Abstract
Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture.
Highlights
Centrosomes are composed of a pair of barrel-shaped, 9-fold symmetric centrioles, surrounded by a proteinaceous matrix collectively referred to as pericentriolar material (PCM).1 Centrosomes are the major microtubule organizing
HERC2 and neuralized-like protein 4 (NEURL4) Are Novel Interactors of CP110 — Through its association with CEP97, CEP76, or CEP290, CP110 is a critical regulator of centrosome biogenesis [13, 15,16,17,18]
We did not follow up any further on the other interacting proteins detected, including SRGAP2, TBK1, PEC1, and KTN1, because our analysis revealed that they were not shared among the network of NEURL4-CEP97-CP110-HERC2
Summary
Cell Culture—U2OS and MG63 cells were grown at 37 °C in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 2 mM L-glutamine under 5% CO2 in a humidified atmosphere and maintained using standard procedures. U2OS cells expressing doxycycline-inducible HERC2 small hairpin RNA were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and supplemented with 1 g/ml puromycin and 5 g/ml blasticidin as described previously [20]. After a 30-min wash with 0.2% PBS/FSG, the cells were incubated with mouse anti-centrin antibodies (1:2000) and either rabbit anti-Cep164 (1:500). The purification was performed in biological replicates from ϳ100 mg of protein (harvested from 5 ϫ 150-mm confluent plates), essentially as described previously [32], with the following modifications: the lysates were precleared with 150 l of packed protein G-Sepharose beads for 3 h at 4 °C; 125 units (total amount) of benzonase was added to lysis buffer prior to harvesting the cells; and the IP was performed using 75 l of packed FLAG-M2-Sepharose beads for 3 h at 4 °C. The other remaining control runs included data from IPs in HEK293 Flp-In T-REx cell lines expressing empty vector where FLAG-M2-agarose resin was used. The ubiquitylation reactions were assessed by subjecting the samples to immunoblot analysis with different ubiquitin antibodies
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