Biology subjects

Pozniakovsky, A.

Publications and source records attributed to Pozniakovsky, A..

2 recordsLinked to original sources

bioRxiv2025

Structural insights into mitotic-centrosome assembly

Centrosomal material assembles rapidly in mitosis. In Drosophila, the coiled-coil protein Cnn forms a scaffold that recruits PCM clients; in C.elegans, SPD-5 plays an analogous role. Here we show that full-length Cnn and SPD-5 can both form spherical condensates in vitro, but that the interactions driving their assembly into scaffolds inside cells appear to diverge. We show that the Cnn PReM adopts a helical hairpin fold that autoinhibits CM2 binding but that phosphorylation appears to increase hairpin breathing to permit CM2 engagement and robust scaffold assembly. Phospho-blocking mutations prevent PReM-CM2 interactions and scaffold formation, whereas phospho-mimetic substitutions partially restore function. The human homologue CDK5RAP2 contains a CM2 domain that can partially substitute for fly CM2 in vivo and we identify a candidate CDK5RAP2 PReM region that forms macromolecular networks with human CM2 in vitro. By contrast, the putative PReM and CM2 regions of SPD-5 cannot substitute for their equivalent fly domains and they do not interact detectably, suggesting a distinct assembly mechanism in worms despite conserved PLK1-dependent control of PCM growth.

cell biology↗

bioRxiv2019

ESI mutagenesis: A one-step method for introducing point mutations into bacterial artificial chromosome transgenes

Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. While homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call ESI (Exogenous/Synthetic Intronization) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI-mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

molecular biology↗