We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly vexing in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review, we explore recent studies in fission yeast that help unravel how different actin structures operate in cells.

F, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or F depends on the nature of the adenine nucleotide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, F and SpoIIAB were barely detectable at t 0 , but their concentrations increased in parallel to reach maxima at about t 1.5 . SpoIIAA-P increased steadily to a maximum at t 3 , but nonphosphorylated SpoIIAA was detectable only from t 1.5 , reached a maximum at t 2.5 , and then declined. Kinetic studies of the phosphorylation of SpoIIAA catalyzed by SpoIIAB suggested that the reaction was limited by a very slow release of one of the products (SpoIIAA-P or ADP) from SpoIIAB, with a turnover of about once per 20 min. This remarkable kinetic property provides an unexpected mechanism for the regulation of F . We propose that when SpoIIE (which dephosphorylates SpoIIAA-P) is active at the same time as SpoIIAB, SpoIIAA cycles repeatedly between the phosphorylated and nonphosphorylated forms. This cycling sequesters SpoIIAB in a long-lived complex and prevents it from inhibiting F .Early in the sporulation of Bacillus subtilis, the cell divides asymmetrically to form two compartments that remain attached to each other, a small prespore and a larger mother cell. Although the two compartments have identical genomes, they show different patterns of gene expression. The prespore gradually becomes transformed into the mature spore, which is eventually released by lysis of the mother cell. Gene expression in the two compartments depends on sigma factors that show both spatial and temporal specificity: in the prespore, F is followed by G , and in the mother cell, E is followed by K (9, 16). The first sporulation-specific sigma factor, F

Budding yeast possesses one myosin-II, Myo1p, whereas fission yeast has two, Myo2p and Myp2p, all of which contribute to cytokinesis. We find that chimeras consisting of Myo2p or Myp2p motor domains fused to the tail of Myo1p are fully functional in supporting budding yeast cytokinesis. Remarkably, the tail alone of budding yeast Myo1p localizes to the contractile ring, supporting both its constriction and cytokinesis. In contrast, fission yeast Myo2p and Myp2p require both the catalytic head domain as well as tail domains for function, with the tails providing distinct functions (Bezanilla and Pollard, 2000). Myo1p is the first example of a myosin whose cellular function does not require a catalytic motor domain revealing a novel mechanism of action for budding yeast myosin-II independent of actin binding and ATPase activity.