Promoter hijack reveals pericentrin functions in mitosis and the DNA damage response.

Centrosomes, the principal microtubule-organizing centers of animal somatic cells, consist of two centrioles embedded in the pericentriolar material (PCM). Pericentrin is a large PCM protein that is required for normal PCM assembly. Mutations in PCNT cause primordial dwarfism. Pericentrin has also been implicated in the control of DNA damage responses. To test how pericentrin is involved in cell cycle control after genotoxic stress, we disrupted the Pcnt locus in chicken DT40 cells. Pericentrin-deficient cells proceeded through mitosis more slowly, with a high level of monopolar spindles, and were more sensitive to spindle poisons than controls. Centriole structures appeared normal by light and electron microscopy, but the PCM did not recruit γ-tubulin efficiently. Cell cycle delays after ionizing radiation (IR) treatment were normal in pericentrin-deficient cells. However, pericentrin disruption in Mcph1-/- cells abrogated centrosome hyperamplification after IR. We conclude that pericentrin controls genomic stability by both ensuring appropriate mitotic spindle activity and centrosome regulation.

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.

Oscillation of APC/C activity during cell cycle arrest promotes centrosome amplification.

Centrosome duplication is licensed by the disengagement, or 'uncoupling', of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the anaphase-promoting complex (APC/C), leading to securin degradation and release of active separase. Although APC/C activation during G2 arrest is dependent on polo-like kinase 1 (Plk1)-mediated degradation of the APC/C inhibitor, early mitotic inhibitor 1 (Emi1), Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulates centriole disengagement in response to hydroxyurea or DNA damage-induced cell-cycle arrest and this leads to centrosome amplification. However, the reduplication of disengaged centrioles is dependent on cyclin-dependent kinase 2 (Cdk2) activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Although release from these arrests leads to mitotic entry, the presence of disengaged and/or amplified centrosomes results in the formation of abnormal mitotic spindles that lead to chromosome mis-segregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability.

A centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage.

DNA damage can induce centrosome overduplication in a manner that requires G2-to-M checkpoint function, suggesting that genotoxic stress can decouple the centrosome and chromosome cycles. How this happens is unclear. Using live-cell imaging of cells that express fluorescently tagged NEDD1/GCP-WD and proliferating cell nuclear antigen, we found that ionizing radiation (IR)-induced centrosome amplification can occur outside S phase. Analysis of synchronized populations showed that significantly more centrosome amplification occurred after irradiation of G2-enriched populations compared with G1-enriched or asynchronous cells, consistent with G2 phase centrosome amplification. Irradiated and control populations of G2 cells were then fused to test whether centrosome overduplication is allowed through a diffusible stimulatory signal, or the loss of a duplication-inhibiting signal. Irradiated G2/irradiated G2 cell fusions showed significantly higher centrosome amplification levels than irradiated G2/unirradiated G2 fusions. Chicken-human cell fusions demonstrated that centrosome amplification was limited to the irradiated partner. Our finding that only the irradiated centrosome can duplicate supports a model where a centrosome-autonomous inhibitory signal is lost upon irradiation of G2 cells. We observed centriole disengagement after irradiation. Although overexpression of dominant-negative securin did not affect IR-induced centrosome amplification, Plk1 inhibition reduced radiation-induced amplification. Together, our data support centriole disengagement as a licensing signal for DNA damage-induced centrosome amplification.

Cellular infrared detector appears to be contained in the centrosome.

Previous experiments have suggested that 3T3 cells were able to extend pseudopodia toward latex particles up to 60 microns away from the cell body if the particles were irradiated by an infrared beam in the range of 700-900 nm [Albrecht-Buehler, 1991: J. Cell Biol. 114:493-502]. The present article reports that this response of cells to infrared light can be inhibited if the cell center is simultaneously irradiated with a beam of the same light. In marked contrast, the cells responded normally to the presence of infrared light scattering particles if the second beam irradiated other parts of the cell body. The results imply that the cellular mechanism of infrared detection is located at the cell center. The infrared sensing mechanism remains intact in enucleated cells and in cells which were incubated in monensin to vesiculate their Golgi apparatus and inhibit their Golgi functions. Accordingly, it is proposed that the centrosome which contains the centrioles is the only remaining candidate in the cell center for a cellular detection device for the direction of infrared signal sources. The results support an earlier suggestion that centrioles may be such detection devices [Albrecht-Buehler, 1981: Cell Motil. Cytoskeleton 1:237-245].

Laser irradiation of centrosomes in newt eosinophils: evidence of centriole role in motility.

Newt eosinophils are motile granulated leukocytes that uniquely display a highly visible centrosomal area. Electron microscope and tubulin antibody fluorescence confirms the presence of centrioles, pericentriolar material, and radiating microtubules within this visible area. Actin antibodies intensely stain the advancing cell edges and tail but only weakly stain pseudopods being withdrawn into the cell. Randomly activated eosinophils follow a roughly consistent direction with an average rate of 22.5 micron/min. The position of the centrosome is always located between the trailing cell nucleus and advancing cell edge. If the cell extends more than one pseudopod, the one closest to or containing the centrosome is always the one in which motility continues. Laser irradiation of the visible centrosomal area resulted in rapid cell rounding. After several minutes following irradiation, most cells flattened and movement continued. However, postirradiation motility was uncoordinated and directionless , and the rate decreased to an average of 14.5 micron/min. Electron microscopy and tubulin immunofluorescence indicated that an initial disorganization of microtubules resulted from the laser microirradiations . After several minutes, organized microtubules reappeared, but the centrioles appeared increasingly damaged. The irregularities in motility due to irradiation are probably related to the damaged centrioles. The results presented in this paper suggest that the centrosome is an important structure in controlling the rate and direction of newt eosinophil motility.

Microtubule-organizing centers abnormal in number, structure, and nucleating activity in x-irradiated mammalian cells.

Microtubule-organizing centers (MTOCs) in x-irradiated cells were visualized by immunofluorescence using antibody against tubulin. From two to ten reassembly sites of microtubules appeared after microtubule depolymerization at low temperature in an irradiated mitotic cell, in contrast to nonirradiated mitotic cells, which predominantly show 2 MTOCs. A time-course examination of MTOCs in synchronously cultured cells revealed that the multiple MTOCs appeared not immediately after irradiation but at the time of mitosis. Those multiple MTOCs formed at mitosis were inherited by the daughter cells in the next generation. The structure and capacity of the centrosomes to nucleate microtubules in vitro were then examined by electron microscopy of whole-mount preparations as well as by dark-field microscopy. About 70-80% of the centrosomes derived from nonirradiated cells were composed of a pair of centrioles and pericentriolar material, which initiated greater than 100 microtubules. The fraction of fully active complete centrosomes decreased with time of incubation after irradiation. These were replaced by disintegrated centrosomal components such as dissociated centrioles and pericentriolar cloud, a nucleating site with a single centriole, or only an amorphous structure of pericentriolar cloud. Assembly of less than 20 microtubules onto the amorphous cloud without centrioles was seen in 54% of the initiating sites in mitotic cells 2 d after irradiation. These results suggest that x-irradiation causes disintegration of centrosomes at mitosis when the structural and functional reorganization of centrosomes is believed to occur.

[Selectivity and localized nature of exposure in laser microirradiation of cells]. / O selektivnosti i lokal'nosti vozdeistviia pri lazernom mikroobluchenii kletok.

Some modern applications of laser microirradiation to studies of relationship between structure and function are reported. The main advantage of the method is the ability to act on various subcellular parts and organelles, both locally and selectively. However, the degrees of laser microirradiation locality and selectivity are not always sufficiently high. These properties of laser microirradiation are analysed in addition to factors of deteriorating and improving the methods. It is assumed that the most important influence in cellular response to microirradiation is exerted by laser on different cellular membrane systems, particularly on the surface membrane and mitochondria.

Evidence for centriolar region RNA functioning in spindle formation in dividing PTK2 cells.

The light-activated, nucleic acid-binding drugs, psoralens, were used in conjunction with a 365-nm laser microbeam to selectively bind to any nucleic acids in the centriolar region. 4'-aminomethyl-4,5',8--trimethyl-psoralen (AMT) has a high affinity for both RNA and DNA and can be shown to cause mitotic abortion when centriolar regions of prophase PTK2 cells and reacted with AMT and 365-nm laser light. Other psoralen derivatives which have a high affinity for DNA and a low affinity for RNA are not effective in blocking mitosis in dividing PTK2 cells. Examination of psoralen-bound centriolar regions by single-cell electron microscopy shows that at various times after treatment, the number of microtubules associated with the irradiated poles is much lower than in normal, dividing cells. Light-activated psoralen binding of the centriolar regions does not seem to affect the condensation or structure of mitotic chromosomes. It is concluded that there is an RNA in the centriolar region that is responsible for the formation of the spindle in dividing cells.