Regulated Activation of the PAR Polarity Network Ensures a Timely and Specific Response to Spatial Cues

How do cells polarize at the correct time and in response to the correct cues? In the C. elegans zygote, the timing and geometry of polarization rely on a single dominant cue-the sperm centrosome-that matures at the end of meiosis and specifies the nascent posterior. Polarization requires that the conserved PAR proteins, which specify polarity in the zygote, be poised to respond to the centrosome. Yet, how and when PAR proteins achieve this unpolarized, but responsive, state is unknown. We show that oocyte maturation initiates a fertilization-independent PAR activation program. PAR proteins are initially not competent to polarize but gradually acquire this ability following oocyte maturation. Surprisingly, this program allows symmetry breaking even in unfertilized oocytes lacking centrosomes. Thus, if PAR proteins can respond to multiple polarizing cues, how is specificity for the centrosome achieved? Specificity is enforced by Polo-like and Aurora kinases (PLK-1 and AIR-1 in C. elegans), which impose a delay in the activation of the PAR network so that it coincides with maturation of the centrosome cue. This delay suppresses polarization by non-centrosomal cues, which can otherwise trigger premature polarization and multiple or reversed polarity domains. Taken together, these findings identify a regulatory program that enforces proper polarization by synchronizing PAR network activation with cell cycle progression, thereby ensuring that PAR proteins respond specifically to the correct cue. Temporal control of polarity network activity is likely to be a common strategy to ensure robust, dynamic, and specific polarization in response to developmentally deployed cues

aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity

The conserved polarity effector proteins PAR-3, PAR-6, CDC-42, and atypical protein kinase C (aPKC) form a core unit of the PAR protein network, which plays a central role in polarizing a broad range of animal cell types. To functionally polarize cells, these proteins must activate aPKC within a spatially defined membrane domain on one side of the cell in response to symmetry-breaking cues. Using the Caenorhabditis elegans zygote as a model, we find that the localization and activation of aPKC involve distinct, specialized aPKC-containing assemblies: a PAR-3-dependent assembly that responds to polarity cues and promotes efficient segregation of aPKC toward the anterior but holds aPKC in an inactive state, and a CDC-42-dependent assembly in which aPKC is active but poorly segregated. Cycling of aPKC between these distinct functional assemblies, which appears to depend on aPKC activity, effectively links cue-sensing and effector roles within the PAR network to ensure robust establishment of polarity.This work was supported by a Faculty Fellowship from Newcastle University and a Royal Society Research Grant (RG2015R2 to J. Rodriguez), a BBSRC PhD fellowship (J.M.), a PhD fellowship from Newcastle University (A.G.G.), Wellcome Trust Senior and Principal Research Fellowships (054523, to J.A.; 080007, to D.StJ.), a University of Cambridge Studentship via the Wellcome Trust PhD Program in Developmental Biology (A.R.F.), and the Francis Crick Institute (N.W.G.), which receives its core funding from Cancer Research UK (FC001086), the UK Medical Research Council (FC001086), and the Wellcome Trust (FC001086). N.W.G. and J. Rodriguez are members of the GENiE network supported by COST Action BM1408 and EMBO

A cell-size threshold limits cell polarity and asymmetric division potential

Reaction-diffusion networks underlie pattern formation in a range of biological contexts, from morphogenesis of organisms to the polarization of individual cells. One requirement for such molecular networks is that output patterns be scaled to system size. At the same time, kinetic properties of constituent molecules constrain the ability of networks to adapt to size changes. Here, we explore these constraints and the consequences thereof within the conserved PAR cell polarity network. Using the stem-cell-like germ lineage of the Caenorhabditis elegans embryo as a model, we find that the behaviour of PAR proteins fails to scale with cell size. Theoretical analysis demonstrates that this lack of scaling results in a size threshold below which polarity is destabilized, yielding an unpolarized system. In empirically constrained models, this threshold occurs near the size at which germ lineage cells normally switch between asymmetric and symmetric modes of division. Consistent with cell size limiting polarity and division asymmetry, genetic or physical reduction in germ lineage cell size is sufficient to trigger loss of polarity in normally polarizing cells at predicted size thresholds. Physical limits of polarity networks may be one mechanism by which cells read out geometrical features to inform cell fate decisions

Regulated Activation of the PAR Polarity Network Ensures a Timely and Specific Response to Spatial Cues

How do cells polarize at the correct time and in response to the correct cues? In the C. elegans zygote, the timing and geometry of polarization rely on a single dominant cue-the sperm centrosome-that matures at the end of meiosis and specifies the nascent posterior. Polarization requires that the conserved PAR proteins, which specify polarity in the zygote, be poised to respond to the centrosome. Yet, how and when PAR proteins achieve this unpolarized, but responsive, state is unknown. We show that oocyte maturation initiates a fertilization-independent PAR activation program. PAR proteins are initially not competent to polarize but gradually acquire this ability following oocyte maturation. Surprisingly, this program allows symmetry breaking even in unfertilized oocytes lacking centrosomes. Thus, if PAR proteins can respond to multiple polarizing cues, how is specificity for the centrosome achieved? Specificity is enforced by Polo-like and Aurora kinases (PLK-1 and AIR-1 in C. elegans), which impose a delay in the activation of the PAR network so that it coincides with maturation of the centrosome cue. This delay suppresses polarization by non-centrosomal cues, which can otherwise trigger premature polarization and multiple or reversed polarity domains. Taken together, these findings identify a regulatory program that enforces proper polarization by synchronizing PAR network activation with cell cycle progression, thereby ensuring that PAR proteins respond specifically to the correct cue. Temporal control of polarity network activity is likely to be a common strategy to ensure robust, dynamic, and specific polarization in response to developmentally deployed cues

S32504, a novel naphtoxazine agonist at dopamine D3/D2 receptors: III. Actions in models of potential antidepressive and anxiolytic activity in comparison with ropinirole.

In forced-swim tests in mice and rats, the novel D(3)/D(2) receptor agonist S32504 [(+)-trans-3,4,4a,5,6,10b-hexahydro-9-carbamoyl-4-propyl-2H-naphth[1,2-b]-1,4-oxazine] dose-dependently (0.04-2.5 mg/kg) and stereospecifically suppressed immobility compared with its enantiomer S32601 [(-)-trans-3,4,4a,5,6,10b-hexahydro-9-carbamoyl-4-propyl-2H-naphth-[1,2-b]-1,4-oxazine]. Ropinirole was less potent than S32504 in this procedure, and it was likewise less potent than S32504 (0.04-2.5 mg/kg) in attenuating motor-suppressant properties of the alpha(2)-adrenoceptor agonist S18616 [(S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2'-(1',2',3',4'-tetrahydronaphthalene)]]. In a learned helplessness paradigm, S32504 (0.08-2.5 mg/kg) suppressed escape failures. Furthermore, in a chronic mild stress model of anhedonia, S32504 (0.16-2.5 mg/kg) rapidly restored the suppression of sucrose consumption. S32504 inhibited marble-burying behavior in mice (0.04-0.16 mg/kg) and aggressive behavior in isolated mice (0.04-2.5 mg/kg): only higher doses of ropinirole mimicked these actions of S32504. In tests of anxiolytic activity, S32504 was more potent (0.0025-0.16 mg/kg) than ropinirole in suppressing fear-induced ultrasonic vocalizations, and S32601 was inactive. Furthermore, in contrast to ropinirole, S32504 modestly enhanced punished responses in a Vogel conflict procedure and increased open-arm entries in a plus-maze. At doses active in the above-described procedures, S32504 did not elicit hyperlocomotion. In the forced-swim, marble-burying, and ultrasonic vocalization models, actions of S32504 were blocked by the D(2)/D(3) antagonists haloperidol and raclopride and by the D(2) antagonist L741,626 [4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol], but not by the D(3) receptor antagonist S33084 [(3aR,9bS)-N-[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl]-(4-phenyl)benzamide. Finally, chronic administration of S32504 did not, in contrast to venlafaxine, modify corticolimbic levels of serotonin(2A) receptors or brain-derived neurotrophic factor. In conclusion, S32504 displays a broad and distinctive profile of activity in models of potential antidepressive and anxiolytic properties. Its actions are more pronounced than those of ropinirole and principally involve engagement of D(2) receptors

S32504, a novel naphtoxazine agonist at dopamine D3/D2 receptors: III. Actions in models of potential antidepressive and anxiolytic activity in comparison with ropinirole.

In forced-swim tests in mice and rats, the novel D(3)/D(2) receptor agonist S32504 [(+)-trans-3,4,4a,5,6,10b-hexahydro-9-carbamoyl-4-propyl-2H-naphth[1,2-b]-1,4-oxazine] dose-dependently (0.04-2.5 mg/kg) and stereospecifically suppressed immobility compared with its enantiomer S32601 [(-)-trans-3,4,4a,5,6,10b-hexahydro-9-carbamoyl-4-propyl-2H-naphth-[1,2-b]-1,4-oxazine]. Ropinirole was less potent than S32504 in this procedure, and it was likewise less potent than S32504 (0.04-2.5 mg/kg) in attenuating motor-suppressant properties of the alpha(2)-adrenoceptor agonist S18616 [(S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2'-(1',2',3',4'-tetrahydronaphthalene)]]. In a learned helplessness paradigm, S32504 (0.08-2.5 mg/kg) suppressed escape failures. Furthermore, in a chronic mild stress model of anhedonia, S32504 (0.16-2.5 mg/kg) rapidly restored the suppression of sucrose consumption. S32504 inhibited marble-burying behavior in mice (0.04-0.16 mg/kg) and aggressive behavior in isolated mice (0.04-2.5 mg/kg): only higher doses of ropinirole mimicked these actions of S32504. In tests of anxiolytic activity, S32504 was more potent (0.0025-0.16 mg/kg) than ropinirole in suppressing fear-induced ultrasonic vocalizations, and S32601 was inactive. Furthermore, in contrast to ropinirole, S32504 modestly enhanced punished responses in a Vogel conflict procedure and increased open-arm entries in a plus-maze. At doses active in the above-described procedures, S32504 did not elicit hyperlocomotion. In the forced-swim, marble-burying, and ultrasonic vocalization models, actions of S32504 were blocked by the D(2)/D(3) antagonists haloperidol and raclopride and by the D(2) antagonist L741,626 [4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol], but not by the D(3) receptor antagonist S33084 [(3aR,9bS)-N-[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl]-(4-phenyl)benzamide. Finally, chronic administration of S32504 did not, in contrast to venlafaxine, modify corticolimbic levels of serotonin(2A) receptors or brain-derived neurotrophic factor. In conclusion, S32504 displays a broad and distinctive profile of activity in models of potential antidepressive and anxiolytic properties. Its actions are more pronounced than those of ropinirole and principally involve engagement of D(2) receptors