Czech National Centre of the European Research Infrastructure Consortium INSTRUCT ERIC

The centromere is a specialized domain that facilitates chromosome segregation during mitosis and serves as the site of kinetochore formation. KINETOCHORE NULL2 (alpha KNL2) is essential for the recognition and loading of the centromeric histone H3 variant CENH3 at centromeres. A yeast two-hybrid screen for alpha KNL2 interactors identified components of the SUMOylation pathway. However, the role of alpha KNL2 SUMOylation in Arabidopsis has not yet been determined. In this study, we demonstrated that the C-terminal region of alpha KNL2 (designated alpha KNL2-C) interacts with small ubiquitin-like modifier 3 (SUMO3) and ULP1d, as shown by bimolecular fluorescence complementation and co-immunoprecipitation assays. Bioinformatic and functional analyses of alpha KNL2-C identified three SUMOylation sites and two SUMO-interacting motifs, which were shown to be critical for growth, fertility, and chromosome alignment. Of the three SUMOylation sites, Lys474 and Lys511 are the most critical for the centromeric localization of alpha KNL2, underscoring the importance of alpha KNL2 SUMOylation for its function. Additionally, both in vitro and in vivo assays showed that alpha KNL2-C undergoes SUMOylation by SUMO1 or SUMO3. The Arabidopsis SUMO protease mutant ulp1d-2 exhibits a mild accumulation of SUMOylated alpha KNL2. We further showed that SUMOylation of alpha KNL2 promotes its binding to CENH3 and controls protein stability. Our findings demonstrate that C-terminal SUMOylation of alpha KNL2 is crucial for its centromeric localization, interaction with CENH3, and kinetochore assembly, emphasizing the significance of post-translational modifications in chromosome segregation and cell division in plants.

Kalidass M. et al.: The C-terminal SuMOylation-dependent regulation of aKNL2 governs its centromere targeting and interaction with CENH3

Plant Communications, DOI: 10.1016/j.xplc.2025.101617

Intracellular crystallization is an emerging approach in structural biology that bypasses the need for protein purification. In 2024, the InCellCryst pipeline was introduced for structural studies of intracellular crystals by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of cells containing intracellular crystals, precluding high-resolution studies on proteins that crystallize only in a few cells. Here we introduce IncelluloED, a method that combines intracellular crystallization with in situ 3D electron diffraction in cells and achieves high-resolution structures from just one crystal inside one cell. Experiments on a microcrystal of the HEX-1 protein from Magnaporthe grisea, grown inside an insect cell, give a structure at 1.9 angstrom resolution from a volume of similar to 1.6 mu m(3) as compared to 1.8 angstrom resolution achieved by serial X-ray crystallography from a combined volume exceeding eleven million mu m(3). IncelluloED uses widely available cryo-EM tools and brings high-resolution structural biology into home laboratories while also advancing a vision for a "single-cell structural laboratory".

Bila S. et al.: Single-cell structural biology with intracellular electron crystallography

Nature Communications, DOI: 10.1038/s41467-026-69205-6

Lysine acetylation within the tandem repeat region of cortactin (CTTN) regulates its actin-binding function and has been linked to cancer cell migration and neuronal development. While several lysine deacetylases (KDACs) have been implicated in modulating CTTN acetylation in cells, their site specificity and direct enzymatic roles remain poorly defined. Here, we use genetic code expansion to generate seven site-specifically acetylated CTTN variants and assess their deacetylation by human KDACs in a fully reconstituted in vitro system. Our results identify HDAC6 as the primary CTTN deacetylase, acting via its second catalytic domain (DD2), and demonstrate that SIRT1 and SIRT2 also directly deacetylate CTTN at overlapping sites in an NAD+-dependent manner. In contrast, other zinc-dependent HDACs, including HDAC8, displayed negligible or very weak activity on full-length CTTN. These findings provide new mechanistic insight into KDAC substrate preferences and highlight the value of biochemical reconstitution for dissecting complex acetylation networks.

Komarek J. et al.: Selective targeting of cortactin tandem repeat acetylation by human lysine deacetylases

FEBS Journal, DOI: 10.1111/febs.70430​