Biology subjects

Allegretti, M.

Publications and source records attributed to Allegretti, M..

13 recordsLinked to original sources

bioRxiv2026

Easymode: general pretrained networks for cellular cryo-ET enable flexible approaches to subtomogram averaging

Cellular cryo-electron tomography (cryo-ET) reveals high-resolution details of macromolecules within their native cellular environment. However, in situ cryo-ET datasets are large and highly heterogeneous, which makes comprehensive identification and extraction of the many different elements of cellular architecture for high-resolution analysis a challenging, time-consuming and often tedious task. Here we present easymode, a library of pretrained general segmentation networks for cryo-ET, trained on over 4,000 tilt series spanning a large and diverse variety of sources. Easymode enables in situ structural determination workflows by rendering tomogram content computationally accessible, without requiring any per-dataset training. Beyond directly facilitating high-resolution subtomogram averaging of a selection of widely prevalent complexes, we show how easymode can be used to leverage cellular context in subtomogram averaging workflows, helping identify, align, or filter particle sets, and enabling automated mapping of the cellular landscape surrounding target proteins. We use easymode to determine the in situ structure of rare inosine monophosphate dehydrogenase (IMPDH) filaments at 4.0 A resolution, and to map and visualize the surrounding cellular environment.

molecular biology↗

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

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↗

bioRxiv2022

Relevance of the viral Spike protein/cellular Estrogen Receptor-α interaction for endothelial-based coagulopathy induced by SARS-CoV-2

Severe coagulopathy has been observed at the level of the microcirculation in several organs including lungs, heart and kidneys in patients with COVID-19, and in a minority of subjects receiving the SARS-CoV-2 vaccine. Various mechanisms have been implicated in these effects, including increases in circulating neutrophil extracellular traps, excessive inflammation, and endothelial dysfunction. Even if a correlation between infection by SARS-CoV-2 and upregulation of coagulation cascade components has been established in the lung, no direct proofs have been yet provided about the transcriptional machinery controlling the expression of these factors. Recent results obtained by us reported a novel transcriptional function of the SARS-CoV-2 Spike (S) viral protein involving a direct protein-protein interaction with the human Estrogen Receptor- (ER). Given the implications of ER in the control of key effectors in the coagulation cascade, we hypothesized that S-protein might increase the pro-coagulation activity of endothelial cells via the transcriptional activity of the ER, thus justifying the enhanced risk of thrombosis. To assess this, we tested the effects of S-protein on the expression of Tissue Factor (TF) and the overall procoagulation activity in a human endothelial cell line and confirmed this finding by overexpressing S-protein by gene transfer in mice. We then designed and tested two-point mutations in the S2 S-protein sequence that abolished the pro-coagulation function of S-protein in vitro and in vivo, without compromising its immunogenicity. In addition to reveal a new potential transcriptional function of S-protein, these results inspire the design of new vaccines with lower risk of thrombogenesis. Indeed, while the benefit/risk ratio remains overwhelming in favor of COVID-19 vaccination, our results shed light on the causal mechanisms of some rare anti-SARS-CoV-2 vaccine adverse events, and are thus essential for current and future vaccination and booster campaigns.

cell biology↗

bioRxiv2022

Targeting C5aR1 signaling reduced neutrophil extracellular traps and ameliorates COVID-19 pathology

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions, and plays immunopathological roles in inflammatory diseases, we investigated whether C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill COVID-19 patients compared to patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular trap (NET)s-dependent immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonist of C5aR1 could be useful for COVID-19 treatment.

immunology↗

bioRxiv2022

The SARS-CoV-2 spike protein binds and modulates estrogen receptors

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ER). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ER binding mode. In cultured cells, S DNA transfection increased ER cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [18F]fluoroestradiol (FES) localized lung pathology with increased ER lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ER expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ER interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.

immunology↗

bioRxiv2021

Characterization of raloxifene as potential pharmacological agent against SARS-CoV-2 and its variants

The new coronavirus that emerged, called SARS-CoV-2, is the causative agent of the COVID-19 pandemic. The identification of potential drug candidates that can rapidly enter clinical trials for the prevention and treatment of COVID-19 is an urgent need, despite the recent introduction of several new vaccines for the prevention and protection of this infectious disease, which in many cases becomes severe. Drug repurposing (DR), a process for studying existing pharmaceutical products for new therapeutic indications, represents one of the most effective potential strategies employed to increase the success rate in the development of new drug therapies. We identified raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a potential pharmacological agent for the treatment of COVID-19 patients. Following a virtual screening campaign on the most relevant viral protein targets, in this work we report the results of the first pharmacological characterization of raloxifene in relevant cellular models of COVID-19 infection. The results obtained on all the most common viral variants originating in Europe, United Kingdom, Brazil, South Africa and India, currently in circulation, are also reported, confirming the efficacy of raloxifene and, consequently, the relevance of the proposed approach. Taken together, all the information gathered supports the clinical development of raloxifene and confirms that the drug can be proposed as a viable new option to fight the pandemic in at least some patient populations. The results obtained so far have paved the way for a first clinical study to test the safety and efficacy of raloxifene, just concluded in patients with mild to moderate COVID-19.

pharmacology and toxicology↗

bioRxiv2020

Nuclear pores constrict upon energy depletion

Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins that form an intricate cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter were reported, but the physiological circumstances and the molecular details remain unknown. Here we combined cryo-electron tomography and subtomogram averaging with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. While actively transporting NPCs adopt a dilated conformation, they strongly constrict upon cellular energy depletion. Fluorescence recovery after photo bleaching experiments show that NPC constriction is concomitant with reduced diffusion and active transport across the nuclear envelope. Our data point to a model where the energy status of cells is linked to the conformation of NPC architecture.

cell biology↗

bioRxiv2020

Cone-shaped HIV-1 capsids are transported through intact nuclear pores

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrate that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, coneshaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

microbiology↗

bioRxiv2020

In cell architecture of the nuclear pore complex and snapshots of its turnover

Nuclear pore complexes (NPCs) mediate exchange across the nuclear envelope. They consist of hundreds of proteins called nucleoporins (Nups) that assemble in multiple copies to fuse the inner and outer nuclear membranes. Elucidating the molecular function and architecture of NPCs imposes a formidable challenge and requires the convergence of in vitro and in situ approaches. How exactly NPC architecture accommodates processes such as mRNA export or NPC assembly and turnover inside of cells remains poorly understood. Here we combine integrated in situ structural biology, correlative light and electron microscopy with yeast genetics to structurally analyze NPCs within the native context of Saccharomyces cerevisiae cells under conditions of starvation and exponential growth. We find an unanticipated in situ layout of nucleoporins with respect to overall dimensions and conformation of the NPC scaffold that could not have been predicted from previous in vitro analysis. Particularly striking is the configuration of the Nup159 complex, which appears critical to spatially accommodate not only mRNA export but also NPC turnover by selective autophagy. We capture structural snapshots of NPC turnover, revealing that it occurs through nuclear envelope herniae and NPC-containing nuclear vesicles. Our study provides the basis for understanding the various membrane remodeling events that happen at the interface of the nuclear envelope with the autophagy apparatus and emphasizes the need of investigating macromolecular complexes in their cellular context.

biophysics↗

bioRxiv2019

An ESCRT-LEM domain inner nuclear membrane protein surveillance system is poised to directly monitor the integrity of the nuclear envelope barrier and nuclear transport system

The integrity of the nuclear envelope membranes coupled to the diffusion barrier and selective transport properties of nuclear pore complexes (NPCs) are a prerequisite for the robust segregation of nucleoplasm and cytoplasm. Recent work supports that mechanical membrane disruption or perturbation to NPC assembly can trigger an ESCRT-dependent surveillance system that seals nuclear envelope pores: how these pores are sensed and sealed remains to be fully defined. Here, we show that the principal components of the nuclear envelope surveillance system in yeast, which includes the ESCRT Chm7 and the integral inner nuclear membrane (INM) protein Heh1, are spatially segregated by the nuclear transport system. Specifically, at steady state Chm7 is actively restricted from the nucleus by Crm1/Xpo1. Consistent with the idea that it is the exposure of the INM that triggers surveillance, the expression of a transmembrane anchor and the winged helix domain of Heh1 is sufficient to recruit and activate Chm7 at a membrane interface. Correlative light electron tomography under conditions of Chm7 hyper-activation further show the formation of an elaborate network of fenestrated sheets at the INM and suggest ER-membrane delivery at sites of nuclear envelope herniation. Our data point to a model in which exposure of Chm7 to Heh1, driven by any perturbation in the nuclear envelope barrier would lead to local nuclear envelope remodeling to promote membrane sealing. Our findings have implications for disease mechanisms associated with defects in NPC assembly and nuclear envelope integrity.

cell biology↗