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

Abstract

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.

Phosphorylation of the F-BAR protein Hof1 drives septin ring splitting in budding yeast

“…Interestingly, Hof1 particles appeared more globular and compact than Hof1-P4E (Fig. 5C ), raising the possibility that mimicking Hof1 phosphorylation might induce a conformational change from a “closed” to more “open” configuration, similar to what has been shown for its Cdc15 ortholog in fission yeast 80 , 81 . The addition of scaling amounts of Hof1 to septins during polymerisation caused the formation of big, interlaced meshes of septin filaments and bundles (Fig.…”

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

“…Our results show that the Mid1 monomer has many modes of membrane binding, which may reflect its multiple interactions with membranes and other cytokinetic ring proteins. Figure 6 shows a model of Mid1 in nodes, which takes into consideration super-resolution data of node proteins [21] (slightly shifted by 10 nm to place Mid1 adjacent to the plasma membrane), as well as the estimated size of Mid1’s IDR region based on coarse-grained MD simulations using methods similar to those in Bhattacharjee et al [38] (data not shown). This figure indicates how a membrane layer of ~8-10 Mid1 per node [21, 39] provides a scaffolding layer for node proteins, together with about twice-as-many membrane-bound Cdc15 and Rng2 [38], as well as Cdr2 (not shown), thus helping anchor formin Cdc12 and type II myosin Myo2.…”

“…Figure 6 shows a model of Mid1 in nodes, which takes into consideration super-resolution data of node proteins [21] (slightly shifted by 10 nm to place Mid1 adjacent to the plasma membrane), as well as the estimated size of Mid1’s IDR region based on coarse-grained MD simulations using methods similar to those in Bhattacharjee et al [38] (data not shown). This figure indicates how a membrane layer of ~8-10 Mid1 per node [21, 39] provides a scaffolding layer for node proteins, together with about twice-as-many membrane-bound Cdc15 and Rng2 [38], as well as Cdr2 (not shown), thus helping anchor formin Cdc12 and type II myosin Myo2. The multiple modes of Mid1 membrane binding may reflect its ability to sustain mechanical forces as Myo2 pulls on Cdc12-nucleated actin filaments, leading to the condensation of the band of nodes into a ring through the opposing steric hindrance by membrane-associated ER [40].…”

“…These membrane attachments should also control the resistance of node movement to applied force, an important biophysical parameter for cytokinetic ring organization. Recent evidence suggests that IDR regions of Cdc15 promote condensation of Cdc15 through forces related to liquid-liquid phase separation [38]. Flexibility in Mid1 membrane binding may thus also be related to its ability to participate into a disordered condensate through its IDR region, which is of comparable size to Cdc15.…”

“…Mid1 is known to be heavily phosphorylated on its N-terminal IDR [7, 34], and regulation by phosphorylation is a common feature of other node proteins [49, 50]. Similar to Cdc15 [38], progressive phosphorylation of the Mid1 IDR may affect the IDR’s association to Mid1’s globular domains. Negatively charged phosphorylated residues could drive the localization of the IDR away from the membrane binding and dimerization interfaces of the C2 domain and towards the PH domain.…”