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.

Antibody purification by affinity chromatography based on small molecule affinity ligands identified by SPR-based screening of chemical microarrays.

Libraries of small molecules were searched for Fc-fragment selective binders to a recombinant human antibody ("MDJ8″, IgG(1)-subtype, κ-light chain) via SPR-based screening of chemical microarrays. Identified hit structures were immobilised on NHS-activated Sepharose for the determination of MDJ8 binding and selectivity versus typical proteineous impurities represented by the spend cell culture supernatant. Columns were packed and the most promising ligands further characterized in terms of binding constants, binding kinetics, as well as dynamic and equilibrium binding capacities. The performance of the best ligand, 2A10, was compared to standard Protein A chromatography. Using ligand 2A10 antibody capture from unprocessed cell culture supernatants was possible at similar recovery yield (>90%), purity (>80%), and eluting concentration (approximately 1 g/L) as with Protein A. Affinity constants (K(d)) of 2A10 were an order of magnitude higher than for the Protein A material, but still in the nM-range, while maximum binding capacities and binding kinetics were in the same order of magnitude. Ligand 2A10 was also able to capture a murine monoclonal antibody, again with similar efficiency as Protein A, as well as a number of humanised therapeutic antibodies. Antibody elution from the 2A10 column was possible using the Protein A standard protocol, i.e. 100mM glycine HCl pH 3.0, but also at near physiological pH, when some organic solvent or modifiers were present. Ligand 2A10 thus constitutes a cheaper, more robust alternative to Protein A as possible generic antibody binder. Moreover, the outlined approach to ligand selection could in principle by used to create suitable affinity ligands for other high value biotech products.

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.

Mutations in centrosomal protein CEP152 in primary microcephaly families linked to MCPH4.

Primary microcephaly is a rare condition in which brain size is substantially diminished without other syndromic abnormalities. Seven autosomal loci have been genetically mapped, and the underlying causal genes have been identified for MCPH1, MCPH3, MCPH5, MCPH6, and MCPH7 but not for MCPH2 or MCPH4. The known genes play roles in mitosis and cell division. We ascertained three families from an Eastern Canadian subpopulation, each with one microcephalic child. Homozygosity analysis in two families using genome-wide dense SNP genotyping supported linkage to the published MCPH4 locus on chromosome 15q21.1. Sequencing of coding exons of candidate genes in the interval identified a nonconservative amino acid change in a highly conserved residue of the centrosomal protein CEP152. The affected children in these two families were both homozygous for this missense variant. The third affected child was compound heterozygous for the missense mutation plus a second, premature-termination mutation truncating a third of the protein and preventing its localization to centrosomes in transfected cells. CEP152 is the putative mammalian ortholog of Drosphila asterless, mutations in which affect mitosis in the fly. Published data from zebrafish are also consistent with a role of CEP152 in centrosome function. By RT-PCR, CEP152 is expressed in the embryonic mouse brain, similar to other MCPH genes. Like some other MCPH genes, CEP152 shows signatures of positive selection in the human lineage. CEP152 is a strong candidate for the causal gene underlying MCPH4 and may be an important gene in the evolution of human brain size.

Gamma-tubulin-containing abnormal centrioles are induced by insufficient Plk4 in human HCT116 colorectal cancer cells.

Cancer cells frequently induce aberrant centrosomes, which have been implicated in cancer initiation and progression. Human colorectal cancer cells, HCT116, contain aberrant centrioles composed of disorganized cylindrical microtubules and displaced appendages. These cells also express unique centrosome-related structures associated with a subset of centrosomal components, including gamma-tubulin, centrin and PCM1. During hydroxyurea treatment, these abnormal structures become more abundant and undergo a change in shape from small dots to elongated fibers. Although gamma-tubulin seems to exist as a ring complex, the abnormal structures do not support microtubule nucleation. Several lines of evidence suggest that the fibers correspond to a disorganized form of centriolar microtubules. Plk4, a mammalian homolog of ZYG-1 essential for initiation of centriole biogenesis, is not associated with the gamma-tubulin-specific abnormal centrosomes. The amount of Plk4 at each centrosome was less in cells with abnormal centrosomes than cells without gamma-tubulin-specific abnormal centrosomes. In addition, the formation of abnormal structures was abolished by expression of exogenous Plk4, but not SAS6 and Cep135/Bld10p, which are downstream regulators required for the organization of nine-triplet microtubules. These results suggest that HCT116 cells fail to organize the ninefold symmetry of centrioles due to insufficient Plk4.

Plk2 regulated centriole duplication is dependent on its localization to the centrioles and a functional polo-box domain.

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The centrosome duplicates once per cell cycle. Polo like kinases (Plks) perform crucial functions in cell cycle progression and during mitosis. The polo-like kinase-2, Plk2, is activated near the G(1)/S phase transition, and plays an important role in the reproduction of centrosomes. In this study, we show that the polo-box of Plk2 is required both for association to the centrosome and centriole duplication. Mutation of critical sites in the Plk2 polo-box prevents centrosomal localization and impairs centriole duplication. Plk2 is localized to centrosomes during early G(1) phase where it only associates to the mother centriole and then distributes equally to both mother and daughter centrioles at the onset of S phase. Furthermore, our results imply that Plk2 mediated centriole duplication is dependent on Plk4 function. In addition, we find that siRNA-mediated downregulation of Plk2 leads to the formation of abnormal mitotic spindles confirming that Plk2 may have a function in the reproduction of centrioles.

Multiple importins function as nuclear transport receptors for the Rev protein of human immunodeficiency virus type 1.

The Rev protein of human immunodeficiency virus type 1 is an RNA-binding protein that is required for nuclear export of unspliced and partially spliced viral mRNAs. Nuclear import of human immunodeficiency virus type 1 Rev has been suggested to depend on the classic nuclear transport receptor importin beta, but not on the adapter protein importin alpha. We now show that, similar to importin alpha, Rev is able to dissociate RanGTP from recycling importin beta, a reaction that leads to the formation of a novel import complex. Besides importin beta, the transport receptors transportin, importin 5, and importin 7 specifically interact with Rev and promote its nuclear import in digitonin-permeabilized cells. A single arginine-rich nuclear localization sequence of Rev is required for interaction with all importins tested so far. In contrast to the importin beta-binding domain of importin alpha, Rev interacts with an N-terminal fragment of importin beta. Transportin contains two independent binding sites for Rev. Hence, the mode of interaction of importin beta and transportin with Rev is clearly distinct from that with their classic import cargoes. Taken together, the viral protein takes advantage of multiple cellular transport pathways for its nuclear accumulation.

Transportin is a major nuclear import receptor for c-Fos: a novel mode of cargo interaction.

c-Fos, a component of the transcription factor AP-1, is rapidly imported into the nucleus after translation. We established an in vitro system using digitonin-permeabilized cells to analyze nuclear import of c-Fos in detail. Two import receptors of the importin beta superfamily, importin beta itself and transportin, promote import of c-Fos in vitro. Under conditions where importin beta-dependent transport was blocked, c-Fos still accumulated in the nucleus in the presence of cytosol. Inhibition of the transportin-dependent pathway, in contrast, abolished import of c-Fos. Furthermore, c-Fos mutants that interact with transportin but not with importin beta were efficiently imported in the presence of cytosol. Hence, transportin appears to be the predominant import receptor for c-Fos. A detailed biochemical characterization revealed that the interaction of transportin with c-Fos is distinct from the interaction with its established import cargoes, the M9 sequence of heterogeneous nuclear ribonucleoprotein A1 or the nuclear localization sequence of some basic proteins. Likewise, the binding sites on importin beta for its classic import cargo and for c-Fos can be separated. In summary, c-Fos employs a novel mode of receptor-cargo interaction. Hence, transportin may be as versatile as importin beta in recognizing different nuclear import cargoes.

The UCS domain protein She4p binds to myosin motor domains and is essential for class I and class V myosin function.

BACKGROUND: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. RESULTS: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. CONCLUSIONS: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.