Functional Analysis of the Drosophila melanogaster condensin subunit Cap-G
Final Report Abstract
The heteropentameric condensin complexes are required for the faithful distribution of the genetic material onto the daughter cells. Metazoans harbor two very similarly structured related complexes, condensin I and condensin II. While both complexes are required for shaping and structuring mitotic chromatin, they clearly differ in their subcellular localization in interphase with condensin II being nuclear and condensin I being cytoplasmic. Drosophila lacks the condensin II-specific subunit Cap-G2. In addition, Drosophila Cap-G behaves differently from the other two condensin I-specific subunits in that it localizes primarily in the nucleus in interphase, which is a hallmark of condensin II. We established that Cap-G, despite its nuclear localization, is not associated with the other condensin II complex components. Nor were we able to identify by a proteomic approach a potential candidate for Cap-G2. Thus, Cap-G appears to act exclusively via its incorporation in condensin I, and condensin II in the fly appears to consist of only four proteins. However, as the other two condensin I complex components Cap-D2 and Cap-H/Barren are primarily localized in the cytoplasm, we tried to elucidate what the functional role is of nuclearly enriched Cap-G. RNAi-mediated knockdown of Cap-G in two different post-mitotic tissues, where Cap-G is expressed, did not lead to any phenotypes; so the functional role of Cap-G in these tissues remains unresolved. We identified the nuclear localization signals in Cap-G and established that a variant with mutated NLS stays in the cytoplasm, but is able to support most of the development of the fly. Thus, nuclear localization of Cap-G is dispensable for all of the zygotic development. However, maternally contributed Cap-G appears to be non-functional, when it is not nuclear. The final experimental proof for this statement is still missing with the demonstration of the rescue of this phenotype by an heterologous functional NLS. The proposed confirmation of the potential Cap-G interactors Brahma and Moira, components of the Drososphila SWI/SNF chromatin remodeling complex, which were initially identified by IP-MassSpec, did not succeed. Nor were we able to induce premature chromosome condensation in early syncytial embryos by injection of the PP2A inhibitor calyculin A. Future studies are required to elucidate the functional relevance of Cap-G nuclear localization in interphase. In quite a few experiments we exploited C-terminal fusions with fluorescent proteins that we had generated via CRISPR/Cas9 using a coupled high efficient screening/tagging approach.
Publications
- 2013. Functional dissection of the Drosophila melanogaster condensin subunit Cap-G reveals its exclusive association with condensin I. PLoS Genet. 9:e1003463
Herzog, S., S. Nagarkar Jaiswal, E. Urban, A. Riemer, S. Fischer, and S. K. Heidmann
(See online at https://doi.org/10.1371/journal.pgen.1003463)
