No abstract

PLK4 is a key regulator of centriole duplication. Here, we show that PLK4 is active beyond the initiation of centriole duplication with the abundance of active kinase increasing to a peak in mitosis. Importantly, we show that differences in PLK4 abundance exist between mother and daughter centrioles and that active PLK4 is restricted to the centrosome.

SummaryPrimary 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.

We studied the effect of inhibition of microsomal triglyceride transfer protein (MTP) on apolipoprotein (apo) B100 translation and secretion using HepG2 cells. The MTP-mediated lipid transfer activity was reduced using a specific MTP inhibitor. ApoB100 translation was synchronized by treatment with puromycin prior to L-[ 35 S]methionine pulse-chase labeling. During the first 4 min of chase, synthesis of apoB polypeptides the size of 100 -200 kDa was insensitive to the inhibitor, suggesting that inhibition of MTP did not affect the initiation of apoB100 translation. After 15 min of chase, the 100 -200-kDa species were chased into polypeptides larger than 320 kDa (i.e. apoB65 or 65% of full-length apoB100) in both control and inhibitor-treated cells. However, the amount of these polypeptides decreased (by 36% for apoB65-75, by 64% for apoB75-85, by 76% for apoB85-95, and by 77% for apoB100) upon MTP inhibition. No accumulation of smaller polypeptides was observed, but total immunoprecipitable apoB radioactivity was decreased suggesting that apoB could undergo cotranslational degradation when MTP activity was reduced. Inhibitors of the multicatalytic proteinase complex (proteasome) such as lactacystin or MG-115 could prevent apoB co-translational degradation. Nevertheless, MG-115 could not avoid the MTP inhibitor decreasing apoB100 secretion but rather induced the accumulation of secretion-incompetent apoB100 in the cell. These results indicate that MTP activity is required during the elongation of apoB100 polypeptides, particularly at the sequences downstream of carboxyl terminus of apoB65. Co-translational degradation might constitute a more general mechanism of early quality control for large or complex proteins.

Triglycerides (TGs), cholesteryl esters (CEs), cholesterol, and phosphatidylcholine have been independently proposed as playing regulatory roles in apoB-100 secretion; the results depended on the cellular model used. In this study, we reinvestigate the role of lipids in apoB-100 production in HepG2 cells and in particular, we clarify the respective roles of intracellular mass and the biosynthesis of lipids in the regulation of apoB-100 production. In a first set of experiments, the pool size of cholesterol, CEs, and TGs was modulated by a 3-day treatment with either lipid precursors or inhibitors of enzymes involved in lipid synthesis. We used simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), 58-035 (an acyl coenzyme A cholesterol acyltransferase inhibitor), 5-tetradecyloxy-2-furancarboxylic acid (TOFA, an inhibitor of fatty acid synthesis), and oleic acid. The secretion rate of apoB-100 was not affected by the large modulation of lipid mass induced by these various pre-treatments. In a second set of experiments, the same lipid modulators were added during a 4-hour labeling period. Simvastatin and 58-035 inhibited cholesterol and CE synthesis without affecting apoB-100 secretion. By contrast, treatment of HepG2 cells with TOFA resulted in the inhibition of TG synthesis and apoB-100 secretion. This effect was highly specific for apoB-100 and was reversed by adding oleic acid, which stimulated both TG synthesis and apoB-100 secretion. Moreover, a combination of oleic acid and 58-035 inhibited CE biosynthesis and increased both TG synthesis and apoB-100 secretion. These results show that in HepG2 cells TG biosynthesis regulates apoB-100 secretion, whereas the rate of cholesterol or CE biosynthesis has no effect.