Science 283, 851C854 [PubMed] [Google Scholar] 11

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Science 283, 851C854 [PubMed] [Google Scholar] 11. Interestingly, inhibition of the proteasome in centrobin-depleted cells restored the cellular and centriolar CPAP expression, suggesting its ubiquitination and proteasome-mediated degradation when centrobin is absent. Intriguingly, however, centrobin-overexpressing cells also showed proteasome-independent accumulation of ubiquitinated CPAP and abnormal, ubiquitin-positive, elongated centrioles. Overall, our results show that centrobin interacts with ubiquitinated CPAP and prevents its degradation for normal centriole elongation function. Therefore, it appears that loss of centrobin expression destabilizes CPAP and triggers its degradation to restrict the centriole length during biogenesis. value was done using Student’s test. The denotes that the results are significant. RESULTS Overexpression of Centrobin Results in Abnormal, Long Centriole-like Structures To understand the mechanism by which centrobin contributes to centriole elongation, U2OS cells were transfected with control or myc-tagged centrobin expression vector for 72 h, and the centriole length was determined in myc-positive cells. For better staining of the centrioles, cells were placed on ice to depolymerize the bulk of cytoskeletal microtubules and then extracted with a detergent-containing buffer as described under Experimental Procedures. Cells were then fixed with ice-cold methanol and stained using anti–tubulin, -myc, and -centrin antibodies for immunofluorescence microscopy. Confocal microscopy imaging revealed that in comparison with the centrioles of control cells, centrobin-overexpressing cells had abnormal, long centriolar structures (Fig. 1depicts the percentage of myc-positive centrioles that showed abnormal elongation. Results represent three independent experiments with 50 cells examined/experiment. < 0.0001. shows that the centrobin-overexpressing, but not control cells, have a massive accumulation of the CPAP protein. On the other hand, the cellular level of CP110 in centrobin-overexpressing cells, albeit relatively higher than control, was not as profoundly different as CPAP levels, suggesting that centrobin-overexpression has a more robust effect on CPAP protein levels. Open in a separate window FIGURE 2. Centrobin overexpression results in increased cellular CPAP but not CP110 and hSAS-6 levels. and shows that endogenous CPAP was undetectable in centrobin-depleted cells, whereas the cellular level of centrin, a centriolar marker, was not affected considerably upon centrobin depletion (Fig. 3demonstrates that centrobin knockdown resulted in the loss of endogenous CPAP, similar to Fig. 3, and demonstrates that although robust expression of the exogenously delivered myc-CPAP was seen in control cells, relatively lower levels of CPAP were detected in the centrobin shRNA-expressing cells. This confirms that centrobin at least partially regulates the stability and persistence of CPAP in cells. In addition, expression of centrobin-365C903 did not restore the CPAP expression in centrobin-depleted cells (Fig. 3abnormal elongation of centrioles can occur upon inhibition of the proteasome activity (57). Because inhibition of the proteasome degradation pathway restored CPAP expression to the centrioles in centrobin-depleted cells (Fig. 4and of Fig. 5using centrobin-365C903 expression vector. shows that although the anti-HA antibody did not stain the centrioles, centrobin-overexpressing cells showed high levels of HA staining on the centrioles indicating centriolar Gamitrinib TPP hexafluorophosphate accumulation of ubiquitinated proteins. Importantly staining using ubiquitin-specific antibody also showed high amounts of ubiquitinated proteins on the elongated centrioles of full-length centrobin (Fig. 5and of this panel. Quantification of mitotic cells in centrobin-depleted cells and centrobin-overexpressing cells are shown in and value <0.001. DISCUSSION Here we have identified the molecular mechanism by which centrobin contributes to the assembly of centrioles. We found that centrobin is critical for stabilizing the cellular and centriolar levels of CPAP. Although depletion of Gamitrinib TPP hexafluorophosphate centrobin leads to cellular CPAP degradation, overexpression of centrobin causes the accumulation of CPAP and abnormal, long centrioles. In association with our previous report that centrobin and CPAP interact directly (48), this study demonstrates Gamitrinib TPP hexafluorophosphate that loss Hpse of centrobin-CPAP connection results in focusing on of ubiquitinated CPAP for proteasome-mediated degradation, a potential mechanism for restricting the centriole size to 500 nm. Gamitrinib TPP hexafluorophosphate Although several centriolar proteins such as STIL, SAS-6, CEP120, SPICE1, and CEP135 can interact with CPAP and positively regulate the centriole duplication and elongation process (30, 32, 45, 46), this is the first study that uncovers the mechanism by which CPAP levels are controlled in the cell to restrict the centriole size. Earlier we showed that centrobin binds to CPAP directly, and this connection is essential for keeping CPAP levels on centrioles (48). Here, we display that centrobin manifestation levels determine the cellular levels of CPAP. Although excessive centrobin causes the persistence of ubiquitinated CPAP and uncontrolled elongation of centrioles due to prolonged connection with centrobin, lack of centrobin prospects to degradation of ubiquitinated CPAP from the Gamitrinib TPP hexafluorophosphate proteasome and inhibition of centriole elongation. It has been demonstrated that CPAP is definitely ubiquitinated from the APC-Cdh1 complex, which is active during.