Kinetic analysis of de novo centriole assembly in heat-shocked mammalian cells.

Mammalian cells are capable of de novo centriole formation after the removal of existing centrioles. This suggests that de novo centriole assembly is repressed in normally duplicating cells to maintain a constant number of centrioles in the cells. However, neither the mechanism of de novo centriole assembly nor that of its hypothesized repression is understood due to the lack of an experimental system. We found that the heat shock (HS; 42°C, 2 h) of mouse embryonic fibroblasts caused the separation of centriole pairs, a transient increase in polo-like kinase (Plk) 4 expression, and the formation of a complex containing γ-tubulin, pericentrin, HS protein (Hsp) 90, and Plk4, in approximately half of the cells. Subsequently, spindle-assembly abnormal protein (Sas) 6, centrosomal protein (Cep) 135, and centrin localized to the complex, and tubulin consequently became polyglutamylated, indicating de novo centriole assembly in the heat-shocked cells. These results suggested that HS-induced de novo centriole assembly could provide an experimental system for further elucidating the regulation of centrosome number in mammalian cells. © 2016 Wiley Periodicals, Inc.

UNC119a bridges the transmission of Fyn signals to Rab11, leading to the completion of cytokinesis.

Src family kinases (SFKs) regulate the completion of cytokinesis through signal transduction pathways that lead to the Rab11-dependent phosphorylation of ERK and its localization to the midbody of cytokinetic cells. We find that UNC119a, a known activator of SFKs, plays essential roles in this signaling pathway. UNC119a localizes to the centrosome in interphase cells and begins to translocate from the spindle pole to the spindle midzone after the onset of mitosis; it then localizes to the intercellular bridge in telophase cells and to the midbody in cytokinetic cells. We show that the midbody localization of UNC119a is dependent on Rab11, and that knocking down UNC119a inhibits the Rab11-dependent phosphorylation and midbody localization of ERK and cytokinesis. Moreover, we demonstrate that UNC119a interacts with a Src family kinase, Fyn and is required for the activation of this kinase. These results suggest that UNC119a plays a key role in the Fyn signal transduction pathway, which regulates the completion of cytokinesis via Rab11.

Coordinate synthesis but discrete localization of homologous N-glycosylated proteins, CLP and CLB, in Naegleria pringsheimi flagellates.

The synchronous amoebae-to-flagellates differentiation of Naegleria pringsheimi has been used as a model system to study the formation of eukaryotic flagella. We cloned two novel genes, Clp, Class I on plasma membrane and Clb, Class I at basal bodies, which are transiently expressed during differentiation and characterized their respective protein products. CLP (2,087 amino acids) and CLB (1,952 amino acids) have 82.9% identity in their amino acid sequences and are heavily N-glycosylated, leading to an ~ 100 × 10(3) increase in the relative molecular mass of the native proteins. In spite of these similarities, CLP and CLB were localized to distinct regions: CLP was present on the outer surface of the plasma membrane, whereas CLB was concentrated at a site where the basal bodies are assembled and remained associated with the basal bodies. Oryzalin, a microtubule toxin, inhibited the appearance of CLP on the plasma membrane, but had no effect on the concentration of CLB at its target site. These data suggest that N. pringsheimi uses separate mechanisms to transport CLP and CLB to the plasma membrane and to the site of basal body assembly, respectively.