bims-micesi 2021-11-21 papers

Protein Sci. 2021 Nov 17.

Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes.

Pim J Huis In 't Veld, Sabine Wohlgemuth, Carolin Koerner, Franziska Müller, Petra Janning, Andrea Musacchio.

As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities of target proteins and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1 activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CAKs or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a ternary complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC). This article is protected by copyright. All rights reserved.

Keywords: CDK1; CKS1; Cell cycle; Cyclin; Cyclin dependent kinase; Cyclin-B; phosphorylation; phostag; processivity; recombinant protein

DOI: https://doi.org/10.1002/pro.4233

Oncogene. 2021 Nov 13.

Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex.

Lan Yu, Yue Lang, Ching-Cheng Hsu, Wei-Min Chen, Jui-Chung Chiang, Jer-Tsong Hsieh, Michael D Story, Zeng-Fu Shang, Benjamin P C Chen, Debabrata Saha.

Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP's interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.

DOI: https://doi.org/10.1038/s41388-021-02106-8

Biochem Soc Trans. 2021 Nov 16. pii: BST20210717. [Epub ahead of print]

Chromosome clustering in mitosis by the nuclear protein Ki-67.

Konstantinos Stamatiou, Paola Vagnarelli.

Ki-67 is highly expressed in proliferating cells, a characteristic that made the protein a very important proliferation marker widely used in the clinic. However, the molecular functions and properties of Ki-67 remained quite obscure for a long time. Only recently important discoveries have shed some light on its function and shown that Ki-67 has a major role in the formation of mitotic chromosome periphery compartment, it is associated with protein phosphatase one (PP1) and regulates chromatin function in interphase and mitosis. In this review, we discuss the role of Ki-67 during cell division. Specifically, we focus on the importance of Ki-67 in chromosome individualisation at mitotic entry (prometaphase) and its contribution to chromosome clustering and nuclear remodelling during mitotic exit.

Keywords: Ki-67; chromosomes; mitosis

DOI: https://doi.org/10.1042/BST20210717

J Cell Biol. 2021 Dec 06. pii: e202110031. [Epub ahead of print]220(12):

A SUMOylation wave to anchor the genome.

Adele L Marston.

Chromatin tethers to the nuclear envelope are lost during mitosis to facilitate chromosome segregation. How these connections are reestablished to ensure functional genome organization in interphase is unclear. Ptak et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202103036) identify a phosphorylation and SUMOylation-dependent cascade that links chromatin to the nuclear membrane during late mitosis.

DOI: https://doi.org/10.1083/jcb.202110031

J Cell Biol. 2021 Dec 06. pii: e202103036. [Epub ahead of print]220(12):

Phosphorylation-dependent mitotic SUMOylation drives nuclear envelope-chromatin interactions.

Christopher Ptak, Natasha O Saik, Ashwini Premashankar, Diego L Lapetina, John D Aitchison, Ben Montpetit, Richard W Wozniak.

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin-nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.

DOI: https://doi.org/10.1083/jcb.202103036

J Cell Biol. 2022 Jan 03. pii: e202106170. [Epub ahead of print]221(1):

Volumetric morphometry reveals spindle width as the best predictor of mammalian spindle scaling.

Tobias Kletter, Sebastian Reusch, Tommaso Cavazza, Nils Dempewolf, Christian Tischer, Simone Reber.

The function of cellular structures at the mesoscale is dependent on their geometry and proportionality to cell size. The mitotic spindle is a good example why length and shape of intracellular organelles matter. Spindle length determines the distance over which chromosomes will segregate, and spindle shape ensures bipolarity. While we still lack a systematic and quantitative understanding of subcellular morphology, new imaging techniques and volumetric data analysis promise novel insights into scaling relations across different species. Here, we introduce Spindle3D, an open-source plug-in that allows for the quantitative, consistent, and automated analysis of 3D fluorescent data of spindles and chromatin. We systematically analyze different mammalian cell types, including somatic cells, stem cells, and one- and two-cell embryos, to derive volumetric relations of spindle, chromatin, and the cell. Taken together, our data indicate that mitotic spindle width is a robust indicator of spindle volume, which correlates linearly with chromatin and cell volume both within single cell types and across mammalian species.

DOI: https://doi.org/10.1083/jcb.202106170