Biology subjects

Knowles, O.

Publications and source records attributed to Knowles, O..

6 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

In-cell structure of a LINC complex reveals the molecular basis for membrane remodelling and head-to-tail coupling in sperm cells

In eukaryotic cells, LINC complexes physically bridge the two nuclear membranes to cytoskeletal filaments, transmitting mechanical forces to the nucleus. Due to their dynamic and membrane-embedded nature, their architecture in native membranes has been hitherto elusive. Here, we combine in-situ electron cryo-tomography, AlphaFold predictions and molecular dynamics simulations to determine the sub-nanometer-resolution architecture of the trimeric [~]130 kDa SUN5-Nesprin3 LINC complex in human spermatozoa. The structure reveals a hexagonal lattice that stitches the nuclear envelope, creating a rigid interface that links the sperm nucleus to the flagellum. Our structure unveils a unique membrane-anchoring mechanism: a SUN5 KASH-lid {beta}-hairpin and a Nesprin3 amphipathic helix insert into the outer nuclear membrane, leading to cooperative remodelling of the nuclear envelope shape by the extended LINC lattice, which results in flattening the caudal sperm nucleus. Our integrative data provides key insights into the architecture and role of a LINC complex in situ, revealing molecular details of a severe male infertility phenotype.

biochemistry↗

bioRxiv2025

Patching up the nucleus: a novel role for PMLII in nuclear envelope rupture repair

The nuclear envelope (NE) is important for cellular health as it protects and organizes the genome. NE dynamics is important for various cellular processes including cell growth, migration and removal of defective NE components. In extreme cases, the NE can rupture leading to exchange of material between the nuclear interior and the cytoplasm. Rapid repair of the NE is initiated to minimize the effect on the genome. While our understanding of the machinery involved in this repair process is increasing, a lot is still unknown about this process including events leading up to NE rupture. Interestingly, biomolecular condensates have recently been found to play important roles in membrane repair and remodelling in cells. Here, we found that promyelocytic leukemia protein isoform II (PMLII), a protein involved in nuclear PML body formation, forms condensates at the NE. These condensates specifically form at sites where the lamina is disrupted. We show that NE rupture often occurs at these sites and that PMLII stays present until rupture repair is initiated suggesting a role in stabilization of the site for effective repair.

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↗

bioRxiv2023

A CD24+CD271+ melanoma cancer stem cell generates a diffuse hierarchy of attributes that promote metastasis and therapeutic resistance

An important role for phenotype switching has been demonstrated in metastasis and therapeutic resistance of both melanoma and epithelial tumours. Phenotype switching in epithelial tumours is driven by a minority cancer stem cell sub-population with lineage plasticity, but such a sub-population has not been identified in melanoma. We investigated whether cell surface markers used to identify cancer stem cells in epithelial tumours could identify a cancer stem cell sub-population with lineage plasticity in melanoma. We identified a CD24+CD271+ minority sub-population in melanoma that possesses the stem cell characteristics of lineage plasticity and self-renewal. This population displayed hybrid characteristics, combining the attributes of discrete CD24+ and CD271+ cellular sub-populations but with heightened sphere formation, lineage plasticity, migratory ability and drug resistance over its single-marker counterparts. CD24+ and CD24+CD271+ stem cell sub-populations were observed in 10% of human melanomas, mainly at the invasive front. The lack of CD24+ and CD24+CD271+ stem cells in the majority of human melanoma specimens led us to conclude that they may be dispensable for melanoma progression. Nevertheless, the enhanced sphere formation, lineage plasticity, migratory ability and drug resistance of the CD24+CD271+ sub-population may signal a contextual requirement for these stem cells when melanomas face challenging environments both clinically and in experimental systems.

cancer biology↗

bioRxiv2020

The effect of acute sleep deprivation on skeletal muscle protein synthesis and the hormonal environment

Chronic sleep loss is a potent catabolic stressor, increasing the risk of metabolic dysfunction and loss of muscle mass and function. To provide mechanistic insight into these clinical outcomes, we sought to determine if acute sleep deprivation blunts skeletal muscle protein synthesis and promotes a catabolic environment. Healthy young adults (N=13; 7 male, 6 female) were subjected to one night of total sleep deprivation (DEP) and normal sleep (CON) in a randomized cross-over design. Anabolic and catabolic hormonal profiles, skeletal muscle fractional synthesis rate and markers of muscle protein degradation were assessed across the following day. Acute sleep deprivation reduced muscle protein synthesis by 18% (CON: 0.072 {+/-} 0.015 vs. DEP: 0.059 {+/-} 0.014 %*h-1, p=0.040). In addition, it increased plasma cortisol by 21% (p=0.030) and decreased plasma testosterone, but not IGF-1, by 22% (p=0.029). A single night of total sleep deprivation is sufficient to induce anabolic resistance and a pro-catabolic environment. These acute changes may represent mechanistic precursors driving the metabolic dysfunction and body composition changes associated with chronic sleep deprivation.

physiology↗