Claudia Pellacani

In animal systems, the only case of third meiotic division without a prior round of DNA replication has been described in Drosophila males carrying mutations in roughex, a gene encoding an inhibitor of Cdk1 activity. Here, we describe another Drosophila gene, terno (teo), that regulates meiotic exit in males. In teo mutants, meiosis I and II are regular, but in many germline cysts, the 64 haploid spermatids undergo an extra division. Additionally, teo mutants are defective in sperm individualization. Teo interacts with cyclin A and B, Fizzy (Cdc20), and the anaphase-promoting complex/cyclosome components Cdc16 and Cdc27. In teo mutant testes, the cyclin A and B levels are higher than in wild type, and downregulation of either cyclin rescues the teo mutant phenotypes. These results suggest that Teo facilitates targeted degradation of the cyclins, so that in teo mutants, there is an increase in Cdk1-CycA and Cdk1-CycB activities, resulting in both the extra meiotic division and the sperm maturation defect.

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and intellectual disability. Mutations in over 30 genes, nearly half linked to centrosome biogenesis or microtubule (MT) dynamics, highlight spindle defects in disease aetiology, yet these alone do not fully explain MCPH. Here, we show that the Drosophila orthologs of ASPM/MCPH5 (asp) and CENPJ/MCPH6 (Sas4) contribute to safeguard nuclear architecture and chromatin organization during brain development. Loss of either gene perturbs MT organization and centromere clustering, leading to reduced Lamin and HP1α levels, and to deformed nuclear lamina. Mutants also display a global reduction in heterochromatin-associated histone marks, H3K9me2/3 and H3K27me3, along with an increase in the euchromatin-associated mark H3K4me3 and elevated DNA damage with delayed repair. Notably, inhibiting demethylases with methylstat restores H3K9me3 and nuclear morphology. These findings suggest a previously unreported role for centrosome proteins in regulating chromatin organization, providing new insights into the mechanisms underlying MCPH pathogenesis.

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and non-progressive intellectual disability. Mutations in over 30 genes have been linked to MCPH. Nearly a half of the genes identified by these mutations encode proteins involved in centrosome biogenesis or microtubule (MT) dynamics, suggesting a central role for mitotic spindle organization and division plane orientation in disease aetiology. However, it has been suggested that disruptions in spindle positioning alone are not sufficient to lead to microcephaly. Here, we investigate the contribution of the Drosophila orthologs of ASPM / MCPH5 ( asp ) and CENPJ/MCPH6 ( Sas4 ) to nuclear architecture, chromatin organization, and genome stability. We show that loss of either Sas4 or Asp leads to aberrant microtubule architecture, mislocalization of the LINC complex, and deformation of the nuclear lamina. These defects are accompanied by reduced levels of both lamin and HP1α and impaired centromere clustering in interphase cells. Sas4 and asp mutants also exhibit a global reduction in heterochromatin-associated histone marks (H3K9me2/3 and H3K27me3) and increased levels of the euchromatin-associated mark H3K4me3. Remarkably, treatment with Methylstat, a demethylase inhibitor, reduced nuclear invaginations by partially restoring H3K9me3 levels. Additionally, Sas4 or Asp depletion leads to DNA damage, increased sensitivity to genotoxic stress, and delayed DNA repair. Together, these findings reveal a previously underappreciated role for Asp and Sas4 in preserving nuclear architecture and chromatin integrity, offering new insight into the pathogenesis of MCPH. GRAPHICAL

INT6/eIF3e is a highly conserved component of the translation initiation complex that interacts with both the 26S proteasome and the COP9 signalosome, two complexes implicated in ubiquitin-mediated protein degradation. The INT6 gene was originally identified as the insertion site of the mouse mammary tumor virus (MMTV), and later shown to be involved in human tumorigenesis. Here we show that depletion of the Drosophila orthologue of INT6 (Int6) results in short mitotic spindles and deformed centromeres and kinetochores with low intra-kinetochore distance. Poleward flux of microtubule subunits during metaphase is reduced, although fluorescence recovery after photobleaching (FRAP) demonstrates that microtubules remain dynamic both near the kinetochores and at spindle poles. Mitotic progression is delayed during metaphase due to the activity of the spindle assembly checkpoint (SAC). Interestingly, a deubiquitinated form of the kinesin Klp67A (a putative orthologue of human Kif18A) accumulates near the kinetochores in Int6-depleted cells. Consistent with this finding, Klp67A overexpression mimics the Int6 RNAi phenotype. Furthermore, simultaneous depletion of Int6 and Klp67A results in a phenotype identical to RNAi of just Klp67A, which indicates that Klp67A deficiency is epistatic over Int6 deficiency. We propose that Int6-mediated ubiquitination is required to control the activity of Klp67A. In the absence of this control, excess of Klp67A at the kinetochore suppresses microtubule plus-end polymerization, which in turn results in reduced microtubule flux, spindle shortening, and centromere/kinetochore deformation. INT6 is an evolutionarily conserved gene originally identified as the insertion site of the mouse mammary tumor virus that causes tumors in mice. INT6 is downregulated in many human cancers, suggesting that it acts as tumor suppressor gene. The INT6 protein is involved in several biological processes, including translation and ubiquitin-mediated protein degradation. We performed RNA interference (RNAi) against the Drosophila homologue of INT6 (Int6) and analyzed the effects of Int6 depletion on mitotic cell division. We found that loss of Int6 results in short spindles, delayed progression though metaphase and abnormally shaped centromeres/kinetochores. We also found that Int6-depleted cells fail to degrade the kinesin Klp67A. This protein, known to attenuate polymerization of microtubule (MTs) plus ends, accumulated at the kinetochores in Int6-depleted cells. We propose that this condition affects MT growth at the kinetochore, which in turn results in centromere/kinetochore deformation and delays satisfaction of the mitotic checkpoint.