Biology subjects

Ariotti, N.

Publications and source records attributed to Ariotti, N..

16 recordsLinked to original sources

bioRxiv2026

A pro-apoptotic selection strategy enables CRISPR screening in mosquitoes and identifies Lachesin as a chikungunya virus entry factor

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that is transmitted primarily by Aedes aegypti and Aedes albopictus mosquitoes. CHIKV infection can result in debilitating arthritis-like symptoms in humans. While determinants of CHIKV host cell entry into mammalian cells are known, their equivalents in mosquito cells remain largely elusive. To identify CHIKV entry factors, we performed a membrane-focused genome-scale CRISPR loss-of-function screen in Aedes albopictus mosquito cells. To enable selection of refractory cells, we engineered CHIKV to express the pro-apoptotic Drosophila Reaper protein. CHIKV-Reaper induced robust cell death in mosquito cells with only modest viral fitness loss. Using this selection strategy together with an Aedes albopictus C6/36-based CRISPR screening platform, we identified glycosylphosphatidylinositol (GPI)-anchored cell surface proteins and multiple enzymes involved in the GPI-anchor biosynthesis pathway as proviral candidates. Ectopic expression of the GPI-anchored cell-adhesion protein Lachesin rendered refractory mammalian cells susceptible to CHIKV and the related arthritogenic alphaviruses Semliki Forest virus and Ross River virus. In contrast, Lachesin-expressing cells remained refractory to the encephalitic alphavirus Venezuelan equine encephalitis virus. We demonstrate that Lachesin is essential for CHIKV infection in both Aedes aegypti and Aedes albopictus cells, as confirmed by gene silencing. Here, we identify Lachesin as a critical candidate entry receptor for CHIKV and establish pro-apoptotic arboviruses as a powerful and versatile strategy for functional CRISPR screening in mosquito cells.

microbiology↗

bioRxiv2026

Mitochondrial morphology and function are tuned by microtubule association

Mitochondrial morphology is regulated through dynamic fission and fusion processes that are critical for cellular metabolism and homeostasis. Microtubules and mitochondria are functionally coupled across many cellular contexts, yet the mechanistic basis of this relationship remains poorly understood. Here, we reveal that microtubule association is a key regulator of mitochondrial morphology and function. Pharmacological or genetic manipulation of microtubule dynamics controls mitochondrial membrane potential and respiratory capacity, with stabilisation driving elongation, and depolymerisation triggering Drp1-dependent fission and reduced respiration. Strikingly, we identify increased microtubule polymerisation as a hallmark of starvation-induced mitochondrial hyperfusion, a key adaptive response to nutrient stress, and show that this expanded microtubule network is an essential prerequisite for the starvation response. We demonstrate that this regulation operates independently of canonical tubulin post-translational modifications that stabilise microtubules. Rather, increased mitochondria-microtubule association, driven by network expansion, is both necessary and sufficient to drive mitochondrial hyperfusion, as demonstrated by forced mitochondria-microtubule tethering. Taken together, our findings establish microtubule binding as a key upstream regulator of mitochondrial network dynamics, with direct implication for understanding how cells coordinate cytoskeletal organisation with metabolic adaptation during physiological stress.

Cell Biology↗

bioRxiv2025

An efficient eukaryotic cell-free expression and correlative cryo-electron tomography platform for structural biology of macromolecular complexes

Cell-free expression using Leishmania tarentolae lysates allows rapid expression of eukaryotic proteins directly from DNA templates. We develop a pipeline that combines cell-free expression system with cryogenic fluorescence microscopy that we term CC-FLEXCET (Correlative Cell-Free Leishmania EXpression and Cryo-Electron Tomography), to target and visualize expressed protein complexes by cryo-electron tomography at high resolution. We demonstrate the utility of this method by structurally characterising the filaments of the full-length apoptosis-associated speck like protein containing CARD (ASC) protein. Cell-free expression of ASC results in a polymeric structure characteristic of its cellular speck assembly. Sub-tomogram averaging allows us to resolve both the pyrin domain (PYD) to medium resolution, and show, for the first time, the arrangement of the flexibly linked caspase recruitment domain (CARD). Finally, we observed an interaction between the ASC filament and the L. tarentolae ribosome. Using template matching and quantitative approaches, we characterise this interaction and determine that there is a random structural association between the filament and the ribosome, with 57% of ribosomes oriented with the LSU toward the ASC polymer. CC-FLEXCET facilitates structural analysis of macromolecules and protein-lipid assemblies without need of purification, providing a pipeline from DNA template to protein expression to cryo-tilt series acquisition, within a single day.

biochemistry↗

bioRxiv2024

Unsaturated lipids as key control points for caveola formation and disassembly

Caveolae are specialized plasma membrane domains with a unique lipid composition. Recent work has implicated lipid peroxidation in triggering caveola disassembly, releasing caveolar proteins to regulate the cellular response to oxidative stress. Here we investigated the role of specific lipid species in caveola formation and response to oxidative stress. Systematic screening of lipid enzymes identified ACSL4, a critical enzyme in the synthesis of polyunsaturated fatty acid-containing lipids, and enzymes involved in the biosynthesis of pro-ferroptotic ether lipids, as key regulators of caveola formation. Omega-6 fatty acids promoted efficient caveola formation, while their oxidation or displacement by omega-3 or monounsaturated fatty acids disrupted this process. Moreover, we found that omega-6 fatty acid-containing membrane lipids were required for oxidative stress-induced caveola disassembly. These findings unveil a new model for caveola formation, highlighting specific unsaturated lipids as essential caveolar components and key control points for caveola disassembly in response to an oxidizing environment.

cell biology↗

bioRxiv2024

A modular encapsulation system for precision delivery of proteins, nucleic acids and therapeutics

Targeted nanoparticles have the potential to revolutionize therapeutics for medical applications. Here, we demonstrate the utility of a flexible precision nanovesicle delivery system for functional delivery of DNA, RNA, proteins and drugs into target cells. Nanovesicles generated by the membrane sculpting protein caveolin, termed caveospheres, can be loaded with RNA, DNA, proteins or drugs post-synthesis or incorporate genetically-encoded cargo proteins during production without the need for protein purification. Functionalized fluorescently-labeled caveospheres form a modular system that shows high stability in biological fluids, specific uptake by target-positive cells, and can deliver proteins, drugs, DNA, and mRNA directly to the cytoplasm and nuclei of only the target cells. The negligible level of off-target transduction and uniform levels of targeted expression demonstrates advantages of the system over lipid-mediated gene delivery. Caveospheres can also be engineered to mimic viral particles by displaying the SARS-CoV-2-RBD protein, enabling targeted delivery to human bronchial epithelial cells. We demonstrate their application as a targeted transfection system for cells in culture, and critically, their efficacy in precision tumor killing in vivo.

bioengineering↗

bioRxiv2024

Molecular basis for the assembly of the Vps5-Vps17 SNX-BAR proteins with Retromer

Retromer mediates endosomal retrieval of transmembrane proteins in all eukaryotes and was first discovered in yeast in complex with the Vps5 and Vps17 sorting nexins (SNXs). Cryoelectron tomography (cryoET) studies of Retromer-Vps5 revealed a pseudo-helical coat on membrane tubules where dimers of the Vps26 subunit bind Vps5 membrane-proximal domains. However, the Vps29 subunit is also required for Vps5-Vps17 association despite being far from the membrane. Here, we show that Vps5 binds both Vps29 and Vps35 subunits through its unstructured N-terminal domain. A Pro-Leu (PL) motif in Vps5 binds Vps29 and is required for association with Retromer on membrane tubules in vitro, and for the proper recycling of the Vps10 cargo in Saccharomyces cerevisiae. CryoET of Retromer tubules with Vps5-Vps17 heterodimers show a similar architecture to the coat with Vps5-Vps5 homodimers, however, the spatial relationship between Retromer units is highly restricted, likely due to more limited orientations for docking. These results provide new mechanistic insights into how Retromer and SNX-BAR association has evolved across species.

cell biology↗

bioRxiv2023

Flexing fish: cell surface plasticity in response to shape change in the whole organism

Plasma membrane rupture can result in catastrophic cell death. The skeletal muscle fibre plasma membrane, the sarcolemma, provides an extreme example of a membrane subject to mechanical stress since these cells specifically evolved to generate contraction and movement. A quantitative model correlating ultrastructural remodelling of surface architecture with tissue changes in vivo is required to understand how membrane domains contribute to the shape changes associated with tissue deformation in whole animals. We and others have shown that loss of caveolae, small invaginations of the plasma membrane particularly prevalent in the muscle sarcolemma, renders the plasma membrane more susceptible to rupture during stretch1-3. While it is thought that caveolae are able to flatten and be absorbed into the bulk membrane to buffer local membrane expansion, a direct demonstration of this model in vivo has been unachievable since it would require measurement of caveolae at the nanoscale combined with detailed whole animal morphometrics under conditions of perturbation. Here, we describe the development and application of the "active trapping model" where embryonic zebrafish are immobilised in a curved state that mimics natural body axis curvature during an escape response. The model is amenable to multiscale, multimodal imaging including high-resolution whole animal three-dimensional quantitative electron microscopy. Using the active trapping model, we demonstrate the essential role of caveolae in maintaining sarcolemmal integrity and quantify the specific contribution of caveolar-derived membrane to surface expansion. We show that caveolae directly contribute to an increase in plasma membrane surface area under physiologically relevant membrane deformation conditions.

cell biology↗

bioRxiv2023

Karyopherin mimicry explains how the HIV capsid penetrates nuclear pores

Summary ParagraphHIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus. Remarkably, the intact HIV capsid is over one thousand times greater than the size-limit prescribed by the nuclear pores diffusion barrier. This barrier is a phase-separated condensate in the central channel of the nuclear pore and is comprised of intrinsically-disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG-interactions, cellular karyopherins and their bound cargoes solubilise in this phase to drive nucleocytoplasmic transport. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG-motifs from multiple nucleoporins and that this interaction licenses capsids to penetrate nucleoporin condensates. This karyopherin mimicry model resolves a key conceptual challenge for the role of the HIV capsid in nuclear entry, and explains how an exogenous entity much larger than any known cellular cargo can non-destructively breach the nuclear envelope.

biochemistry↗

bioRxiv2022

Precision super-resolution cryo-correlative light and electron microscopy for rapid in situ structural analyses of optogenetically-positioned organelles

Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a novel cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes in the cell by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. We have developed a protocol where cells can be frozen, imaged by cryo-fluorescence microscopy and ready for batch cryo-ET within a day.

cell biology↗

bioRxiv2021

Antidepressants are complex regulators of lipoprotein(a) macropinocytosis

We recently found that plasminogen receptors regulate the plasma membrane binding and uptake of Lp(a) via macropinocytosis. In this study, we sought to further define lipoprotein(a) [Lp(a)] macropinocytosis, discovering an unexpected role for antidepressants and serotonin in the regulation of this process. We found that the tricyclic antidepressant imipramine enhanced Lp(a) uptake, in contradiction of its published role as a macropinocytosis inhibitor. Extending these experiments to the commonly used serotonin uptake inhibitors (SSRIs) citalopram and sertraline, we found that citalopram stimulated, while sertraline inhibited, Lp(a) uptake. Imipramine and citalopram enhanced cell surface binding of Lp(a) rather than upregulating macropinocytosis. Consistent with imipramine and citalopram boosting extracellular serotonin levels, serotonin itself also enhanced Lp(a) surface binding and uptake. Imipramine and serotonin increased expression of the plasminogen receptor with a C-terminal lysine (PlgRKT), a receptor known to enhance cell surface binding of Lp(a), likely accounting for their effects on Lp(a) uptake. Finally, imipramine and citalopram increased Lp(a) delivery into Rab11 recycling endosomes, but not degradative pathways in the cell. These findings indicate citalopram and imipramine may have utility as a potential Lp(a)-lowering therapeutic in people suffering from depression who often have elevated Lp(a) levels and an increased risk of cardiovascular disease.

cell biology↗

bioRxiv2021

Muscle-specific Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems: mouse and zebrafish. In both models Cavin4 localized to T-tubules and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule-associated BAR domain protein, Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.

cell biology↗

bioRxiv2020

APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. The method allows detection of endogenously expressed proteins in genome-edited cells. We make use of a cell-free expression system to generate membrane particles with a defined quantum of an APEX-fusion protein. These particles can be added to cells to provide an internal standard for estimating absolute density of expressed APEX-fusion proteins.

cell biology↗

bioRxiv2020

An inverted Caveolin-1 topology defines a novel exosome secreted from prostate cancer cells

Caveolin-1 (Cav1) expression and secretion is associated with prostate cancer (PCa) disease progression but the mechanisms underpinning Cav1 release remain poorly understood. Numerous studies have shown Cav1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) Cav1 topology. Here we show that Cav1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive Cav1 secretion. Characterization of the isolated vesicles by electron microscopy, single molecule fluorescent microscopy and proteomics reveals they represent a novel class of exosomes [~]40 nm in diameter containing [~]50-60 copies of Cav1 and strikingly, are released via a non-canonical secretory autophagy pathway. This study provides novel insights into a mechanism whereby Cav1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression.

cell biology↗

bioRxiv2020

An Endocytic Capture Model for Skeletal Muscle T-tubule Formation

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction that shows dysmorphology in a number of genetically inherited muscle diseases. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we have used the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging with three-dimensional electron microscopy we derived a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo we developed a quantitative screen for proteins that associate with and modulate early T-tubule formation including an overexpression screen of the entire zebrafish Rab protein family. We propose a new endocytic capture model involving i) formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma ii) stabilization by myofibrils/sarcoplasmic reticulum and iii) delivery of membrane from the recycling endosome and Golgi complex.

cell biology↗

bioRxiv2020

Dissecting the nanoscale lipid profile of caveolae

Caveolae are specialized domains of the vertebrate cell surface with a well-defined morphology and crucial roles in cell migration and mechanoprotection. Unique compositions of proteins and lipids determine membrane architectures. The precise caveolar lipid profile and the roles of the major caveolar structural proteins, caveolins and cavins, in selectively sorting lipids have not been defined. Here we used quantitative nanoscale lipid mapping together with molecular dynamic simulations to define the caveolar lipid profile. We show that caveolin1 (CAV1) and cavin1 individually sort distinct plasma membrane lipids. Intact caveolar structures composed of both CAV1 and cavin1 further generate a unique lipid nano-environment. The caveolar lipid sorting capability includes selectivities for lipid headgroups and acyl chains. Because lipid headgroup metabolism and acyl chain remodelling are tightly regulated, this selective lipid sorting may allow caveolae to act as transit hubs to direct communications among lipid metabolism, vesicular trafficking and signalling.

biochemistry↗

bioRxiv2019

Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions (DR1-3) that flank positively charged -helical regions (HR1 and HR2). Here we show that the three DR domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between DR and HR regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1 (CAV1), and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and CAV1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

cell biology↗