-coupled selection for optimal adhE * expression levels using MoClo-mediated randomized construct assembly. (A) Schematic of E. coli central metabolism depicting the catabolism of ethanol by AdhE*—the acetaldehyde intermediate is not depicted. (B) Workflow of the selection experiment starting with randomized Golden Gate Assembly of promoters, RBSs and destination vectors, yielding a diverse population of plasmids. Subsequent transformation and passaging on M9 minimal medium with 1 mM glucose + 300 mM ethanol results in selection for optimal expression of adhE *. (C) Number of sequenced clones carrying a certain adhE * expression construct before selection on ethanol medium was imposed. (D) Number of sequenced clones carrying a certain adhE * expression construct after 1, 2, or 3 passages on M9 minimal medium with 1 mM glucose + 300 mM ethanol. Three independent populations were transformed and passaged, four colonies of each population were picked at each step (pre-selection and passage 1–3) and the resulting construct composition counts were combined in the heatmaps shown in panels C and D. See Table S5 for raw counts and expression construct composition per independent population and per step. Created with Biorender.com.

Article Snippet: The destination vectors described in this study are publicly available on Addgene (catalog #217582–217609).

Techniques: Selection, Expressing, Construct, Transformation Assay, Passaging, Clone Assay