Organization. Telomere clustering defects in CEP63 deficient cells As a direct role for CEP63/CEP152 in

Organization. Telomere clustering defects in CEP63 deficient cells As a direct role for CEP63/CEP152 in meiotic recombination seemed unlikely given the lack of detectable nuclear localization, we asked whether or not aberrant centrosomes in Cep63T/T cells impair chromosome movements required for recombination. Such a defect may be triggered by loss of chromosome attachment for the nuclear envelope or lack of tethering towards the microtubule cytoskeleton by LINC complexes40, 42, 43. In Cep63T/T spermatocytes, SUN1, the telomeric SUN domain protein that mediates attachment of chromosomes to the inner nuclear envelope, was localized to telomeres, and the distribution of TRF1 (telomeric marker) was constant with nuclear envelope localization (Fig. 7a), suggesting that chromosome attachment was intact. We next analyzed the outcome of chromosome tethering to the microtubule cytoskeleton by quantifying “bouquets”, a leptotene/zygotene chromosome configuration that arises from transient clustering of chromosome termini39. Staining with ACA antibodies to label the centromeric ends of mouse acrocentric chromosomes and SCP3 antibodies for staging revealed a substantial reduction in telomere clustering in Cep63T/T spermatocytes of juvenile mice in comparison to WT (Fig. 7b and 7c). Together our data suggests that in spermatocytes, CEP63 loss triggers cell death independently of p53, ATM or CHK2, mostly as a consequence of defective meiotic recombination, in lieu of mitotic spindle errors. Contemplating that CEP63 and CEP152 are physically separated in the chromosomes and may only be detected at centrosomes throughout prophase I (Fig. 6a), and that PCM organization is impaired in CEP63 deficient cells (Fig. 6e ), we propose that these errors are probably caused by aberrant centrosomes that impair standard intranuclear chromosome movements (Fig. 7d).Author Manuscript Author Manuscript Author Manuscript Author ManuscriptDiscussionCep63 deficient mice recapitulate two key elements of human Seckel syndrome associated with CEP63 mutations (SCKL6); growth retardation and microcephaly5. Our analysis suggests that the etiology of microcephaly in Cep63T/T animals is partially distinct from thatNat Commun. Author manuscript; offered in PMC 2016 January 09.Marjanovi et al.Pagereported for Atr or Mcph1. Atr deficiency causes much more extreme facial dysmorphia and runting than either Mcph1 or Cep63 deficiency24, 44. B7-2 Inhibitors products Additionally, deletion of p53 in Atr deficient animals exacerbates the Triallate Cancer neurodevelopmental phenotypes, whereas deletion of p53 in Cep63T/T mice rescued microcephaly (Fig. 3e ). Rescue of microcephaly following p53 deletion has also been reported in other models with far more extreme centriole loss25. Microcephaly in Mcph1 deficient mice has been linked to abrogated CHK1 recruitment to centrosomes and premature CDK1 activation that compromises the timing of division modes and self-renewal capacity of NPCs, resulting in their premature decline27. Even though we observed lowered neurosphere forming capacity of NPCs from Cep63T/T embryos, we did not observe a progressive decline, as was reported in Mcph1 mutants, suggesting that selfrenewal defects are most likely not the key reason for NPC depletion in Cep63T/T mice (Supplementary Fig. 7). Moreover, previous work has implicated CEP63 in CDK1 recruitment to centrosomes, arguing against a mechanism of premature centrosomal CDK1 activation27, 45. On the other hand, this situation will need further investigation considering the fact that some of the CDK1 antibodies applied.