The Rho1 GTPase controls anillo-septin assembly to facilitate contractile ring closure during cytokinesis.

Animal cell cytokinesis requires activation of the GTPase RhoA (Rho1 in Drosophila), which assembles an F-actin- and myosin II-dependent contractile ring (CR) at the equatorial plasma membrane. CR closure is poorly understood, but involves the multidomain scaffold protein, Anillin. Anillin binds many CR components including F-actin and myosin II (collectively actomyosin), RhoA and the septins. Anillin recruits septins to the CR but the mechanism is unclear. Live imaging of Drosophila S2 cells and HeLa cells revealed that the Anillin N-terminus, which scaffolds actomyosin, cannot recruit septins to the CR. Rather, septin recruitment required the ability of the Anillin C-terminus to bind Rho1-GTP and the presence of the Anillin PH domain, in a sequential mechanism occurring at the plasma membrane, independently of F-actin. Anillin mutations that blocked septin recruitment, but not actomyosin scaffolding, slowed CR closure and disrupted cytokinesis. Thus, CR closure requires coordination of two Rho1-dependent networks: actomyosin and anillo-septin.

Animal Cell Cytokinesis: The Rho-Dependent Actomyosin-Anilloseptin Contractile Ring as a Membrane Microdomain Gathering, Compressing, and Sorting Machine.

Cytokinesis is the last step of cell division that partitions the cellular organelles and cytoplasm of one cell into two. In animal cells, cytokinesis requires Rho-GTPase-dependent assembly of F-actin and myosin II (actomyosin) to form an equatorial contractile ring (CR) that bisects the cell. Despite 50 years of research, the precise mechanisms of CR assembly, tension generation and closure remain elusive. This hypothesis article considers a holistic view of the CR that, in addition to actomyosin, includes another Rho-dependent cytoskeletal sub-network containing the scaffold protein, Anillin, and septin filaments (collectively termed anillo-septin). We synthesize evidence from our prior work in Drosophila S2 cells that actomyosin and anillo-septin form separable networks that are independently anchored to the plasma membrane. This latter realization leads to a simple conceptual model in which CR assembly and closure depend upon the micro-management of the membrane microdomains to which actomyosin and anillo-septin sub-networks are attached. During CR assembly, actomyosin contractility gathers and compresses its underlying membrane microdomain attachment sites. These microdomains resist this compression, which builds tension. During CR closure, membrane microdomains are transferred from the actomyosin sub-network to the anillo-septin sub-network, with which they flow out of the CR as it advances. This relative outflow of membrane microdomains regulates tension, reduces the circumference of the CR and promotes actomyosin disassembly all at the same time. According to this hypothesis, the metazoan CR can be viewed as a membrane microdomain gathering, compressing and sorting machine that intrinsically buffers its own tension through coordination of actomyosin contractility and anillo-septin-membrane relative outflow, all controlled by Rho. Central to this model is the abandonment of the dogmatic view that the plasma membrane is always readily deformable by the underlying cytoskeleton. Rather, the membrane resists compression to build tension. The notion that the CR might generate tension through resistance to compression of its own membrane microdomain attachment sites, can account for numerous otherwise puzzling observations and warrants further investigation using multiple systems and methods.

Rho-dependent control of the Citron kinase, Sticky, drives midbody ring maturation.

Rho-dependent proteins control assembly of the cytokinetic contractile ring, yet it remains unclear how those proteins guide ring closure and how they promote subsequent formation of a stable midbody ring. Citron kinase is one important component required for midbody ring formation but its mechanisms of action and relationship with Rho are controversial. Here, we conduct a structure-function analysis of the Drosophila Citron kinase, Sticky, in Schneider's S2 cells. We define two separable and redundant RhoGEF/Pebble-dependent inputs into Sticky recruitment to the nascent midbody ring and show that each input is subsequently required for retention at, and for the integrity of, the mature midbody ring. The first input is via an actomyosin-independent interaction between Sticky and Anillin, a key scaffold also required for midbody ring formation. The second input requires the Rho-binding domain of Sticky, whose boundaries we have defined. Collectively, these results show how midbody ring biogenesis depends on the coordinated actions of Sticky, Anillin, and Rho.

IPIP27 Coordinates PtdIns(4,5)P2 Homeostasis for Successful Cytokinesis.

During cytokinesis, an actomyosin contractile ring drives the separation of the two daughter cells. A key molecule in this process is the inositol lipid PtdIns(4,5)P2, which recruits numerous factors to the equatorial region for contractile ring assembly. Despite the importance of PtdIns(4,5)P2 in cytokinesis, the regulation of this lipid in cell division remains poorly understood. Here, we identify a role for IPIP27 in mediating cellular PtdIns(4,5)P2 homeostasis. IPIP27 scaffolds the inositol phosphatase oculocerebrorenal syndrome of Lowe (OCRL) by coupling it to endocytic BAR domain proteins. Loss of IPIP27 causes accumulation of PtdIns(4,5)P2 on aberrant endomembrane vacuoles, mislocalization of the cytokinetic machinery, and extensive cortical membrane blebbing. This phenotype is observed in Drosophila and human cells and can result in cytokinesis failure. We have therefore identified IPIP27 as a key modulator of cellular PtdIns(4,5)P2 homeostasis required for normal cytokinesis. The results indicate that scaffolding of inositol phosphatase activity is critical for maintaining PtdIns(4,5)P2 homeostasis and highlight a critical role for this process in cell division.