The dynamic regulation of actin polymerisation plays crucial roles in cell morphology and endocytosis. The mechanistic details of these processes and the proteins involved are not fully understood, especially in neurons. PICK1 is a PDZ-BAR-domain protein involved in regulated AMPAR endocytosis in neurons. Here, we demonstrate that PICK1 binds F-actin and the actinnucleating Arp2/3 complex, and potently inhibits Arp2/3-mediated actin polymerisation. RNAi knockdown of PICK1 in neurons induces a reorganisation of the actin cytoskeleton resulting in aberrant cell morphology. Wild-type PICK1 rescues this phenotype, but a mutant PICK1 (W413A) that does not bind or inhibit Arp2/3 has no effect. Furthermore, this mutant also blocks NMDA-induced AMPAR internalisation. This study identifies PICK1 as a new negative regulator of Arp2/3-mediated actin polymerisation that is critical for a specific form of vesicle trafficking and also for the development of neuronal architecture.The dynamic actin cytoskeleton plays multiple roles in eukaryotic cells to regulate cell morphology, cell motility and vesicle trafficking by exerting mechanical forces that alter the shape of the plasma membrane. The Arp2/3 complex is the major catalyst for the formation of branched actin networks that mediate changes in membrane geometry. Proteins such as N-WASP, WAVE and cortactin bind and activate the Arp2/3 complex so that changes in cell morphology or vesicle trafficking occur at appropriate times and subcellular locations1-3.Regulation of the actin cytoskeleton is crucial for neuronal morphogenesis1,4,5. Recent reports have suggested a role for the Arp2/3 activator N-WASP in neurite elongation and branching in neuronal development6,7. However, negative regulators of the Arp2/3 complex in these processes have not yet been identified. A role for Arp2/3-mediated actin dynamics in the regulation of endocytosis in mammalian cell lines is becoming increasingly well established. Localised alterations in actin turnover are proposed to provide mechanical forces that contribute to plasma membrane curvature, vesicle scission, and propulsion of nascent vesicles away from the membrane2,8-10. The specific molecular mechanisms for regulating actin to control endocytosis in neurons are unknown. Dendritic spines, the sites of excitatory synapses in neurons, are particularly enriched in filamentous (F-) actin11-13, which is extremely dynamic, rapidly cycling between globular (G-) actin and F-actin14. Since designated endocytic sites are found in dendritic spines15,16, it seems likely that the densely packed, dynamic actin filaments influence endocytosis. PICK1 is a PDZ and BAR-* correspondence: e-mail: jon.hanley@bristol.ac.uk, tel: +44 (0)117 331 1944, fax: +44 (0)117 929 1687. AUTHOR CONTRIBUTIONS D.L.R. planned and performed the biochemistry and some imaging experiments. S.M. supervised generation of shRNA, planned and performed some imaging experiments. E.L.J. generated shRNA constructs. J.G.H planned and performed imaging experiments, mutagenesis/cl...

Quantitative microscopy has proven a versatile and powerful phenotypic screening technique. Recently, image-based profiling has shown promise as a means for broadly characterizing molecules’ effects on cells in several drug-discovery applications, including target-agnostic screening and predicting a compound’s mechanism of action (MOA). Several profiling methods have been proposed, but little is known about their comparative performance, impeding the wider adoption and further development of image-based profiling. We compared these methods by applying them to a widely applicable assay of cultured cells and measuring the ability of each method to predict the MOA of a compendium of drugs. A very simple method that is based on population means performed as well as methods designed to take advantage of the measurements of individual cells. This is surprising because many treatments induced a heterogeneous phenotypic response across the cell population in each sample. Another simple method, which performs factor analysis on the cellular measurements before averaging them, provided substantial improvement and was able to predict MOA correctly for 94% of the treatments in our ground-truth set. To facilitate the ready application and future development of image-based phenotypic profiling methods, we provide our complete ground-truth and test datasets, as well as open-source implementations of the various methods in a common software framework.

We describe seven patients with KDM6A (located on Xp11.3 and encodes UTX) mutations, a rare cause of Kabuki syndrome (KS2, MIM 300867) and report, for the first time, germ-line missense and splice-site mutations in the gene. We demonstrate that less than 5% cases of Kabuki syndrome are due to KDM6A mutations. Our work shows that similar to the commoner Type 1 Kabuki syndrome (KS1, MIM 147920) caused by KMT2D (previously called MLL2) mutations, KS2 patients are characterized by hypotonia and feeding difficulties during infancy and poor postnatal growth and short stature. Unlike KS1, developmental delay and learning disability are generally moderate-severe in boys but mild-moderate in girls with KS2. Some girls may have a normal developmental profile. Speech and cognition tend to be more severely affected than motor development. Increased susceptibility to infections, join laxity, heart, dental and ophthalmological anomalies are common. Hypoglycaemia is more common in KS2 than in KS1. Facial dysmorphism with KDM6A mutations is variable and diagnosis on facial gestalt alone may be difficult in some patients. Hypertrichosis, long halluces and large central incisors may be useful clues to an underlying KDM6A mutation in some patients.