Journal: The Journal of Biological Chemistry

Article Title: Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae

doi: 10.1016/j.jbc.2024.105766

Figure Lengend Snippet: CA-binding site mutants alter but do not block endocytic actin assembly. A , widefield fluorescence micrographs of budding yeast strains harboring Abp1-TagRFP-T. Brightness and contrast were adjusted individually for each image to optimize visualization of endocytic actin patches. Orange arrowheads mark aberrant strand-like actin structures. Scale bar represents 2 μm. B , plot of the maximum number of Abp1-TagRFP-T molecules at endocytic sites in mutant and control strains. Statistical significance was measured with an ordinary one-way ANOVA with indicated p values for comparison to control strain ( black ) and Abp1Δacidic strain ( green ) calculated with a Dunnet’s test. Error bars represent standard deviation. Dashed horizontal black line marks the average maximum number of molecules of Abp1-TagRFP-T in the control strain. Each data point represents a single endocytic punctum. C , plot of the Abp1 assembly rate for mutant and control strains. Statistical significance for plots was measured as described in B .

Article Snippet: Samples were imaged on a DeltaVision Ultra High-Resolution Widefield Fluorescence microscope (Cytiva) equipped with a pco.edge 4.2 sCMOS camera, an Olympus 100× 1.4 numerical aperture Oil UPlanSApo objective and B-G-O-FR polychroics.

Techniques: Binding Assay, Blocking Assay, Fluorescence, Mutagenesis, Control, Comparison, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae

doi: 10.1016/j.jbc.2024.105766

Figure Lengend Snippet: Mutations in both Arp3 and Arp2–ARPC1 binding sites cause endocytic actin internalization defects. A , widefield fluorescence microscopy images of control and mutant Saccharomyces cerevisiae strains expressing mNG-Las17 and Abp1-TagRFP-T. Scale bar represents 2 μm. Plots of the trajectories of three representative endocytic events are shown to the right , based on the position of Abp1-TagRFP-T over time. The start of each trajectory is marked with a black circle , the end with a red circle , and the color gradient ( red , green , cyan , blue , violet , and magenta ) of the line indicates the time within the trajectory. B , analysis of the maximum distance traveled by Abp1-TagRFP-T puncta from their initial position on the membrane. Punta that moved more than 0.25 μm ( gray bar ) from their initial position were considered internalized. Each data point represents a single endocytic punctum. Data were collected from at least three separate videos. Error bars represent standard deviation. Statistical significance was measured with an ordinary one-way ANOVA with indicated p values for comparison to control strain calculated with a Dunnet’s test. C , schematic of procedure for generating average number of molecules versus time plots. Plots of the average number of mNG-Las17 or Abp1-TagRFP-T molecules versus time for the Arp3(K363D) and Arp2(V142A, Y146A) mutants are shown at the bottom of the panel. D , widefield fluorescence microscopy images of mutant S . cerevisiae strains expressing mNG-Las17 and Abp1-TagRFP-T. Scale bar represents 2 μm. To the right are kymographs of individual representative endocytic events. Scale bar for distance (d) represents 1 μm, and scale bar for time (t) represents 5 s.

Article Snippet: Samples were imaged on a DeltaVision Ultra High-Resolution Widefield Fluorescence microscope (Cytiva) equipped with a pco.edge 4.2 sCMOS camera, an Olympus 100× 1.4 numerical aperture Oil UPlanSApo objective and B-G-O-FR polychroics.

Techniques: Binding Assay, Fluorescence, Microscopy, Control, Mutagenesis, Expressing, Membrane, Standard Deviation, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae

doi: 10.1016/j.jbc.2024.105766

Figure Lengend Snippet: Mutations at both CA-binding sites cause defects in actin-based bead motility. A , schematic of actin-based bead motility assay. B , widefield fluorescence microscopy images of bead motility assays using WT and mutant Arp2/3 complexes. Two of the most defective point mutants are shown. The brightness and contrast of the second row of images is adjusted to show actin filament bundles more clearly. All other images are adjusted to same brightness and contrast values. Scale bar represents 20 μm. C , average bead velocity versus reaction time for reactions with WT or the most defective point mutants. Shaded area shows the standard deviation (n = 63–145 beads). D , plot of the bead velocity at 50 min. Statistical significance was measured with an ordinary one-way ANOVA with indicated p values for comparison to control calculated using a Dunnet’s test. Each data point represents an individually tracked bead. E , plot of the relative actin polymerization rate at the surface 50 min into the motility reaction for mutant and WT complexes. The relative polymerization rate was calculated by multiplying the velocity of the bead by the intensity of actin at the bead surface . Statistical significance was measured with an ordinary one-way ANOVA with p values as described in D . F , angular intensity plots showing the average actin fluorescence intensities within an annulus centered on the bead. Scale bar represents 2 μm. Beads were aligned based on their direction of motion ( black arrow ) to calculate average fluorescence. Error bars represent standard deviation (n = 90–112).

Article Snippet: Samples were imaged on a DeltaVision Ultra High-Resolution Widefield Fluorescence microscope (Cytiva) equipped with a pco.edge 4.2 sCMOS camera, an Olympus 100× 1.4 numerical aperture Oil UPlanSApo objective and B-G-O-FR polychroics.

Techniques: Binding Assay, Motility Assay, Fluorescence, Microscopy, Mutagenesis, Standard Deviation, Comparison, Control

Journal: The Journal of Cell Biology

Article Title: Functional Analysis of a Human Homologue of the Drosophila Actin Binding Protein Anillin Suggests a Role in Cytokinesis

doi:

Figure Lengend Snippet: Characterization of an anillin homologue. (A) An alignment of the conserved COOH-terminal region of anillin homologues found in blast searches of the databases. The aligned sequences are human anillin (this report; GenBank accession No. AF273437); Drosophila anillin ( ; GenBank accession No. X89858); the products of the C . elegans genes K10B2.5 (GenBank accession No. T16604), Y43F8C.14 (GenBank accession No. T26874), and Y49E10.19 (GenBank accession No. T27053); and the product of the Drosophila gene CG4530 (GenBank accession No. AAF47044). The sequences were aligned with ClustalX using the default settings. The asterisk indicate positions where the amino acid is identical in all six sequences; a single dot indicates positions where the amino acids of all sequences fall into a weak similarity group; and a double dot indicates positions where the amino acids of all sequences fall into a strong similarity group. The PH domain is underlined. (B) A schematic comparison of Drosophila and human anillin homologues. The overall level of identity between the two proteins is 25%. The COOH-terminal third of the protein is the most conserved (∼36% identity). This region includes a PH domain (human, amino acids 985–1,110; Drosophila , amino acids 1,069–1,194), which is 54% identical between the two proteins. The minimal sequences required for actin binding (amino acids 258–340, indicated here in black) and bundling (amino acids 246–371) have been mapped for the Drosophila protein . A larger region of the Drosophila protein (amino acids 127–371, shown here in medium gray) was found to stabilize actin binding and bundling. The locations of potential nuclear localization sequences, identified using the program PSORT II , are indicated with black triangles (human, amino acids 64, 66, 195, 786, 896, and 1,021; Drosophila , amino acids 996 and 1,105). Also conserved is a potential SH3 binding motif. There is one SH3 binding consensus in the human and two in the Drosophila sequence (indicated here with asterisks). (C) An affinity-purified antibody against hsanillin recognizes a single band in extracts of mammalian cells. A Western blot of cell extracts probed with affinity-purified antibodies to hsanillin. Extracts of BHK-21, BS-C-1, and HeLa cells were loaded. In all cases, a single band running at ∼180 kD is detected. (D) hsanillin, like the Drosophila protein, has a dynamic cell cycle–dependent localization pattern. Methanol-fixed BHK cells were stained for hsanillin. Three-dimensional widefield data was collected and deconvolved. The interphase cell is a projection of the entire stack; the other images are selected sections. In interphase cells, hsanillin is found primarily in the nucleus, upon nuclear envelope breakdown, hsanillin becomes enriched at the cortex. During cytokinesis, the protein localizes to the cleavage furrow. In late telophase, anillin is concentrated in the cortex surrounding the midbody.

Article Snippet: Three-dimensional images were acquired on a widefield microscope (DeltaVision; Applied Precision Inc.).

Techniques: Binding Assay, Sequencing, Affinity Purification, Western Blot, Staining

Journal: The Journal of Cell Biology

Article Title: Functional Analysis of a Human Homologue of the Drosophila Actin Binding Protein Anillin Suggests a Role in Cytokinesis

doi:

Figure Lengend Snippet: Anillin and Hcdc10 are found in punctate foci that localize along actin cables during mitosis in BHK cells. BHK cells were fixed and stained for actin, Hcdc10, anillin, and DNA. Three-dimensional widefield data sets were collected and deconvolved. Shown are projections of entire cells. During metaphase and early anaphase, actin is found in cables that often appear to encircle the cell. During late anaphase, as the cell initiates contraction, the actin cables appear to align parallel to the spindle axis. As contraction proceeds, more actin cables within the cleavage furrow are observed perpendicular to the spindle axis but, within the actively contracting region near the base of the cell, the actin cables are still parallel to the spindle axis. During all phases of mitosis, anillin and Hcdc10 colocalize to foci that align along actin cables.

Article Snippet: Three-dimensional images were acquired on a widefield microscope (DeltaVision; Applied Precision Inc.).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Functional Analysis of a Human Homologue of the Drosophila Actin Binding Protein Anillin Suggests a Role in Cytokinesis

doi:

Figure Lengend Snippet: Anillin and the septin Hcdc10 colocalize on the cortex of latrunculin-treated cells but do not accumulate in the cleavage furrow. (A) A typical field of BHK cells fixed and stained for DNA (top), anillin (bottom, red), and Hcdc10 (bottom, green). Three-dimensional widefield data sets were collected and deconvolved. A projection of the field is shown. During interphase (I) Hcdc10 is present in punctate cortical structures on the cortex. Anillin is in the nuclei of most, but not all, interphase cells. Note that the nuclear staining is somewhat weaker in formaldehyde-fixed cells compared with the methanol-fixed cells shown in . At metaphase (M), anillin concentrates at the cortex where it colocalizes with septin foci. At telophase (T), foci containing anillin and Hcdc10 are concentrated in the cleavage furrow. (B) BHK cells grown on coverslips were treated with 2 μg/ml latrunculin A (a gift from Miranda Sanders and Phil Crews, University of California, Santa Cruz) or latrunculin B (Calbiochem) in normal culture media for 60 min before processing for immunofluorescence. In the left panel, a merge of DNA (red) and actin (green) is shown. In the right panels, a merge of anillin (red) and the septin Hcdc10 (green) is shown. Images were collected as described for and are projections of entire cells. When the actin is depolymerized by latrunculin treatment, neither Hcdc10 nor anillin are recruited to the cell equator during mitosis; instead, they colocalize to large cortical spots.

Article Snippet: Three-dimensional images were acquired on a widefield microscope (DeltaVision; Applied Precision Inc.).

Techniques: Staining, Immunofluorescence