The DIAPH3 linker specifies a ß-actin network that maintains RhoA and Myosin-II at the cytokinetic furrow.

Cytokinesis is the final step of the cell division cycle that leads to the formation of two new cells. Successful cytokinesis requires significant remodelling of the plasma membrane by spatially distinct ß- and γ-actin networks. These networks are generated by the formin family of actin nucleators, DIAPH3 and DIAPH1 respectively. Here we show that ß- and γ-actin perform specialized and non-redundant roles in cytokinesis and cannot substitute for one another. Expression of hybrid DIAPH1 and DIAPH3 proteins with altered actin isoform specificity relocalized cytokinetic actin isoform networks within the cell, causing cytokinetic failure. Consistent with this we show that ß-actin networks, but not γ-actin networks, are required for the maintenance of non-muscle myosin II and RhoA at the cytokinetic furrow. These data suggest that independent and spatially distinct actin isoform networks form scaffolds of unique interactors that facilitate localized biochemical activities to ensure successful cell division.

An anillin-CIN85-SEPT9 complex promotes intercellular bridge maturation required for successful cytokinesis.

Cleavage of one cell into two is the most dramatic event in the life of a cell. Plasma membrane fission occurs within a narrow intercellular bridge (ICB) between the daughter cells, but the mechanisms underlying ICB formation and maturation are poorly understood. Here we identify CIN85 as an ICB assembly factor and demonstrate its requirement for robust and timely cytokinesis. CIN85 interacts directly with the N-terminal region of anillin and SEPT9 and thereby facilitates SEPT9-containing filament localization to the plasma membrane of the ICB. In contrast, the C-terminal pleckstrin homology (PH) domain of anillin binds to septin units lacking SEPT9 but enriched in SEPT11. Anillin's interactions with distinct septin units are required to promote ICB elongation and maturation that, we propose, generate the physical space into which the abscission machinery is recruited to drive the final membrane scission event releasing two independent daughter cells.

Inhibition of polar actin assembly by astral microtubules is required for cytokinesis.

During cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a ß-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the ß- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies ß-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a ß-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.

CDK11p58-cyclin L1ß regulates abscission site assembly.

Rigorous spatiotemporal regulation of cell division is required to maintain genome stability. The final stage in cell division, when the cells physically separate (abscission), is tightly regulated to ensure that it occurs after cytokinetic events such as chromosome segregation. A key regulator of abscission timing is Aurora B kinase activity, which inhibits abscission and forms the major activity of the abscission checkpoint. This checkpoint prevents abscission until chromosomes have been cleared from the cytokinetic machinery. Here we demonstrate that the mitosis-specific CDK11p58 kinase specifically forms a complex with cyclin L1ß that, in late cytokinesis, localizes to the stem body, a structure in the middle of the intercellular bridge that forms between two dividing cells. Depletion of CDK11 inhibits abscission, and rescue of this phenotype requires CDK11p58 kinase activity or inhibition of Aurora B kinase activity. Furthermore, CDK11p58 kinase activity is required for formation of endosomal sorting complex required for transport III filaments at the site of abscission. Combined, these data suggest that CDK11p58 kinase activity opposes Aurora B activity to enable abscission to proceed and result in successful completion of cytokinesis.