Anillin-related Mid1 as an adaptive and multimodal contractile ring anchoring protein: A simulation study.

Cytokinesis of animal and fungi cells depends crucially on the anillin scaffold proteins. Fission yeast anillin-related Mid1 anchors cytokinetic ring precursor nodes to the membrane. However, it is unclear if both of its Pleckstrin Homology (PH) and C2 C-terminal domains bind to the membrane as monomers or dimers, and if one domain plays a dominant role. We studied Mid1 membrane binding with all-atom molecular dynamics near a membrane with yeast-like lipid composition. In simulations with the full C terminal region started away from the membrane, Mid1 binds through the disordered L3 loop of C2 in a vertical orientation, with the PH away from the membrane. However, a configuration with both C2 and PH initially bound to the membrane remains associated with the membrane. Simulations of C2-PH dimers show extensive asymmetric membrane contacts. These multiple modes of binding may reflect Mid1's multiple interactions with membranes, node proteins, and ability to sustain mechanical forces.

Design principles of Cdr2 node patterns in fission yeast cells.

Pattern-forming networks have diverse roles in cell biology. Rod-shaped fission yeast cells use pattern formation to control the localization of mitotic signaling proteins and the cytokinetic ring. During interphase, the kinase Cdr2 forms membrane-bound multiprotein complexes termed nodes, which are positioned in the cell middle due in part to the node inhibitor Pom1 enriched at cell tips. Node positioning is important for timely cell cycle progression and positioning of the cytokinetic ring. Here, we combined experimental and modeling approaches to investigate pattern formation by the Pom1-Cdr2 system. We found that Cdr2 nodes accumulate near the nucleus, and Cdr2 undergoes nucleocytoplasmic shuttling when cortical anchoring is reduced. We generated particle-based simulations based on tip inhibition, nuclear positioning, and cortical anchoring. We tested model predictions by investigating Pom1-Cdr2 localization patterns after perturbing each positioning mechanism, including in both anucleate and multinucleated cells. Experiments show that tip inhibition and cortical anchoring alone are sufficient for the assembly and positioning of nodes in the absence of the nucleus, but that the nucleus and Pom1 facilitate the formation of unexpected node patterns in multinucleated cells. These findings have implications for spatial control of cytokinesis by nodes and for spatial patterning in other biological systems.

Design principles of Cdr2 node patterns in fission yeast cells.

Pattern forming networks have diverse roles in cell biology. Rod-shaped fission yeast cells use pattern formation to control the localization of mitotic signaling proteins and the cytokinetic ring. During interphase, the kinase Cdr2 forms membrane-bound multiprotein complexes termed nodes, which are positioned in the cell middle due in part to the node inhibitor Pom1 enriched at cell tips. Node positioning is important for timely cell cycle progression and positioning of the cytokinetic ring. Here, we combined experimental and modeling approaches to investigate pattern formation by the Pom1-Cdr2 system. We found that Cdr2 nodes accumulate near the nucleus, and Cdr2 undergoes nucleocytoplasmic shuttling when cortical anchoring is reduced. We generated particle-based simulations based on tip inhibition, nuclear positioning, and cortical anchoring. We tested model predictions by investigating Pom1-Cdr2 localization patterns after perturbing each positioning mechanism, including in both anucleate and multinucleated cells. Experiments show that tip inhibition and cortical anchoring alone are sufficient for the assembly and positioning of nodes in the absence of the nucleus, but that the nucleus and Pom1 facilitate the formation of unexpected node patterns in multinucleated cells. These findings have implications for spatial control of cytokinesis by nodes and for spatial patterning in other biological systems.

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast.

The F-BAR protein Cdc15 is essential for cytokinesis in Schizosaccharomyces pombe and plays a key role in attaching the cytokinetic ring (CR) to the plasma membrane (PM). Cdc15's abilities to bind to the membrane and oligomerize via its F-BAR domain are inhibited by phosphorylation of its intrinsically disordered region (IDR). Multiple cell polarity kinases regulate Cdc15 IDR phosphostate, and of these the DYRK kinase Pom1 phosphorylation sites on Cdc15 have been shown in vivo to prevent CR formation at cell tips. Here, we compared the ability of Pom1 to control Cdc15 phosphostate and cortical localization to that of other Cdc15 kinases: Kin1, Pck1, and Shk1. We identified distinct but overlapping cohorts of Cdc15 phosphorylation sites targeted by each kinase, and the number of sites correlated with each kinases' abilities to influence Cdc15 PM localization. Coarse-grained simulations predicted that cumulative IDR phosphorylation moves the IDRs of a dimer apart and toward the F-BAR tips. Further, simulations indicated that the overall negative charge of phosphorylation masks positively charged amino acids necessary for F-BAR oligomerization and membrane interaction. Finally, simulations suggested that dephosphorylated Cdc15 undergoes phase separation driven by IDR interactions. Indeed, dephosphorylated but not phosphorylated Cdc15 undergoes liquid-liquid phase separation to form droplets in vitro that recruit Cdc15 binding partners. In cells, Cdc15 phosphomutants also formed PM-bound condensates that recruit other CR components. Together, we propose that a threshold of Cdc15 phosphorylation by assorted kinases prevents Cdc15 condensation on the PM and antagonizes CR assembly.

Anillin Related Mid1 as an Adaptive and Multimodal Contractile Ring Anchoring Protein: A Simulation Study.

The organization of the cytokinetic ring at the cell equator of dividing animal and fungi cells depends crucially on the anillin scaffold proteins. In fission yeast, anillin related Mid1 binds to the plasma membrane and helps anchor and organize a medial broad band of cytokinetic nodes, which are the precursors of the contractile ring. Similar to other anillins, Mid1 contains a C terminal globular domain with two potential regions for membrane binding, the Pleckstrin Homology (PH) and C2 domains, and an N terminal intrinsically disordered region that is strongly regulated by phosphorylation. Previous studies have shown that both PH and C2 domains can associate with the membrane, preferring phosphatidylinositol-(4,5)-bisphosphate (PIP 2 ) lipids. However, it is unclear if they can simultaneously bind to the membrane in a way that allows dimerization or oligomerization of Mid1, and if one domain plays a dominant role. To elucidate Mid1's membrane binding mechanism, we used the available structural information of the C terminal region of Mid1 in all-atom molecular dynamics (MD) near a membrane with a lipid composition based on experimental measurements (including PIP 2 lipids). The disordered L3 loop of C2, as well as the PH domain, separately bind the membrane through charged lipid contacts. In simulations with the full C terminal region started away from the membrane, Mid1 binds through the L3 loop and is stabilized in a vertical orientation with the PH domain away from the membrane. However, a configuration with both C2 and PH initially bound to the membrane remains associated with the membrane. These multiple modes of binding may reflect Mid1's multiple interactions with membranes and other node proteins, and ability to sustain mechanical forces.

A mechanism with severing near barbed ends and annealing explains structure and dynamics of dendritic actin networks.

Single molecule imaging has shown that part of actin disassembles within a few seconds after incorporation into the dendritic filament network in lamellipodia, suggestive of frequent destabilization near barbed ends. To investigate the mechanisms behind network remodeling, we created a stochastic model with polymerization, depolymerization, branching, capping, uncapping, severing, oligomer diffusion, annealing, and debranching. We find that filament severing, enhanced near barbed ends, can explain the single molecule actin lifetime distribution, if oligomer fragments reanneal to free ends with rate constants comparable to in vitro measurements. The same mechanism leads to actin networks consistent with measured filament, end, and branch concentrations. These networks undergo structural remodeling, leading to longer filaments away from the leading edge, at the +/-35° orientation pattern. Imaging of actin speckle lifetimes at sub-second resolution verifies frequent disassembly of newly-assembled actin. We thus propose a unified mechanism that fits a diverse set of basic lamellipodia phenomenology.

Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers.

Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.

Insights into Actin Polymerization and Nucleation Using a Coarse-Grained Model.

We studied actin filament polymerization and nucleation with molecular dynamics simulations and a previously established coarse-grained model having each residue represented by a single interaction site located at the Cα atom. We approximate each actin protein as a fully or partially rigid unit to identify the equilibrium structural ensemble of interprotein complexes. Monomers in the F-actin configuration bound to both barbed and pointed ends of a short F-actin filament at the anticipated locations for polymerization. Binding at both ends occurred with similar affinity. Contacts between residues of the incoming subunit and the short filament were consistent with expectation from models based on crystallography, x-ray diffraction, and cryo-electron microscopy. Binding at the barbed and pointed end also occurred at an angle with respect to the polymerizable bound structure, and the angle range depended on the flexibility of the D-loop. Additional barbed end bound states were seen when the incoming subunit was in the G-actin form. Consistent with an activation barrier for pointed end polymerization, G-actin did not bind at an F-actin pointed end. In all cases, binding at the barbed end also occurred in a configuration similar to the antiparallel (lower) dimer. Individual monomers bound each other in a short-pitch helix complex in addition to other configurations, with several of them apparently nonproductive for polymerization. Simulations with multiple monomers in the F-actin form show assembly into filaments as well as transient aggregates at the barbed end. We discuss the implications of these observations on the kinetic pathway of actin filament nucleation and polymerization and possibilities for future improvements of the coarse-grained model.