An Autonomous Oscillation Times and Executes Centriole Biogenesis.

The accurate timing and execution of organelle biogenesis is crucial for cell physiology. Centriole biogenesis is regulated by Polo-like kinase 4 (Plk4) and initiates in S-phase when a daughter centriole grows from the side of a pre-existing mother. Here, we show that a Plk4 oscillation at the base of the growing centriole initiates and times centriole biogenesis to ensure that centrioles grow at the right time and to the right size. The Plk4 oscillation is normally entrained to the cell-cycle oscillator but can run autonomously of it-potentially explaining why centrioles can duplicate independently of cell-cycle progression. Mathematical modeling indicates that the Plk4 oscillation can be generated by a time-delayed negative feedback loop in which Plk4 inactivates the interaction with its centriolar receptor through multiple rounds of phosphorylation. We hypothesize that similar organelle-specific oscillations could regulate the timing and execution of organelle biogenesis more generally.

Drosophila Sas-6, Ana2 and Sas-4 self-organise into macromolecular structures that can be used to probe centriole and centrosome assembly.

Centriole assembly requires a small number of conserved proteins. The precise pathway of centriole assembly has been difficult to study, as the lack of any one of the core assembly proteins [Plk4, Ana2 (the homologue of mammalian STIL), Sas-6, Sas-4 (mammalian CPAP) or Asl (mammalian Cep152)] leads to the absence of centrioles. Here, we use Sas-6 and Ana2 particles (SAPs) as a new model to probe the pathway of centriole and centrosome assembly. SAPs form in Drosophila eggs or embryos when Sas-6 and Ana2 are overexpressed. SAP assembly requires Sas-4, but not Plk4, whereas Asl helps to initiate SAP assembly but is not required for SAP growth. Although not centrioles, SAPs recruit and organise many centriole and centrosome components, nucleate microtubules, organise actin structures and compete with endogenous centrosomes to form mitotic spindle poles. SAPs require Asl to efficiently recruit pericentriolar material (PCM), but Spd-2 (the homologue of mammalian Cep192) can promote some PCM assembly independently of Asl. These observations provide new insights into the pathways of centriole and centrosome assembly.

Discovery of BRD4 bromodomain inhibitors by fragment-based high-throughput docking.

Bromodomains (BRDs) recognize acetyl-lysine modified histone tails mediating epigenetic processes. BRD4, a protein containing two bromodomains, has emerged as an attractive therapeutic target for several types of cancer as well as inflammatory diseases. Using a fragment-based in silico screening approach, we identified two small molecules that bind to the first bromodomain of BRD4 with low-micromolar affinity and favorable ligand efficiency (0.37 kcal/mol per non-hydrogen atom), selectively over other families of bromodomains. Notably, the hit rate of the fragment-based in silico approach is about 10% as only 24 putative inhibitors, from an initial library of about 9 million molecules, were tested in vitro.

Centriole growth is limited by the Cdk/Cyclin-dependent phosphorylation of Ana2/STIL.

Centrioles duplicate once per cell cycle, but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. Here, we show that in Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6, and Sas-4 are low, but remain constant throughout the assembly process-indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly toward the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit this diffusion change and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of Ana2 seems to reduce the efficiency of daughter centriole assembly toward the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis.

A combined 3D-SIM/SMLM approach allows centriole proteins to be localized with a precision of ∼4-5 nm.

Centrioles are small barrel-shaped structures that form centrosomes and cilia [1]. Centrioles assemble around a central cartwheel comprising the Sas-6 and Ana2/STIL proteins. The amino termini of nine Sas-6 dimers form a central hub of ∼12 nm radius from which nine dimer spokes radiate, placing the Sas-6 carboxyl termini at the outer edge of the ∼60 nm radius cartwheel [2]. Several centriole proteins are distributed in a toroid around the cartwheel, and super-resolution light microscopy studies have measured the average radii of these ∼100-200 nm radius toroids with a 'precision' - or standard deviation (s.d. or 1σ) - of ±âˆ¼10-40 nm. The organization of Ana2/STIL within the cartwheel, however, has not been resolvable. Here, we develop methods to calculate the average toroidal radius of centriolar proteins in the ∼20-60 nm range with a s.d. of just ±âˆ¼4-5 nm, revealing that the amino and carboxyl termini of Ana2 are located in the outer cartwheel region.

Cdk1 Phosphorylates Drosophila Sas-4 to Recruit Polo to Daughter Centrioles and Convert Them to Centrosomes.

Centrosomes and cilia are organized by a centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic "centriole conversion" is regulated, but it requires Plk1/Polo kinase. Here we show that in flies, Cdk1 phosphorylates the conserved centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block centriole disengagement during mitosis, but block efficient centriole conversion and lead to embryonic lethality. These observations can explain why daughter centrioles have to pass through mitosis before they can duplicate and organize a centrosome.