## 3 Results
Three independent experiments were performed. Error bars show the SD. The \(p\)-value was calculated with a two-tailed Student’s \(t\)-test. \({}^{**}\) indicates \(p<0.01\), \({}^{***}\)\(p<0.005\), \({}^{***}\)\(p<0.001\). (**F**) Representative images showing \(\upgamma\)-tubulin and CDKSPAP2 signals in WT and _cep135_ KO cells. Scale bar: 10 \(\upmu\)m or 2 \(\upmu\)m (for enlarged areas). Centrosome splitting was previously observed as a consequence of reduced pericentriolar material [18; 19; 20]. To analyze if this was a cause for frequent, premature centrosome splitting in _cep135_ KO cells, we quantified centrosomal signals of \(\gamma\)-tubulin, as well as CDK5 RAP2, as markers of the PCM in _cep135_ KO cells (Figure 4D,E). _cep135_ KO cells showed significantly diminished \(\gamma\)-tubulin signals when cells were restricted to G\({}_{0}\)-phase upon starvation and, although to a lesser extent, to S-phase (Figure 4D, compare WT and _cep135_ KO unspl). Centrosomal localization of CDK5 RAP2 was significantly reduced in starved _cep135_ KO cells compared to WT (Figure 4E, compare WT and _cep135_ KO, Figure 4F). Importantly, both marker proteins were more dramatically reduced in _cep135_ KO cells that had undergone centrosome splitting (Figure 4D-F, compare WT and _cep135_ KO split single), albeit when the signal of the two separated centrosomes was summed (Figure 4D,E, compare WT and _cep135_ KO split pair). Previous publications have demonstrated the crucial role of PCM proteins directly functioning as centrosomal linker proteins (CLPs) in centrosome cohesion [21; 22]. Therefore, we next examined whether loss of CEP135 may lead to reduced recruitment of CLPs, C-NAP1, and rootletin. When we visualized and quantified centrosomal rootletin and C-NAP1 by indirect immunofluorescence, we detected a significant loss of both proteins even in cells bearing a single centrosome, i.e., before centrosome splitting (Figure 5A,B). The reduction was stronger in cells that showed split centrosomes (Figure 5A,B). The total expression of C-NAP1 in cell lysates was diminished in _cep135_ KO cells, while rootletin and the CLP-dissolving kinase NEK2A [23] remained at similar expression levels (Figure 5C). These data show that stable centrosomal loading of linker proteins rootletin and C-NAP1 is strongly reduced in a fraction of _cep135_ KO cells, which may be due to reduced expression or stability of C-NAP1. Taken together, this suggests that the compromised physical linkage between centrosomes, as a consequence of compromised PCM establishment, precedes premature splitting in _cep135_ KO cells. Our observation is consistent with previous reports based on RNAi knockdowns which showed that CEP135 is required for centrosomal targeting of C-NAP1 [20]. To prove that the observed centrosome splitting was solely due to the loss of CEP135, we generated _cep135_ KO rescue cells by stably expressing full-length human _CEP135_ cDNA fused to GFP. The frequently observed splitting of centrosomes was completely complemented when full-length CEP135 was expressed in _cep135_ KO cells but not when we expressed the N-terminus of CEP135 alone, or to a much lesser extent, when cells expressed the C-terminal PACT domain of CEP135 (Figure 6A,B). This proves that the observed centrosome splitting defect in human cells was due to the loss of CEP135. Consistently, the observed reduction in PCM proteins \(\gamma\)-tubulin and CDK5 RAP2 was fully restored in _cep135_ KO cells expressing full-length human _CEP135_ (Figure 6C). We next rationalized that premature centrosome splitting and reduced PCM may lead to diminished MT nucleating functions in _cep135_ KO cells. Using a MT regrowth assay, we tested centrosomal MT reassembly after MT depolymerization in the cold (Figure 7A) and quantified situations of expected \"normal\" MT asters (Figure 7A, I) and situations of dispersed MT organization centers (Figure 7A, II), split and asymmetrical MTOCs (Figure 7A, III), or two symmetrical MTOCs (Figure 7A, IV).