Anillin Phosphorylation Controls Timely Membrane Association and Successful Cytokinesis

Abstract

During cytokinesis, a contractile ring generates the constricting force to divide a cell into two daughters. This ring is composed of filamentous actin and the motor protein myosin, along with additional structural and regulatory proteins, including anillin. Anillin is a required scaffold protein that links the actomyosin ring to membrane and its organizer, RhoA. However, the molecular basis for timely action of anillin at cytokinesis remains obscure. Here, we find that phosphorylation regulates efficient recruitment of human anillin to the equatorial membrane. Anillin is highly phosphorylated in mitosis, and is a substrate for mitotic kinases. We surveyed function of 46 residues on anillin previously found to be phosphorylated in human cells to identify those required for cytokinesis. Among these sites, we identified S635 as a key site mediating cytokinesis. Preventing S635 phosphorylation adjacent to the AH domain disrupts anillin concentration at the equatorial cortex at anaphase, whereas a phosphomimetic mutant, S635D, partially restores this localization. Time-lapse videomicroscopy reveals impaired recruitment of S635A anillin to equatorial membrane and a transient unstable furrow followed by ultimate failure in cytokinesis. A phosphospecific antibody confirms phosphorylation at S635 in late cytokinesis, although it does not detect phosphorylation in early cytokinesis, possibly due to adjacent Y634 phosphorylation. Together, these findings reveal that anillin recruitment to the equatorial cortex at anaphase onset is enhanced by phosphorylation and promotes successful cytokinesis.

Fig 1. Anillin is highly phosphorylated in mitosis.

(A) Schematic of anillin domains (Myo = myosin binding; RBD = Rho Binding Domain; AH = anillin homology; PH = pleckstrin homology). (B) Immunoblot analysis of electrophoretic shift of anillin. HeLa cells were arrested using nocodazole (0.2 μg/ml) for 12 h (M) and released into fresh drug-free medium for the indicated time. S phase cells by double thymidine block serves as control. Cyclin B loss confirms mitotic exit. λppase = lambda phosphatase, vanadate = sodium orthovanadate (Na₃VO₄). (C) The mitotic hyperphosphorylation of anillin is a common feature in various cell lines. RPE1 is a non-transformed human retinal pigment epithelial cell line; 786-O and ACHN are human renal cancer cell lines. Loading control: a non-specific band detected by anillin antibody. (D) In vitro kinase assays with indicated kinases and GST-tagged anillin fragments. Protein phosphorylation is visualized by autoradiography (³²P, top panel) and equal protein loading by Coomassie blue staining (bottom panel). *: Bovine serum albumin.

Fig 2. Efficient cytokinesis requires phosphorylation in or near the Anillin Homology (AH) domain.

(A) Schematic of GFP-tagged non-phosphorylatable anillin constructs with siRNA-resistant (siR) silent mutations. Non-phosphorylatable mutants were generated by either gBlock/Gibson assembly (A1-A5) or site-directed mutagenesis (A6 and A7). Mutated phosphorylation sites of each construct were listed on the right (Ser/Thr/Tyr mutated to Ala/Val/Phe, respectively). (B-D) RNAi-mediated rescue experiment with non-phosphorylatable mutants. (B) Experimental protocol. HeLa cells were arrested by thymidine for 16 h, followed by a 1 h release to fresh medium and then transfected with anillin siRNA and the indicated GFP anillin constructs. After 48 h, cells were fixed and processed for immunofluorescence. (C) Multinucleation in GFP-positive interphase cells 48 h after transfection. Error bars, mean ± s.e. of three experiments (n > 200 cells each). Statistical comparison between WT and A5 was made with a two-tailed Student’s t-test (*p< 0.05). (D) Immunoblot analysis of protein extracts prepared from B. Endogenous anillin and transgenic GFP anillin are indicated by open and filled arrowheads, respectively. The loading control is a ~300 kDa non-specific band detected with anillin antibody. (E) Quantitative immunoblotting revealing degree of knockdown and addback in control and A5 conditions. ± SD is shown.

Fig 3. Phospho-deficient A5 mutant has impaired equatorial localization and is unable to fix RhoA.

(A) IF analysis of transiently expressed A5 in formaldehyde-fixed anaphase cells. GFP-A5 and endogenous anillin were detected using anti-GFP and anillin antibodies, respectively. (B) Quantification of furrow localization of GFP-anillin and GFP-A5. Error bars, mean ± s.e. from three experiments (n > 30 cells each). p<0.05 by Student’s t-test. Scale bar, 10 μm. (C) Mean intensity ratio (GFP fluorescence at the equatorial cortex:pole) of individual cells were plotted for each GFP anillin construct. Red bars indicate median values. p<0.0001 by Student’s t-test. Right: diagram showing locations of regions of interest. Note: used cytoplasmic GFP levels at the poles. (D-E) IF analysis of furrow RhoA after rescue experiment with A5. HeLa cells simultaneously transfected with anillin siRNA and the indicated GFP anillin constructs. Transfected cells were enriched in anaphase by monastrol block and release. (D) Representative images of furrow RhoA at either early or late stage of cytokinesis. (E) Quantification of RhoA at equatorial furrow in TCA-fixed anaphase cells. Error bars, mean ± s.e. from three experiments (n > 20 cells each; * and **, p<0.05).

Fig 4. Identification of Ser635 as a critical site for the equatorial localization of anillin at furrow.

(A) IF analysis of transiently expressed GFP-anillin Y634F/S635A double mutant. GFP anillin mutants and endogenous anillin were detected using anti-GFP and anillin antibodies, respectively. (B) Localization of Y634F and S634 mutant anillin by IF. (C) Mean intensity ratio of GFP fluorescence at the equatorial cortex:pole for the indicated GFP anillin mutants. Red bars indicate median values. P values by Student’s t-test. (D) Quantification of GFP anillin mutants at equatorial furrow. Summary of 8 single non-phosphorylatable GFP mutants’ localization. Error bars, mean ± s.e. from three experiments. *p<0.05 (E) Sequence alignment of the region surrounding S635, revealing conservation. (F) IF analysis of transiently expressed phosphomimetic anillin, S635D. (G) Quantification of phosphomimetic S635 Anillin furrow localization by observer (H) Quantitative immunofluorescence with representative cell from panel F showing regions quantified in violet (equator) and yellow (pole). Red bars in H indicate median values. P values by Student’s t-test. Scale bars, 5 μm. See also S2 and S3 Figs.

Fig 5. Timelapse videomicroscopy of anillin mutants.

To assess contractile rings in live cells, timelapse videomicroscopy was performed in HeLa cells in which endogenous anillin was depleted and the GFP-tagged constructs were transiently transfected. The merge of DIC and GFP are shown. GFP signal is scaled equivalently among all constructs. Times from anaphase onset is reported (hours:min:sec) in the upper right of each panel. Scale bar, 10 μm. See also S4 Fig and Supplementary Videos.

Fig 6. Ser635A anillin exhibits a destabilized furrow, and phosphomimetic does not alter binding to Ect2, RhoA, or selectivity for specific phospholipids.

(A) Knockdown/addback revealed eccentric furrows seen in anaphase cells after depletion of anillin. Dashed line marks approximate region of furrow. Scale bar, 5 μm. (B) Asynchronous Hela cells were treated by knockdown/addback and analyzed for furrow location at 48 hours. Fraction of eccentric furrows was quantified in anaphase and telophase cells (n = 3; ≥50 cells per condition). *ANOVA detected differences between wild-type rescue and other conditions. (C) Western blot analysis of pulled down RhoA and Ect2 with GST AH-PH anillin. HeLa extracts from transfected with GFP Q63L RhoA (top panel) or untransfected (bottom) were used in pull-down assays with wild-type or phosphomimetic S635 GST AH-PH. (D) Coomassie blue staining of recombinant GST and GST AH-PH anillin (left panel). GST fusion proteins were expressed in E. coli and purified using Glutathione Sepharose 4B Fast Flow. Lipid arrays (middle panel) were incubated with the indicated recombinant proteins before being probed by anti-GST (right panel). Key for lipid array: TG = triglyceride; DAG = diacylglycerol; PA = phosphatidyl acid; PS = phosphatidylserine; PE = phosphatidylethanolamide, PC = phosphatidylcholine; PG = phosphatidylglycerol; CL = cardiolipin; PI = phosphatidylinositol; PIP = PI 4-phosphate; PIP₂ = PI 4,5-bisphosphate; PIP₃ = PI 3,4,5-triphosphate; Chol = cholesterol; SM = sphingomyelin; Sulf: sulfatide; - = Blank.

Fig 7. Anillin Ser635 is an in vivo phosphorylation site.

(A-D) Validation of phosphospecific antibody raised against pS635. (A) Known amounts of phosphorylated (pS635) or unphosphorylated (S635) peptides were spotted on PVDF and immunoblotted with anti-pS635 antibody to assess phosphoselectivity. Phosphopeptide sequence for immunization; pS635 in red. (B) IF staining of cells in early (top) or late (bottom) anaphase by affinity purified anti-pS635 antibody (phospho-S635 Ab). Antibody purified with non-phosphorylated immunizing peptide (non-phospho Ab) was used as control. Scale bar, 10 μm. (C) Knockdown/addback experiment demonstrating that phospho-anillin staining fails to detect anillin S635A. Scale bar, 5 μm (D) Fraction of GFP positive (GFP+) cells that have localized pS635 signal. (E) Phosphospecific S635 antibody detects anillin at late stage of cytokinesis. Representative images of cells are shown from metaphase to late cytokinesis. Cells were stained by anti-pS635 antibody (green), anti-α-tubulin (red) and DAPI (blue). Scale bar, 10 μm. See also S5–S7 Figs.