Cytokinesis requires coordination of actomyosin ring (AMR) contraction with rearrangements of the plasma membrane and extracellular matrix. In Saccharomyces cerevisiae, new membrane, the chitin synthase Chs2 (which forms the primary septum [PS]), and the protein Inn1 are all delivered to the division site upon mitotic exit even when the AMR is absent. Inn1 is essential for PS formation but not for Chs2 localization. The Inn1 C-terminal region is necessary for localization, and distinct PXXP motifs in this region mediate functionally important interactions with SH3 domains in the cytokinesis proteins Hof1 (an F-BAR protein) and Cyk3 (whose overexpression can restore PS formation in inn1Δ cells). The Inn1 N terminus resembles C2 domains but does not appear to bind phospholipids; nonetheless, when overexpressed or fused to Hof1, it can provide Inn1 function even in the absence of the AMR. Thus, Inn1 and Cyk3 appear to cooperate in activating Chs2 for PS formation, which allows coordination of AMR contraction with ingression of the cleavage furrow.

SummaryAsymmetric cell division plays a crucial role in cell differentiation, unequal replicative senescence, and stem cell maintenance. In budding yeast, the identities of mother and daughter cells begin to diverge at bud emergence when distinct plasma-membrane domains are formed and separated by a septin ring. However, the mechanisms underlying this transformation remain unknown. Here, we show that septins recruited to the site of polarization by Cdc42-GTP inhibit Cdc42 activity in a negative feedback loop, and this inhibition depends on Cdc42 GTPase-activating proteins. Combining live-cell imaging and computational modeling, we demonstrate that the septin ring is sculpted by polarized exocytosis, which creates a hole in the accumulating septin density and relieves the inhibition of Cdc42. The nascent ring generates a sharp boundary that confines the Cdc42 activity and exocytosis strictly to its enclosure and thus clearly delineates the daughter cell identity. Our findings define a fundamental mechanism underlying eukaryotic cell fate differentiation.

Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.

AimsTo investigate within-and between-subject variability of the pharmacodynamics and pharmacokinetics of (R)-and (S)-methadone in methadone maintenance subjects at steady-state. MethodsSix non-holder subjects were studied on three occasions at 7-16 day intervals; doses (20-170 mg/day) remained unchanged. Blood samples and pharmacodynamic data were collected 10-12 times over a 24-h inter-dosing interval. All pharmacodynamic data were expressed as the area under the end-point versus time curve. Using analyses of variance with mixed effects, best estimates were made of the ratio of between-to within-subject variation, with corresponding 95% confidence intervals (CI) for within-subject variation at the average value. ResultsSubjects were relatively consistent between occasions, whereas there was much greater between-subject variability ( P < 0.02) for all measures. Estimates of the ratio of between-to within-subject variation ranged from 2.2-12.8 for pharmacodynamic measures, and 1.3-7.9 for pharmacokinetic parameters. For pain, total mood disturbance, withdrawal, pupil size and respiration rate, 95% CI for within-subject measures ranged £ 2-fold, while this was greater for subjective direct opioid effects (4.2-fold). For CL/F of the active (R)-methadone, the variance ratio was 4.9 ( P < 0.0003), with 95% CI for within-subject measures ranging £ 2-fold. (S)-methadone CL/F demonstrated greater within-subject variability (3.4-fold), possibly contributing to a smaller (2.7; P < 0.0003) ratio of between-to within-subject variance. ConclusionsNon-holder methadone maintenance treatment participants appear to respond consistently with respect to pharmacokinetics and pharmacodynamics over a 1-2 month period. Such knowledge may help prescribers to determine whether alternative dosing regimens or treatments might be more appropriate in this population. Correspondence

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