A compact and simple method of achieving differential transgene expression by exploiting translational readthrough.

The development of multicistronic vectors enabling differential transgene expression is a goal of gene therapy and poses a significant engineering challenge. Current approaches rely on the insertion of long regulatory sequences that occupy valuable space in vectors, which have a finite and limited packaging capacity. Here we describe a simple method of achieving differential transgene expression by inserting stop codons and translational readthrough motifs (TRMs) to suppress stop codon termination.

Primary ciliogenesis requires the distal appendage component Cep123.

Primary cilium formation is initiated at the distal end of the mother centriole in a highly co-ordinated manner. This requires the capping of the distal end of the mother centriole with a ciliary vesicle and the anchoring of the basal body (mother centriole) to the cell cortex, both of which are mediated by the distal appendages. Here, we show that the distal appendage protein Cep123 (Cep89/CCDC123) is required for the assembly, but not the maintenance, of a primary cilium.

Assessing the localization of centrosomal proteins by PALM/STORM nanoscopy.

The structure of the centrosome was resolved by EM many years ago to reveal a pair of centrioles embedded in a dense network of proteins. More recently, the molecular composition of the centrosome was catalogued by mass spectroscopy and many novel components were identified. Determining precisely where a novel component localizes to within the centrosome remains a challenge, and until now it has required the use of immuno-EM.

Polo-like kinase 4: the odd one out of the family.

Polo-like kinase 4 (PLK4) is a unique member of the Polo-like family of kinases that shares little homology with its siblings and has an essential role in centriole duplication. The turn-over of this kinase must be strictly controlled to prevent centriole amplification. This is achieved, in part, by an autoregulatory mechanism, whereby PLK4 autophosphorylates residues in a PEST sequence located carboxy-terminal to its catalytic domain.

Autophosphorylation of polo-like kinase 4 and its role in centriole duplication.

Centrosome duplication occurs once every cell cycle in a strictly controlled manner. Polo-like kinase 4 (PLK4) is a key regulator of this process whose kinase activity is essential for centriole duplication. Here, we show that PLK4 autophosphorylation of serine S305 is a consequence of kinase activation and enables the active fraction to be identified in the cell.

Chromatin remodeling proteins interact with pericentrin to regulate centrosome integrity.

Pericentrin is an integral centrosomal component that anchors regulatory and structural molecules to centrosomes. In a yeast two-hybrid screen with pericentrin we identified chromodomain helicase DNA-binding protein 4 (CHD4/Mi2beta). CHD4 is part of the multiprotein nucleosome remodeling deacetylase (NuRD) complex.

Centrosomal anchoring of the protein kinase CK1delta mediated by attachment to the large, coiled-coil scaffolding protein CG-NAP/AKAP450.

Protein kinase CK1 (formerly termed casein kinase I) is ubiquitous in eukaryotic cells and comprises a family of as many as 14 isoforms (including splice variants) in mammalian cells. Mammalian CK1delta and CK1epsilon, which are highly related to each other, are enriched at the centrosomes in interphase cells and at the spindle during mitosis. In the present study we have isolated, using the yeast two-hybrid system, a 182 amino acid residue fragment of the centrosomal and golgi N-kinase anchoring protein (CG-NAP, also known as AKAP450), which specifically interacts with CK1delta and CK1epsilon, but not with other CK1 isoforms.