I-Ju Lee

Assembly and architecture of precursor nodes during fission yeast cytokinesis

Mapping of fission yeast precursor node interaction modules and assembly reveals important steps in contractile ring assembly.

Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30-50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. alpha-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis.

Contractile‐ring assembly in fission yeast cytokinesis: Recent advances and new perspectives

The fission yeast Schizosaccharomyces pombe is an excellent model organism to study cytokinesis. Here, we review recent advances on contractile‐ring assembly in fission yeast. First, we summarize the assembly of cytokinesis nodes, the precursors of a normal contractile ring. IQGAP Rng2 and myosin essential light chain Cdc4 are recruited by the anillin‐like protein Mid1, followed by the addition of other cytokinesis node proteins. Mid1 localization on the plasma membrane is stabilized by interphase node proteins. Second, we discuss proteins and processes that contribute to the search, capture, pull, and release mechanism of contractile‐ring assembly. Actin filaments nucleated by formin Cdc12, the motor activity of myosin‐II, the stiffness of the actin network, and severing of actin filaments by cofilin all play essential roles in contractile‐ring assembly. Finally, we discuss the Mid1‐independent pathway for ring assembly, and the possible mechanisms underlying the ring maturation and constriction. Collectively, we provide an overview of the current understanding of contractile‐ring assembly and uncover future directions in studying cytokinesis in fission yeast.

Characterization of Mid1 domains for targeting and scaffolding in fission yeast cytokinesis

Summary Division-site selection and contractile-ring assembly are two crucial steps in cytokinesis. In fission yeast, the anillin-like Mid1 protein specifies the division site at the cell equator by assembling cortical nodes, the precursors of the contractile ring. Thus, Mid1 is essential for linking the positional cues for the cleavage site to contractile-ring formation. However, how Mid1 domains cooperate to regulate cytokinesis is poorly understood. Here we unravel the functions of different Mid1 domains (motifs) by a series of truncations. We report that the conserved PH domain stabilizes Mid1 in nodes by binding to lipids and is required for Mid1 cortical localization during interphase in the absence of Cdr2 kinase. Mid1 lacking an internal region that is approximately one third of the full-length protein has higher nuclear and cortical concentration and suppresses the division-site positioning defects in cells with a deletion of the dual-specificity tyrosine-regulated kinase Pom1. The N-terminus of Mid1 physically interacts with cytokinesis node proteins. When fused to cortical node protein Cdr2, Mid1(1–100) is sufficient to assemble cytokinesis nodes and the contractile ring. Collectively, our study recognizes domains regulating Mid1 cortical localization and reveals domains sufficient for contractile-ring assembly.

Roles of putative Rho-GEF Gef2 in division-site positioning and contractile-ring function in fission yeast cytokinesis

How Rho-GEFs and Rho GTPases regulate division-site selection during cytokinesis in fission yeast is unknown. The Rho-GEF Gef2 interacts with the anillin Mid1 to regulate contractile-ring positioning and assembly in coordination with the polo kinase Plo1. In addition, Gef2 is involved in contractile-ring stability and disassembly.

SOAX: a software for quantification of 3D biopolymer networks.

Filamentous biopolymer networks in cells and tissues are routinely imaged by confocal microscopy. Image analysis methods enable quantitative study of the properties of these curvilinear networks. However, software tools to quantify the geometry and topology of these often dense 3D networks and to localize network junctions are scarce. To fill this gap, we developed a new software tool called “SOAX”, which can accurately extract the centerlines of 3D biopolymer networks and identify network junctions using Stretching Open Active Contours (SOACs). It provides an open-source, user-friendly platform for network centerline extraction, 2D/3D visualization, manual editing and quantitative analysis. We propose a method to quantify the performance of SOAX, which helps determine the optimal extraction parameter values. We quantify several different types of biopolymer networks to demonstrate SOAXs potential to help answer key questions in cell biology and biophysics from a quantitative viewpoint.

Regulation of spindle-pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

A previous model suggested doubling of Sfi1 as the first step of SPB assembly. Here it is shown that Sfi1 is gradually recruited to SPBs throughout the cell cycle. Conserved tryptophans in Sfi1 are required for its equal partitioning during mitosis, and unequal partitioning of Sfi1 underlies SPB assembly and mitotic defects in the next cell cycle.

Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis

The cleavage-furrow tip adjacent to the actomyosin contractile ring is believed to be the predominant site for plasma-membrane insertion through exocyst-tethered vesicles during cytokinesis. Here we found that most secretory vesicles are delivered by myosin-V on linear actin cables in fission yeast cytokinesis. Surprisingly, by tracking individual exocytic and endocytic events, we found that vesicles with new membrane are deposited to the cleavage furrow relatively evenly during contractile-ring constriction, but the rim of the cleavage furrow is the main site for endocytosis. Fusion of vesicles with the plasma membrane requires vesicle tethers. Our data suggest that the transport particle protein II (TRAPP-II) complex and Rab11 GTPase Ypt3 help to tether secretory vesicles or tubulovesicular structures along the cleavage furrow while the exocyst tethers vesicles at the rim of the division plane. We conclude that the exocyst and TRAPP-II complex have distinct localizations at the division site, but both are important for membrane expansion and exocytosis during cytokinesis.

Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis

The regulation of Rho-GAP localization is not well understood. A novel coiled-coil protein Rng10 is characterized that localizes the Rho-GAP Rga7 in fission yeast. Rng10 and Rga7 physically interact and work together to regulate the accumulation and dynamics of glucan synthases for successful septum formation during cytokinesis.