Integrins are required for synchronous ommatidial rotation in the Drosophila eye linking planar cell polarity signalling to the extracellular matrix.

Integrins mediate the anchorage between cells and their environment, the extracellular matrix (ECM), and form transmembrane links between the ECM and the cytoskeleton, a conserved feature throughout development and morphogenesis of epithelial organs. Here, we demonstrate that integrins and components of the ECM are required during the planar cell polarity (PCP) signalling-regulated cell movement of ommatidial rotation in the Drosophila eye. The loss-of-function mutations of integrins or ECM components cause defects in rotation, with mutant clusters rotating asynchronously compared to wild-type clusters. Initially, mutant clusters tend to rotate faster, and at later stages they fail to be synchronous with their neighbours, leading to aberrant rotation angles and resulting in a disorganized ommatidial arrangement in adult eyes. We further demonstrate that integrin localization changes dynamically during the rotation process. Our data suggest that core Frizzled/PCP factors, acting through RhoA and Rho kinase, regulate the function/activity of integrins and that integrins thus contribute to the complex interaction network of PCP signalling, cell adhesion and cytoskeletal elements required for a precise and synchronous 90° rotation movement.

Prickle is phosphorylated by Nemo and targeted for degradation to maintain Prickle/Spiny-legs isoform balance during planar cell polarity establishment.

Planar cell polarity (PCP) instructs tissue patterning in a wide range of organisms from fruit flies to humans. PCP signaling coordinates cell behavior across tissues and is integrated by cells to couple cell fate identity with position in a developing tissue. In the fly eye, PCP signaling is required for the specification of R3 and R4 photoreceptors based upon their positioning relative to the dorso-ventral axis. The 'core' PCP pathway involves the asymmetric localization of two distinct membrane-bound complexes, one containing Frizzled (Fz, required in R3) and the other Van Gogh (Vang, required in R4). Inhibitory interactions between the cytosolic components of each complex reinforce asymmetric localization. Prickle (Pk) and Spiny-legs (Pk-Sple) are two antagonistic isoforms of the prickle (pk) gene and are cytoplasmic components of the Vang complex. The balance between their levels is critical for tissue patterning, with Pk-Sple being the major functional isoform in the eye. Here we uncover a post-translational role for Nemo kinase in limiting the amount of the minor isoform Pk. We identified Pk as a Nemo substrate in a genome-wide in vitro band-shift screen. In vivo, nemo genetically interacts with pkpk but not pksple and enhances PCP defects in the eye and leg. Nemo phosphorylation limits Pk levels and is required specifically in the R4 photoreceptor like the major isoform, Pk-Sple. Genetic interaction and biochemical data suggest that Nemo phosphorylation of Pk leads to its proteasomal degradation via the Cullin1/SkpA/Slmb complex. dTAK and Homeodomain interacting protein kinase (Hipk) may also act together with Nemo to target Pk for degradation, consistent with similar observations in mammalian studies. Our results therefore demonstrate a mechanism to maintain low levels of the minor Pk isoform, allowing PCP complexes to form correctly and specify cell fate.

The Drosophila selectin furrowed mediates intercellular planar cell polarity interactions via frizzled stabilization.

Establishment of planar cell polarity (PCP) in a tissue requires coordination of directional signals from cell to cell. It is thought that this is mediated by the core PCP factors, which include cell-adhesion molecules. Here, we demonstrate that furrowed, the Drosophila selectin, is required for PCP generation. Disruption of PCP in furrowed-deficient flies results from a primary defect in Fz levels and cell adhesion. Furrowed localizes at or near apical junctions, largely colocalizing with Frizzled and Flamingo (Fmi). It physically interacts with and stabilizes Frizzled, and it mediates intercellular Frizzled-Van Gogh (Vang)/Strabismus interactions, similarly to Fmi. Furrowed does so through a homophilic cell-adhesion role that is distinct from its known carbohydrate-binding function described during vertebrate blood-cell/endothelial cell interactions. Importantly, the carbohydrate function is dispensable for PCP establishment. In vivo studies suggest that Furrowed functions partially redundantly with Fmi, mediating intercellular Frizzled-Vang interactions between neighboring cells.

Implementation of a two-specimen requirement for verification of ABO/Rh for blood transfusion.

BACKGROUND: This study presents our implementation of a two-specimen requirement with no prior record of ABO/Rh to verify patients' blood type before transfusion. MATERIALS AND METHODS: Blood type verification was introduced, discussed, approved, and implemented over a 12-month period (May 2007 to May 2008). Potential barriers and impact on benchmark indicators were identified and tracked. RESULTS: Inpatient identification and/or specimen labeling for nursing and laboratory phlebotomists baseline corrected error rates were 1:467 and 1:5555, respectively. This study therefore sought and obtained approval to initiate a new policy of blood type verification before blood transfusion. Compliance in turnaround time (TAT) before and after implementation for completion of STAT type and screen/crossmatch within 60 minutes worsened marginally, from 90% to 80%. The impact on use of O-, uncrossmatched blood was found to be manageable. Seven (of 25 total) recorded electronic complaints were received after implementation. The corrected error rate for nurse phlebotomy draws after implementation was 1:630. CONCLUSION: Despite the lack of an instigating event, verification of blood type before blood transfusion was successfully implemented. An impact on resources and benchmark indicators such as TAT can be anticipated and managed. Further process improvement efforts will be needed to ensure safety (e.g., at time of blood transfusion) for patients receiving blood transfusions. ABO/Rh verification may be necessary even after future implementation of bar coding and/or RFID chips, because human errors continue to occur even with systems improvements.

Impairment of ubiquitylation by mutation in Drosophila E1 promotes both cell-autonomous and non-cell-autonomous Ras-ERK activation in vivo.

Ras signaling can promote proliferation, cell survival and differentiation. Mutations in components of the Ras pathway are found in many solid tumors and are associated with developmental disorders. We demonstrate here that Drosophila tissues containing hypomorphic mutations in E1, the most upstream enzyme in the ubiquitin pathway, display cell-autonomous upregulation of Ras-ERK activity and Ras-dependent ectopic proliferation. Ubiquitylation is widely accepted to regulate receptor tyrosine kinase (RTK) endocytosis upstream of Ras. However, although the ectopic proliferation of E1 hypomorphs is dramatically suppressed by removing one copy of Ras, removal of the more upstream components Egfr, Grb2 or sos shows no suppression. Thus, decreased ubiquitylation may lead to growth-relevant Ras-ERK activation by failing to regulate a step downstream of RTK endocytosis. We further demonstrate that Drosophila Ras is ubiquitylated. Our findings suggest that Ras ubiquitylation restricts growth and proliferation in vivo. We also report our intriguing observation that complete inactivation of E1 causes non-autonomous activation of Ras-ERK in adjacent tissue, mimicking oncogenic Ras overexpression. We demonstrate that maintaining sufficient E1 function is required both cell autonomously and non-cell autonomously to prevent inappropriate Ras-ERK-dependent growth and proliferation in vivo and may implicate loss of Ras ubiquitylation in developmental disorders and cancer.