Cytokinesis requires localized β-actin filament production by an actin isoform specific nucleator

Fig. 1. Anillin enhances RhoA-mediated release of DIAPH3 intramolecular auto-inhibition interaction. a Schematic of DIAPH3 domain organization in the auto-inhibited and activated states (not to scale). b Immunoblots and quantification to determine the amount of GST-DIAPH3-CT and MBP-DIAPH3-NT bound to amylose beads in the presence of increasing concentrations of GST, GST-RhoA, and 6× His-anillin(1–151 aa). Values normalized to the amount of MBP-DIAPH3-NT immobilized on beads. Each binding assay was repeated at least three times. Error bars indicate ± s.e.m. *p = 0.014, **p = 0.0009 compared to GST control, Student’s t test. n = 3. c MBP-DIAPH3-NT and GST-DIAPH3-CT were pre-mixed, then incubated with either GST-RhoA, 6× His-anillin1–151, or both. MBP-DIAPH3-NT was re-isolated on amylose beads and immunoblots performed to detect co-purifying RhoA and d anillin

Fig. 2. Anillin (ANLN) enhances RhoA-mediated activation of DIAPH3 actin nucleation activity. Pyrene-labeled actin polymerization assays comparing the relative actin polymerization activity of GST-DIAPH3-CT and a complex of GST-DIAPH3-CT + MBP-DIAPH3-NT in the presence of a GST-RhoA, b 6× His-anillin(1–151), and c GST-RhoA and 6× His-anillin(1–151)

Fig. 3. A direct interaction between anillin and DIAPH3 enhances RhoA-mediated activation of DIAPH3 actin nucleation activity and the successful completion of cytokinesis. a MBP-anillin(1–151), mutants, and wild type were incubated with GST-DIAPH3-NT, then the GST fusion proteins re-isolated on glutathione agarose beads and immunoblotted to detect and quantify the relative amount of co-purifying MBP-anillin mutants and wild type. n = 3. Error bars indicate ± s.e.m. *p = 0.0072, Student’s t test, comparing wt to RQPL 41–44 AAAA. b Pyrene-labeled actin nucleation assays in the presence of DIAPH3-CT-NT, GST-RhoA, and either 6× His-anillin wild type (1–151) or 6× His-anillin 4xA (41-RQPL-44 to AAAA). c Lysates from stable HeLa cell lines with a single integrated cDNA encoding GFP-anillin-wt (GFP-ANLN) or GFP-anillin 41-RQPL-44 to AAAA (GFP-4xA) under the control of the tet regulator were probed with an anti-anillin antibody. GFP fusion protein expression was induced by adding 1 μg/ml doxycyclin (Dox). d GFP-anillin-wt (GFP-ANLN) and GFP-anillin-41-RQPL-44 to AAAA (GFP-4xA) were isolated from stable cell lines using a GFP-TRAP and co-purifying DIAPH3 detected by immunoblotting. e The number…

Fig. 4. The anillin–DIAPH3 interaction is required for the early stages of furrow organization and β-actin polymerization at the cleavage furrow. a HeLa cells in anaphase stained with DAPI (blue) to mark chromosomes and phalloidin (white) to mark F-actin and the cell cortex. b Comparative furrow ingression indices, a measure of the degree of ingression of the furrow on each side of the cell where 1 = symmetrical ingression, in the presence and absence of endogenous anillin, GFP-ANLN, and GFP-4xA. Inter-chromosomal mass distance 8 μm + 0.1. Red bar = average, gray box = 25–75 percentile, and whiskers indicate data extremes. At least 100 cells were analyzed in each of three experimental repeats. *p = 0.013, **p = 0.003, ***p = 0.0025, Student’s t test. c Comparative furrow instability indices, a measure of furrow position, where 1 = furrow ingression in the middle of the cell in the presence and absence of endogenous anillin, GFP-ANLN, and GFP-4xA. Red bar = average, gray box = 25–75 percentile, and whiskers indicate data extremes. At least 100 cells were analyzed in each of three experimental repeats. *p = 0.004, **p = 0.003, ***p = 0.002, Student’s t test. d Comparative distribution of…

Fig. 5. DIAPH3 preferentially nucleates β-actin filaments over γ-actin filaments. a Actin pelleting assay in the presence and absence of GST-DIAPH3-CT using actin isolated from human platelets or chicken gizzard. The polymerization reaction was carried out for 30 min. P = pellet fraction, polymerized actin. S = actin in the supernatant, unpolymerized actin. n = 3. Error bars indicate ± s.e.m. b Immunoblots of the actin pelleting assay in A, probed with β- and γ-actin-specific antibodies. P = pellet fraction, polymerized actin. S = actin in the supernatant, unpolymerized actin. c On glass actin nucleation assay, where actin filaments are fixed and visualized by phalloidin staining. d Actin isolated from human platelets nucleated on glass by GST-DIAPH3-CT, fixed, and stained with β- and γ-actin-specific antibodies. e Actin isolated from chicken gizzard nucleated on glass by GST-DIAPH3-CT, fixed, and stained with β- and γ-actin-specific antibodies. f Quantitation of the number of filaments per field relative to the number of filaments generated upon incubation of human platelet actin in buffer. n = 3. Error bars indicate ± s.e.m. *p < 0.01, Student’s t test. All scale bars are 10 μm