Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP.

Centrosomes have many important functions and comprise a 'mother' and 'daughter' centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic 'S-CAP' complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes.PTC was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (105653/Z/14/Z) and by an Issac Newton Trust Research Grant from the University of Cambridge awarded to TTW (RG78799). AW, ZN and JWR were supported by a Senior Investigator Award awarded to JWR and funded by the Wellcome Trust (104575/Z/14/Z). The OMX microscope used in this study is part of the Oxford Micron Advanced Bioimaging Unit supported by a Wellcome Trust Strategic Award (091911).This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.0848

Structural Basis for Mitotic Centrosome Assembly in Flies.

In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.Z.F. and A.F.M.H. were supported by Sir William Dunn School EPA PhD studentships and also a Clarendon Scholarship and a Santander Graduate Award to A.F.M.H; A.C., A.W., M.A.C., P.T.C., and J.W.R. were supported by a Wellcome Trust Senior Investigator Award (104575); S.J. and S.M.L. were supported by a Wellcome Trust Senior Investigator Award (100298); A.W. was also partially supported by a Wellcome Trust Strategic Award to the Micron Oxford Advanced Bioimaging Unit (107457)

Evidence that a positive feedback loop drives centrosome maturation in fly embryos.

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. Here, we show that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. Our findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos

Home and Online Management and Evaluation of Blood Pressure (HOME BP) using a digital intervention in poorly controlled hypertension: randomised controlled trial

Objective: The HOME BP (Home and Online Management and Evaluation of Blood Pressure) trial aimed to test a digital intervention for hypertension management in primary care by combining self-monitoring of blood pressure with guided self-management. Design: Unmasked randomised controlled trial with automated ascertainment of primary endpoint. Setting: 76 general practices in the United Kingdom. Participants: 622 people with treated but poorly controlled hypertension (>140/90 mm Hg) and access to the internet. Interventions: Participants were randomised by using a minimisation algorithm to self-monitoring of blood pressure with a digital intervention (305 participants) or usual care (routine hypertension care, with appointments and drug changes made at the discretion of the general practitioner; 317 participants). The digital intervention provided feedback of blood pressure results to patients and professionals with optional lifestyle advice and motivational support. Target blood pressure for hypertension, diabetes, and people aged 80 or older followed UK national guidelines. Main outcome measures: The primary outcome was the difference in systolic blood pressure (mean of second and third readings) after one year, adjusted for baseline blood pressure, blood pressure target, age, and practice, with multiple imputation for missing values. Results: After one year, data were available from 552 participants (88.6%) with imputation for the remaining 70 participants (11.4%). Mean blood pressure dropped from 151.7/86.4 to 138.4/80.2 mm Hg in the intervention group and from 151.6/85.3 to 141.8/79.8 mm Hg in the usual care group, giving a mean difference in systolic blood pressure of −3.4 mm Hg (95% confidence interval −6.1 to −0.8 mm Hg) and a mean difference in diastolic blood pressure of −0.5 mm Hg (−1.9 to 0.9 mm Hg). Results were comparable in the complete case analysis and adverse effects were similar between groups. Within trial costs showed an incremental cost effectiveness ratio of £11 ($15, €12; 95% confidence interval £6 to £29) per mm Hg reduction. Conclusions: The HOME BP digital intervention for the management of hypertension by using self-monitored blood pressure led to better control of systolic blood pressure after one year than usual care, with low incremental costs. Implementation in primary care will require integration into clinical workflows and consideration of people who are digitally excluded. Trial registration: ISRCTN13790648