SummaryMany microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.

The clustered protocadherins (Pcdhs) comprise 58 cadherin-related proteins encoded by three tandemly arrayed gene clusters, -α,-β, and -γ (, , and, respectively). Pcdh isoforms from different clusters are combinatorially expressed in neurons. They form multimers that interact homophilically and mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, axonal tiling, and dendritic self-avoidance. Most studies have analyzed clusters individually. Here, we assessed functional interactions between and clusters. To circumvent neonatal lethality associated with deletion of , we used Crispr-Cas9 genome editing in mice to combine a constitutive mutant allele with a conditional allele. We analyzed roles of Pcdhas and Pcdhgs in the retina and cerebellum from mice (both sexes) lacking one or both clusters. In retina, Pcdhgs are essential for survival of inner retinal neurons and dendritic self-avoidance of starburst amacrine cells, whereas Pcdhas are dispensable for both processes. Deletion of both and clusters led to far more dramatic defects in survival and self-avoidance than deletion alone. Comparisons of an allelic series of mutants support the conclusion that Pcdhas and Pcdhgs function together in a dose-dependent and cell-type-specific manner to provide a critical threshold of Pcdh activity. In the cerebellum, Pcdhas and Pcdhgs also cooperate to mediate self-avoidance of Purkinje cell dendrites, with modest but significant defects in either single mutant and dramatic defects in the double mutant. Together, our results demonstrate complex patterns of redundancy between Pcdh clusters and the importance of Pcdh cluster diversity in postnatal CNS development. The formation of neural circuits requires diversification and combinatorial actions of cell surface proteins. Prominent among them are the clustered protocadherins (Pcdhs), a family of ∼60 neuronal recognition molecules. Pcdhs are encoded by three closely linked gene clusters called -α,-β, and -γ. The Pcdhs mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, and spatial patterning of axons and dendrites. Most studies to date have been limited to single clusters. Here, we used genome editing to assess interactions between-α and -γ gene clusters. We examined two regions of the CNS, the retina and cerebellum and show that the 14 α-Pcdhs and 22 γ-Pcdhs act synergistically to mediate neuronal survival and dendrite patterning.

␤-Catenin-independent Wnt signaling pathways have been implicated in the regulation of planar cell polarity (PCP) and convergent extension (CE) cell movements. Prickle, one of the core proteins of these pathways, is known to asymmetrically localize proximally at the adherens junction of Drosophila melanogaster wing cells and to locally accumulate within plasma membrane subdomains in cells undergoing CE movements during vertebrate development. Using mass spectrometry, we have identified the Ste20 kinase Mink1 as a Prickle-associated protein and found that they genetically interact during the establishment of PCP in the Drosophila eye and CE in Xenopus laevis embryos. We show that Mink1 phosphorylates Prickle on a conserved threonine residue and regulates its Rab5-dependent endosomal trafficking, a process required for the localized plasma membrane accumulation and function of Prickle. Mink1 also was found to be important for the clustering of Vangl within plasma membrane puncta. Our results provide a link between Mink and the Vangl-Prickle complex and highlight the importance of Prickle phosphorylation and endosomal trafficking for its function during Wnt-PCP signaling. P lanar cell polarity (PCP), which is orthogonal to the cell apicobasal axis, is important to coordinate cellular orientation at the tissue level (15,41,56,57,63). The stereotypical orientation of the hairs at the distal tip of Drosophila melanogaster wing cells and the organization of the photoreceptor cells in the fly compound eye are two particularly striking manifestations of PCP that have been exploited as model systems for its study. Genetic screens have identified a core set of proteins that are important for the establishment and maintenance of PCP. These proteins include Frizzled (Fz) (1, 55), Flamingo (Fmi) (53), Van Gogh (Vangl) (50, 61), Dishevelled (Dsh) (39), Diego (16), and Prickle (Pk) (18). The underlying molecular mechanisms of how the core PCP proteins regulate cell polarity are poorly understood. At the cellular level, the asymmetric localization of the PCP core proteins on the apical cortex is thought to be key for PCP establishment (45). In the Drosophila wing, the seven-transmembrane protein Fz along with the cytosolic proteins Dsh and Diego are distally localized, whereas the four-transmembrane protein Vangl and the membrane-associated protein Pk are proximally localized. The asymmetric localization of these core proteins is thought to result from intracellular feedback interactions between proximal Vangl-Pk and distal Fz-Dsh protein complexes. One emerging mechanism that has been proposed to control the asymmetric distribution of PCP proteins involves the polarized control of membrane-trafficking events. The microtubule-dependent polarized transport of Fmi and Fz (42,46), as well as the regulation of Fz and Fmi endocytosis (32,46,47), have been shown to play key roles in their localized accumulation on the cortex. Furthermore, it has been demonstrated recently that the cytoplasmic PCP core proteins Dsh, Pk, and Dgo are req...