The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ∼40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1–DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models.

The contractile ring is essential for cytokinesis in most fungal and animal cells. In fission yeast, cytokinesis nodes are precursors of the contractile ring and mark the future cleavage site. However, their assembly and architecture have not been well described. We found that nodes are assembled stoichiometrically in a hierarchical order with two modules linked by the positional marker anillin Mid1. Mid1 first recruits Cdc4 and IQGAP Rng2 to form module I. Rng2 subsequently recruits the myosin-II subunits Myo2 and Rlc1. Mid1 then independently recruits the F-BAR protein Cdc15 to form module II. Mid1, Rng2, Cdc4, and Cdc15 are stable node components that accumulate close to the plasma membrane. Both modules recruit the formin Cdc12 to nucleate actin filaments. Myo2 heads point into the cell interior, where they efficiently capture actin filaments to condense nodes into the contractile ring. Collectively, our work characterizing the assembly and architecture of precursor nodes defines important steps and molecular players for contractile ring assembly.

The actomyosin contractile ring assembles through the condensation of a broad band of nodes that forms at the cell equator in fission yeast cytokinesis. The condensation process depends on actin filaments that interconnect nodes. By mutating or titrating actin cross-linkers α-actinin Ain1 and fimbrin Fim1 in live cells, we reveal that both proteins are involved in node condensation. Ain1 and Fim1 stabilize the actin cytoskeleton and modulate node movement, which prevents nodes and linear structures from aggregating into clumps and allows normal ring formation. Our computer simulations modeling actin filaments as semiflexible polymers reproduce the experimental observations and provide a model of how actin cross-linkers work with other proteins to regulate actin-filament orientations inside actin bundles and organize the actin network. As predicted by the simulations, doubling myosin II Myo2 level rescues the node condensation defects caused by Ain1 overexpression. Taken together, our work supports a cooperative process of ring self-organization driven by the interaction between actin filaments and myosin II, which is progressively stabilized by the cross-linking proteins.

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. α-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.

The fission yeastSchizosaccharomyces pombeis 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. © 2012 Wiley Periodicals, Inc