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SummaryAt the onset of mitosis, cells need to break down their nuclear envelope, form a bipolar spindle and attach the chromosomes to microtubules via kinetochores. Previous studies have shown that spindle bipolarization can occur either before or after nuclear envelope breakdown. In the latter case, early kinetochore-microtubule attachments generate pushing forces that accelerate centrosome separation. However, until now, the physiological relevance of this prometaphase kinetochore pushing force was unknown. We investigated the depletion phenotype of the kinetochore protein CENP-L, which we find to be essential for the stability of kinetochore microtubules, for a homogenous poleward microtubule flux rate and for the kinetochore pushing force. Loss of this force in prometaphase not only delays centrosome separation by 5-6 minutes, it also causes massive chromosome alignment and segregation defects due to the formation of syntelic and merotelic kinetochore-microtubule attachments. By contrast, CENP-L depletion has no impact on mitotic progression in cells that have already separated their centrosomes at nuclear envelope breakdown. We propose that the kinetochore pushing force is an essential safety mechanism that favors amphitelic attachments by ensuring that spindle bipolarization occurs before the formation of the majority of kinetochore-microtubule attachments.

The mitotic spindle drives chromosome movement during mitosis and attaches to chromosomes at dedicated genomic loci named centromeres. Centromeres are epigenetically specified by their histone composition, namely the presence of the histone H3 variant CENP-A, which is regulated during the cell cycle by its dynamic expression and localization. Here, we combined biochemical methods and quantitative imaging approaches to investigate a new function of CUL4-RING E3 ubiquitin ligases (CRL4) in regulating CENP-A dynamics. We found that the core components CUL4 and DDB1 are required for centromeric loading of CENP-A, but do not influence CENP-A maintenance or pre-nucleosomal CENP-A levels. Interestingly, we identified RBBP7 as a substrate-specific CRL4 adaptor required for this process, in addition to its role in binding and stabilizing soluble CENP-A. Our data thus suggest that the CRL4 complex containing RBBP7 (CRL4 RBBP7 ) might regulate mitosis by promoting ubiquitin-dependent loading of newly synthesized CENP-A during the G1 phase of the cell cycle.

Efficient duplication of the genome and its equal distribution into both daughter cells is an essential process for all dividing organisms. To ensure that DNA replication occurs only once during the S phase of the cell cycle, initiation of replication is tightly controlled. Initiation factors are responsible for the recruitment of the replisome, the large molecular machine carrying out DNA synthesis, to origins of replication and license them to start DNA duplication. Remarkably, most of the currently known initiators have been classified as members of the family of AAA(+) ATPases. In our recent study we identified an additional AAA(+) ATPase, CDC-48, to be essential for proper DNA replication in Caenorhabditis elegans. Here, we speculate on the function of CDC-48 (also known as Cdc48p in yeast and p97 in vertebrates) during DNA replication initiation, addressing its ubiquitin-selective chaperone activity.

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