Biology subjects

Kolata, P.

Publications and source records attributed to Kolata, P..

4 recordsLinked to original sources

bioRxiv2025

Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

Proteasomes are proteolytic machines essential for proteostasis and genome integrity. They consist of a 20S core particle capped by regulatory complexes that confer substrate selectivity (Voges, 1999). Despite evidence that proteasome function is necessary for successful spermatogenesis and fertilisation (Sutovsky, 2011; Xiong et al., 2022), the presence, composition, and role of proteasomes within the sperm nucleus have remained a subject of debate. Here, we used in situ electron cryo-tomography (cryo-ET) in human sperm cells to elucidate the molecular architecture of nuclear proteasomes, which cluster in DNA-free, nuclear cavities within the sperm nucleus. Subtomogram averaging revealed that the main population of proteasomes consists of 20S core particles, with a smaller fraction of 20S capped by PA200 activator, solved at sub-nanometre resolution. Subsequent single-particle cryo-EM analysis of purified native human sperm proteasomes further elucidated atomic features of the essential testis-specific subunit 4s, showing the presence of a unique splice variant. Notably, our reconstruction resolved a native peptide in the catalytic {beta}2 subunit, providing novel insights into the proteolysis mechanism and PA200-mediated enhancement of trypsin activity. Finally, we show nuclear enrichment of proteasomes during sperm cell differentiation in human testis tissue, with 20S and PA200 clustering following meiosis, at the spermatid stage. Our findings shed light on the unique organisation and compositional diversity of nuclear proteasomes in human sperm cells, disclosing novel molecular insights into their catalytic function.

cell biology↗

bioRxiv2025

Nuclear Disruption as a Mechanism of the Chemotherapeutic Drug Paclitaxel in Interphase

Regulation of Lamin A/C levels and distribution is crucial for nuclear integrity and mechanotransduction via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Dysregulation of Lamin A/C correlates with poor cancer prognosis, and its levels determine sensitivity to the microtubule-stabilising drug paclitaxel. Paclitaxel is well-known for disrupting mitosis, yet it also reduces tumour size in slow-dividing tumours, indicating an additional, poorly characterised interphase mechanism. Here, we reveal that paclitaxel induces nuclear aberrations in interphase through SUN2-dependent Lamin A/C disruption. Using advanced optical imaging and electron cryo-tomography, we show the formation of aberrant microtubule-vimentin bundles during paclitaxel treatment, which coincides with nuclear deformation and altered Lamin A/C protein levels and organisation at the nuclear envelope. SUN2 is required for Lamin A/C reduction in paclitaxel and is in turn regulated by polyubiquitination. Furthermore, Lamin A/C expression levels determine not only cell survival during treatment but also recovery after drug removal. Our findings support a model in which paclitaxel acts through both defective mitosis and interphase nuclear-cytoskeletal disruption, providing additional mechanistic insights into a widely used anticancer drug.

cell biology↗

bioRxiv2024

Human spermatogenesis leads to a reduced nuclear pore structure and function

Nuclear pore complexes (NPCs) are nuclear gateways which regulate the transit of molecules larger than 40kDa through a Ran-dependent transport1. The somatic human NPC scaffold consists of three stacked rings: the cytoplasmic (CR), the nucleoplasmic (NR), and the inner ring (IR), which define a central channel approximately 55nm wide2. Although many studies have investigated human NPC architecture3, it remains largely unknown how the NPC accommodates the different functions of non-somatic cells. Here, we reveal the in-cell architecture of the human sperm NPC. We show that it exhibits a central channel less than 40nm-wide, outlined exclusively by the IR. This structural alteration is accompanied by a six-fold reduction in nuclear diffusion rate and mis-localization of Ran-dependent transport components. Additionally, we identify a network of septin filaments interconnecting NPCs within the inter-membrane space of the nuclear envelope (NE), suggesting a potential mechanical role in channel constriction. Furthermore, our human tissue imaging data indicate meiosis as a pivotal differentiation stage driving architectural changes to the NPC. Given the critical role of the IR for successful spermatogenesis4,5, our work offers important insight into this fundamental biological process. Our study also demonstrates the power of an integrative approach by combining electron-cryo- tomography, super-resolution-light-microscopy and biochemical analysis, to elucidate macromolecular structure-function relations in human physiological contexts.

cell biology↗

bioRxiv2021

Structure of Escherichia coli respiratory complex I reconstituted into lipid nanodiscs reveals an uncoupled conformation

Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against trans-membrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further mechanistic studies to understand the enzyme coupling mechanism. Here we describe the single particle cryo-electron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the cytoplasmic and membrane domains suggests an uncoupled conformation of the complex. Based on these structural data we suggest a new simple and testable coupling mechanism for the molecular machine.

biochemistry↗