Centrosomal and Non-centrosomal Functions Emerged through Eliminating Centrosomes.

Centrosomes are highly conserved organelles that act as the major microtubule-organizing center (MTOC) in animal somatic cells. Through their MTOC activity, centrosomes play various roles throughout the cell cycle, such as supporting cell migration in interphase and spindle organization and positioning in mitosis. Various approaches for removing centrosomes from somatic cells have been developed and applied over the past few decades to understand the precise roles of centrosomes. Centrinone, a reversible and selective PLK4 (polo-like kinase 4) inhibitor, has recently emerged as an efficient approach to eliminate centrosomes. In this review, we describe the latest findings on centrosome function that have been revealed using various centrosome-eliminating approaches. In addition, we discuss our recent findings on the mechanism of centrosome-independent spindle bipolarization, discovered through the use of centrinone.Key words: centrosome, centrinone, mitotic spindle, bipolarity, NuMA.

NuMA assemblies organize microtubule asters to establish spindle bipolarity in acentrosomal human cells.

In most animal cells, mitotic spindle formation is mediated by coordination of centrosomal and acentrosomal pathways. At the onset of mitosis, centrosomes promote spindle bipolarization. However, the mechanism through which the acentrosomal pathways facilitate the establishment of spindle bipolarity in early mitosis is not completely understood. In this study, we show the critical roles of nuclear mitotic apparatus protein (NuMA) in the generation of spindle bipolarity in acentrosomal human cells. In acentrosomal human cells, we found that small microtubule asters containing NuMA formed at the time of nuclear envelope breakdown. In addition, these asters were assembled by dynein and the clustering activity of NuMA. Subsequently, NuMA organized the radial array of microtubules, which incorporates Eg5, and thus facilitated spindle bipolarization. Importantly, in cells with centrosomes, we also found that NuMA promoted the initial step of spindle bipolarization in early mitosis. Overall, these data suggest that canonical centrosomal and NuMA-mediated acentrosomal pathways redundantly promote spindle bipolarity in human cells.

Feedback loops in the Plk4-STIL-HsSAS6 network coordinate site selection for procentriole formation.

Centrioles are duplicated once in every cell cycle, ensuring the bipolarity of the mitotic spindle. How the core components cooperate to achieve high fidelity in centriole duplication remains poorly understood. By live-cell imaging of endogenously tagged proteins in human cells throughout the entire cell cycle, we quantitatively tracked the dynamics of the critical duplication factors: Plk4, STIL and HsSAS6. Centriolar Plk4 peaks and then starts decreasing during the late G1 phase, which coincides with the accumulation of STIL at centrioles. Shortly thereafter, the HsSAS6 level increases steeply at the procentriole assembly site. We also show that both STIL and HsSAS6 are necessary for attenuating Plk4 levels. Furthermore, our mathematical modeling and simulation suggest that the STIL-HsSAS6 complex in the cartwheel has a negative feedback effect on centriolar Plk4. Combined, these findings illustrate how the dynamic behavior of and interactions between critical duplication factors coordinate the centriole-duplication process.This article has an associated First Person interview with the first author of the paper.

[Tracheal intubation in patients with Williams syndrome].

Williams syndrome is caused by the deletion of genetic material from chromosome 7, including the elastin gene. Affected individuals frequently have connective tissue disorders and skeletal hypoplasia, resulting in micrognathia and mandibular retrusion. These conditions hinder adequate visualization of the larynx and render intubation difficult. We used different methods of tracheal intubation for two patients with Williams syndrome. The first case was a 35-year-old woman with micrognathia, mandibular retrusion, and a Mallampati class III airway, scheduled for mitral valve replacement. She had a three fingerbreadth oral aperture, 3.0 cm mandibulohyoid distance, and 30 degree neck flexion. Since she had previously been successfully intubated at age 18 for patch enlargement of the aorta and extended aortoplasty, we elected to use intravenous anesthetic agents for this intubation. After administration of diazepam and fentanyl, mask ventilation was difficult and the direct laryngoscopic view was Cormack grade IV. Fortunately, blind orotracheal intubation using a Mallinckrodt tracheal tube with direct laryngoscopy was accomplished on the first attempt. The second case was a 71-year-old woman with micrognathia, slight mandibular retrusion, macroglossia, and a Mallampati class IV airway, scheduled for sigmoidectomy and insertion of a ureteral stent. She had a 2.5 fingerbreadth oral aperture, 4.0 cm mandibulohyoid distance, and 30 degree neck flexion. From our experience with the first patient, we expected difficult ventilation and intubation. After 8% lidocaine spray to the larynx, and then 4% lidocaine spray to the vocal cords and trachea using a bronchoscope, we accomplished awake, fibreoptic-guided orotracheal intubation easily and quickly on the first attempt using a Parker Flex-Tip tube. Difficult tracheal intubation should be anticipated in Williams syndrome patients. Awake, fibreoptic-guided technique is easier and safer than direct laryngoscopy for intubating such patients.