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. TRMs reduced downstream transgene expression ∼sixfold to ∼140-fold, depending on the combination of stop codon and TRM used. We show that a TRM can facilitate the controlled secretion of the highly potent cytokine IL-12 at therapeutically beneficial levels in an aggressive immunocompetent mouse melanoma model to prevent tumor growth. Given their compact size (6 bp) and ease of introduction, we envisage that TRMs will be widely adopted in recombinant DNA engineering to facilitate differential transgene expression.

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. In the absence of Cep123 ciliary vesicle formation fails, suggesting that it functions in the early stages of primary ciliogenesis. Consistent with such a role, Cep123 interacts with the centriolar satellite proteins PCM-1, Cep290 and OFD1, all of which play a role in primary ciliogenesis. These interactions are mediated by a domain in the C-terminus of Cep123 (400-783) that overlaps the distal appendage-targeting domain (500-600). Together, the data implicate Cep123 as a new player in the primary ciliogenesis pathway and expand upon the role of the distal appendages in this process.

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. This technique is both time-consuming and unreliable, as it often fails due to problems with antigen accessibility. We have investigated the use of two nanoscopic techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), as alternative techniques for localizing centrosomal proteins. The localization of a known centrosomal component, the distal appendage protein Cep164 was investigated by direct STORM (dSTORM) and resolved with a high spatial resolution. We further validated the use of nanoscopic PALM imaging by showing that the previously uncharacterized centrosomal protein CCDC123 (Cep123) localizes to the distal appendages, forming ring-like structures with a diameter of 500 nm. Our results demonstrate that both PALM and STORM imaging have great potential as alternatives to 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. Phosphorylated PLK4 is subsequently recognized by the SCF complex, ubiquitinylated and targeted to the proteasome for degradation. Recent data have also shown that active PLK4 is restricted to the centrosome, a mechanism that could serve to prevent aberrant centriole assembly elsewhere in the cell. While significant advances have been made in understanding how PLK4 is regulated it is certain that additional regulatory mechanisms exist to safeguard the fidelity of centriole duplication. Here, we overview past and present data discussing the regulation and functions of PLK4.

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. Active PLK4 is detectable on the replicating mother centriole in G1/S, with the proportion of active kinase increasing through interphase to reach a maximum in mitosis. Activation of PLK4 at the replicating daughter centriole is delayed until G2, but a level equivalent to the replicating mother centriole is achieved in M phase. Active PLK4 is regulated by the proteasome, because either proteasome inhibition or mutation of the degron motif of PLK4 results in the accumulation of S305-phosphorylated PLK4. Autophosphorylation probably plays a role in the process of centriole duplication, because mimicking S305 phosphorylation enhances the ability of overexpressed PLK4 to induce centriole amplification. Importantly, we show that S305-phosphorylated PLK4 is specifically sequestered at the centrosome contrary to the nonphosphorylated form. These data suggest that PLK4 activity is restricted to the centrosome to prevent aberrant centriole assembly and sustained kinase activity is required for centriole duplication.

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. The 182 amino acid residue CG-NAP fragment, or full length CG-NAP, co-immunoprecipitates with CK1delta and CK1epsilon from mammalian cells. Consistent with this association, endogenous CG-NAP/AKAP450 and CK1delta co-localize in cells. Moreover, when expressed in the presence of CK1delta the 182 amino acid residue CG-NAP fragment adopts the same sub-cellular localization as CK1delta. Strikingly, attachment of the CG-NAP fragment to the plasma membrane is sufficient to re-localize a significant level of CK1delta to the membrane. These findings support a model in which sub-cellular localization of CK1delta/epsilon molecules at the centrosome is mediated, at least in part, through the action of CG-NAP/AKAP450 and provide a potential mechanism by which the contribution to cell cycle progression by CK1delta/epsilon may be regulated.