Cep78 is a new centriolar protein involved in Plk4-induced centriole overduplication.

Centrioles are core components of centrosomes, the major microtubule-organizing centers of animal cells, and act as basal bodies for cilia formation. Control of centriole number is therefore crucial for genome stability and embryogenesis. Centriole duplication requires the serine/threonine protein kinase Plk4. Here, we identify Cep78 as a human centrosomal protein and a new interaction partner of Plk4. Cep78 is mainly a centriolar protein that localizes to the centriolar wall. Furthermore, we find that Plk4 binds to Cep78 through its N-terminal domain but that Cep78 is not an in vitro Plk4 substrate. Cep78 colocalizes with Plk4 at centrioles and is required for Plk4-induced centriole overduplication. Interestingly, upon depletion of Cep78, newly synthesized Plk4 is not localized to centrosomes. Our results suggest that the interaction between Cep78 and the N-terminal catalytic domain of Plk4 is a new and important element in the centrosome overduplication process.

GCP6 is a substrate of Plk4 and required for centriole duplication.

Centriole duplication occurs once per cell cycle and requires Plk4, a member of the Polo-like kinase family. A key component of the centrosome is the γ-tubulin ring complex (γ-TuRC) that nucleates microtubules. GCP6 is a member of the γ-TuRC, but its role in human cells and the regulation of its functions remain unclear. Here we report that depletion of human GCP6 prevents assembly of the γ-TuRC and induces a high percentage of monopolar spindles. These spindles are characterized by a loss of centrosomal γ-tubulin and reduced centriole numbers. We found that GCP6 is localized in the pericentriolar material but also at distal portions of centrioles. In addition, GCP6 is required for centriole duplication and Plk4-induced centriole overduplication. GCP6 interacts with and is phosphorylated by Plk4. Moreover, we find that Plk4-dependent phosphorylation of GCP6 regulates centriole duplication. These data suggest that GCP6 is a target of Plk4 in centriole biogenesis.

Quantitative analysis of cytoskeletal reorganization during epithelial tissue sealing by large-volume electron tomography.

The closure of epidermal openings is an essential biological process that causes major developmental problems such as spina bifida in humans if it goes awry. At present, the mechanism of closure remains elusive. Therefore, we reconstructed a model closure event, dorsal closure in fly embryos, by large-volume correlative electron tomography. We present a comprehensive, quantitative analysis of the cytoskeletal reorganization, enabling separated epidermal cells to seal the epithelium. After establishing contact through actin-driven exploratory filopodia, cells use a single lamella to generate 'roof tile'-like overlaps. These shorten to produce the force, 'zipping' the tissue closed. The shortening overlaps lack detectable actin filament ensembles but are crowded with microtubules. Cortical accumulation of shrinking microtubule ends suggests a force generation mechanism in which cortical motors pull on microtubule ends as for mitotic spindle positioning. In addition, microtubules orient filopodia and lamellae before zipping. Our 4D electron microscopy picture describes an entire developmental process and provides fundamental insight into epidermal closure.

Cep152 acts as a scaffold for recruitment of Plk4 and CPAP to the centrosome.

Both gain and loss of function studies have identified the Polo-like kinase Plk4/Sak as a crucial regulator of centriole biogenesis, but the mechanisms governing centrosome duplication are incompletely understood. In this study, we show that the pericentriolar material protein, Cep152, interacts with the distinctive cryptic Polo-box of Plk4 via its N-terminal domain and is required for Plk4-induced centriole overduplication. Reduction of endogenous Cep152 levels results in a failure in centriole duplication, loss of centrioles, and formation of monopolar mitotic spindles. Interfering with Cep152 function prevents recruitment of Plk4 to the centrosome and promotes loss of CPAP, a protein required for the control of centriole length in Plk4-regulated centriole biogenesis. Our results suggest that Cep152 recruits Plk4 and CPAP to the centrosome to ensure a faithful centrosome duplication process.

Characterization of a novel type of adherens junction in meningiomas and the derived cell line HBL-52.

Adhering junctions are generally grouped into desmosomes and adherens junctions based on their ultrastructural appearance and molecular composition. The armadillo-protein plakoglobin is common to both types of junctions, which are otherwise composed of mutually exclusive proteins. This view is based on observations in epithelial tissues but cannot easily be transferred to other cell types and tissues, as has become apparent during the last decade with the identification of new junctional proteins and the investigation of further non-epithelial junctions. Using a broad array of well-characterized specific antibodies against key junctional proteins in immunoblot reactions, high-resolution double-label laser scanning confocal microscopy, and immunoelectron microscopy, we describe a new type of adherens junction in human meningiomas and the human meningioma cell line HBL-52. This novel junction has a unique composition of proteins not found in any other tissue; it contains the desmosomal armadillo-protein plakophilin 2 together with the classic proteins of "epithelial" adherens junctions, i.e., E-cadherin (in some instances replaced by N-cadherin), alpha-catenin, beta-catenin, plakoglobin, and p120(ctn). Ultrastructurally, it is formed between two or three neighboring cells. For pragmatic reasons, we suggest the name "meningeal junction" for this new structure.

Plastic-embedded protein crystals.

Rapid vitrification followed by the replacement of the vitrified water by a solvent (freeze substitution) and then resin is a widely used procedure for preparing biological samples for electron microscopy. The resulting plastic-embedded samples permit convenient room-temperature sectioning (microtomy) and can yield well preserved cellular structures. Here this procedure has been applied to crystalline protein samples, and it is shown that it is possible to freeze-substitute vitrified crystals while preserving some of their original diffraction properties. The plastic-embedded crystals were used to collect a series of complete room-temperature data sets at a powerful macromolecular crystallography synchrotron beamline. Whereas one normally observes specific damage to disulfide bonds upon X-ray radiation, no such damage was seen for the plastic-embedded sample. The X-ray diffraction data allowed an initial atomic analysis to be made of the effects of freeze-substitution and plastic embedding on biological samples.