Mechanistic insights into the anchorage of the contractile ring by anillin and Mid1

Abstract

Anillins and Mid1 are scaffold proteins that play key roles in anchorage of the contractile ring at the cell equator during cytokinesis in animals and fungi, respectively. Here, we report crystal structures and functional analysis of human anillin and S. pombe Mid1. The combined data show anillin contains a cryptic C2 domain and a Rho-binding domain. Together with the tethering PH domain, three membrane-associating elements synergistically bind to RhoA and phospholipids to anchor anillin at the cleavage furrow. Surprisingly, Mid1 also binds to the membrane through a cryptic C2 domain. Dimerization of Mid1 leads to high affinity and preference for PI(4,5)P2, which stably anchors Mid1 at the division plane, bypassing the requirement for Rho GTPase. These findings uncover the unexpected general machinery and the divergent regulatory logics for the anchorage of the contractile ring through the anillin/Mid1 family proteins from yeast to humans.

Figure 1. Anillin Contains a Cryptic Lipid-binding C2 Domain

(A) Domain organization of human anillin. The N-terminus of anillin (residue 1–420) contains a formin-binding domain (FBD), a myosin-binding domain (MBD) and an actin-binding domain (ABD). (B) Overall structure of the AHD. The RBD (blue) and the C2 domain (orange). The lipid-binding motif is disordered in the crystals, and shown as dotted line. The N- and C-termini are indicated. (C) The AHD domain shows preference for binding to negatively charged lipids. The data points are connected with a line to show the trend of binding. (D) Multiple sequence alignments of anillins around the lipid-binding motif. Positively charged residues (blue); hydrophobic residues (magenta). (E) The L3 loop is responsible for the lipid binding of the AHD. Assays were done as in Figure 1C. AHD (WT): AHD of anillin (712–981); AHD (L3-del): AHD mutant with the fifteen residues in the L3 loop (residues 895–909) deleted. Quantification of cleavage furrow localization of varied constructs of anillin in HeLa cells (F) and Drosophila S2 cells (G). Error bars show SD, *p <0.01. Scale bar, 2 \xm. The distribution profiles of various proteins during cytokinesis in S2 cells were shown in Fig. G (bottom panel). (H) Quantification of binuclear cell formation. HeLa cells were treated with siRNA to knockdown the endogenous anillin, and rescued with various constructs of anillin (full-length). Error bars show SD. AHD-PH (A740D), AHD-PH (E758K) and AHD-PH (L3-7KA): anillin (712–1124) with Ala 740 mutated to aspartate, Glu 758 mutated to lysine, seven lysine residues in the L3 loop replaced with alanine, respectively.

Figure 2. Structure of Anillin in Complex with RhoA

(A) Structure of the anillin-RhoA complex. Anillin is colored as Figure 1. RhoA is colored green. The bound GTP and Mg is colored magenta, and shown as stick and sphere, respectively. (B) Expended view of the anillin-RhoA interactions (boxed region in A). (C) Sequence alignments at the RBD region of anillins in animals. Alignments were done with EBI Clustal-omega server. Residues involved in RhoA interaction (yellow); conserved residues (*); highly similar residues (:); similar residues (.). The mutated residues are labeled with red triangles.

Figure 3. Synergistic Actions of Multiple Weak Elements Stably Anchor Anillin and Mid1 at the Plasma Membrane

(A) SPR measurements of the binding affinity of anillin to PI(4,5)P2 vesicle. RhoA* indicated lipid-modified RhoA. (B) ITC analysis of the binding of anillin to RhoA. ΔS=37.1 cal/mol/deg and ΔH=3.97 kcal/mol. (C) SPR measurements of the binding of WT and mutant Mid1 to PI(4,5)P2 and PS. (D) Disassociation constants of anillin and Mid1 to PI(4,5)P2 and PS vesicles (Kd, µM). Relative fold enhancement of the binding affinities of anillin and Mid1 is shown in parenthesis.

Figure 4. Anillin Links the Contractile Ring to the Division Plane

(A) Domain organizations of the fusion proteins. CH-AHD-PH, MLC-AHD-PH and Mid1-AHD-PH: the C-terminal anillin in fusion with the tandem CH domain of filamin, myosin regulatory light chain (MLC) and the N-terminal domain of Mid1 (residues 1–550), respectively. Representative cell images (B) and quantification (C) of binuclear cells rescued with different fusion proteins. Experiments were done as in Figure 1G. White arrows indicated binuclear/multinuclear cells. (D-F) In vitro reconstitution of F-actin-membrane linkage mediated by anillin. PI(4,5)P2-containing GUVs were labeled with TopFluor-488 (green), and actin filaments were labeled with Alexa 647-phalloidin (purple). Scale bars, 2 µm.

Figure 5. Mid1 Shares the Same Core Lipid-binding Element as Anillin

(A) Domain organization of Mid1. (B) Overall structure of the C-terminal Mid1 (residues 579–920). The C2 domain, the connector domain and the PH domain are colored cyan, red and yellow, respectively. The lipid-binding loop of Mid1 is shown as dotted lines. (C) Structural alignment of the C2 domains between human anillin (orange) and S. pombe Mid1 (cyan). (D) Multiple sequence alignments of Mid1 homologue proteins in fungi around the lipid-binding motif.

Figure 6. Dimerizaion of Mid1 Restricts Its Localization at the Mid Cell during Cytokinesis

(A) MALS analysis of the C-terminal domain of Mid1. Mid1 (579–920), WT (black line); Met616, Leu618 and Pro619 mutated to alanine (Mid1^3A, red); Met616 mutated glutamate (Mid1^M616E, green); replacing the hydrophobic loop, Mid1 (579–920rep, blue). (B) AUC (left) and MALS (right) independently show that the N-terminal region of Mid1 (1–452) is a monomer. AUC analysis yielded sedimentation coefficient of 1.2S, frictional ratio of 4.08 and the molecular weight ~50 kDa. (C) Structure of Mid1 dimer. (D) Close-up view of the dimerization interface. (E) Dimerization of Mid1 is important for its cellular localization. Yellow arrowheads mark the spreading of the mutant protein to the cell poles. (F) Quantification of cytokinesis defect in the WT and mutant cells. (G) Differential Interference Contrast (DIC) images of WT, gef2A mid1^3A and gef2A mid1^3A cells grown at 36 oC for 4 h. Arrowheads, aberrant septa. Scale bars, 5 um. (H) mid1 mutant has synthetic interactions with gef2A. gef2A mid1^3A mutant cells were darker than WT and single mutants when incubated at 36 oC for 30 h on a YE5S+Phloxin B plate. Phloxin B is a dye that will accumulate in dead or unhealthy cells.

Figure 7. Models for the anchorage of contractile ring at division plane through anillin (A) and Mid1 (B)

Anillin, RhoA and Mid1 are shown as cartoon models. The plasma membranes are schematically shown as double lines. The disordered lipid-binding loops and the amphipathic sequence are shown as a dotted line and a cylinder, respectively. The C-terminal tail of RhoA is indicated by a red line. The N-terminal domain of anillin (orange oval) interacts with actin filament (green line) and myosin (blue). Gef2 (grey circle) interacts with the N-terminus of Mid1. Additional contractile ring components (grey ovals).