Biology subjects

Perez-Sala, D.

Publications and source records attributed to Perez-Sala, D..

12 recordsLinked to original sources

bioRxiv2026

Mimicking two posttranslational modifications associated with oxidative stress affords phase separation of vimentin

Biomolecular condensates are membraneless compartments critical for the functional organization of cellular macromolecules in essential processes such as cell division, gene transcription or stress responses. We previously reported that vimentin filaments remodel into phase separated biomolecular condensates upon oxidative stress. This process requires vimentin single cysteine, C328, suggesting the involvement of oxidative modifications of this residue. Here, we aimed to generate vimentin condensates by inserting mutations mimicking posttranslational modifications associated with oxidative stress. In vimentin deficient cells, a cysteine oxidation mimetic mutant, vimentin C328D, formed only elongated particles or short filaments that evolved towards droplets upon serum deprivation or treatment with the oxidant diamide. Among vimentin posttranslational modifications rapidly responding to these stimuli, glycosylation confers filament stability whereas phosphorylation promotes disassembly. We observed that the O-deglycosylation inhibitor thiamet G, and the kinase inhibitors staurosporine and H-89, attenuated diamide-elicited vimentin C328D droplet formation, suggesting a potential glycosylation/phosphorylation interplay in this effect. Indeed, introducing phosphomimetic residues at certain single vimentin glycosylation and/or phosphorylation sites induced the formation of droplets, only if combined with the C328D mutation. In particular, the vimentin S49D,C328D mutant formed condensates that were reversibly dispersed by dilution through hypotonic shock. Therefore, mimicking C328 oxidation and S49 phosphorylation was sufficient to elicit vimentin phase separation. In vitro, purified vimentin S49D,C328D polymerized into a mixture of aberrant filaments and aggregates, which, in the presence of crowders, evolved towards paracrystals or clusters of beaded assemblies depending on pH. These findings highlight the role of C328 perturbations in the formation of biomolecular condensates and suggest a modulatory role of glycosylation/phosphorylation, thus shedding light on the processes regulating vimentin phase separation.

cell biology↗

bioRxiv2025

The pathogenic mutant vimentin L387P disrupts endoplasmic reticulum organization and proteostasis

The organization and dynamic regulation of the vimentin intermediate filament network are essential for diverse cellular processes, including cell division, cytoskeletal crosstalk, and organelle positioning. In particular, the vimentin network is required to maintain a compact perinuclear endoplasmic reticulum (ER) that supports the ER quality-control compartment. Here, we show that the vimentin variant c.1160T>C (L387P), identified in a progeroid syndrome, disrupts ER organization and function. In SW13/cl.2 cells, vimentin L387P forms aggregates containing cisternal-like structures of predominant perinuclear localization that associate with pronounced nuclear distortion. These aggregates sequester chaperones, the SEL1L/HRD1 complex, and the 20S proteasomal subunit, while excluding RNF26 and VCP/p97. This extensive redistribution of ER-associated degradation components coincides with impaired proteostasis and reduced free Ca2+ in intra-organelle compartments, indicating compromised ER functionality. Altogether, these findings suggest that the L387P mutation drives extensive ER disorganization and highlight the importance of an intact vimentin network for ER homeostasis.

cell biology↗

bioRxiv2024

Lysosomal function, resistance to stress and repair are compromised by expression of the Alexander disease GFAP R239C mutant

Intermediate filaments are critical regulators of cell responses and organizers of cellular structures. GFAP (glial fibrillary acidic protein) is an intermediate filament protein mainly expressed in astrocytes. GFAP mutations are associated with Alexander disease (AxD), a type of leukodystrophy that causes degeneration of astrocytes and ultimately neurodegeneration. AxD astrocytes display protein aggregation, proteostasis defects and altered organelle homeostasis. We previously showed that expression of GFAP AxD mutants in astrocytes provoked mitochondrial alterations and oxidative stress. Here we have used an astrocytoma cell model to explore the impact of GFAP AxD mutants on the lysosomal degradation pathway. Expression of GFAP AxD mutants in this model elicits marked alterations in lysosomal distribution. Cells expressing the GFAP R239C mutant display defective lysosomal activity and intraluminal acidification. Lysosomes are primary sites of oxidative damage. Moreover, expression of GFAP R239C increases susceptibility of lysosomes to oxidative stress, resulting in a greater loss of lysosomal "mass" and compromised membrane integrity, as revealed by increased intraluminal recruitment of galectins, with respect to cells expressing GFAP wt. Notably, lysosomes in GFAP R239C expressing cells are also more vulnerable to chemically-induced rupture. Interestingly, lysosomes of cells expressing GFAP wt are able to rapidly recover after removal of the damaging agent. In sharp contrast, recovery of acidic vesicles is severely impaired in cells expressing GFAP R239C, suggesting a defect in lysosomal repair. Taken together, our results show that expression of the GFAP AxD mutant is sufficient to deeply perturb lysosomal distribution, function and repair. These alterations could contribute to proteostasis defects and cellular toxicity in AxD.

cell biology↗

bioRxiv2024

Formation of vimentin biomolecular condensate-like structures under oxidative stress

The intermediate filament protein vimentin performs a key role in cytoskeletal interplay and dynamics, and in cellular responses to stress. The vimentin monomer possesses a central -helical rod domain flanked by N- and C-terminal low complexity domains. Interactions between this type of domains play an important function in the formation of phase-separated biomolecular condensates, which in turn are critical for the organization of cellular components. Vimentin filaments undergo distinct and versatile reorganizations in response to diverse stimuli. Here we show that certain oxidants and electrophiles, including hydrogen peroxide and diamide, elicit the remodeling of vimentin filaments into small particles. Diamide in particular, induces a fast conversion of filaments into circular, motile dots, for which the presence of the single vimentin cysteine residue, C328, is critical. This effect is reversible, and filament reassembly can be noticed within minutes of removal of the oxidant. Diamide-elicited structures can recover fluorescence after photobleaching. Moreover, fusion of cells expressing differentially tagged vimentin allows the detection of dots positive for both tags, suggesting that vimentin dots can merge upon cell fusion. The aliphatic alcohol 1,6-hexanediol, known to alter interactions between low complexity domains, readily dissolves diamide-elicited vimentin dots at low concentrations, whereas at high concentrations it disrupts vimentin filaments. Taken together, these results indicate that vimentin oxidation can promote a fast and reversible filament remodeling into biomolecular condensate-like structures. Moreover, we hypothesize that this reorganization into droplet-like structures could play a protective role against irreversible damage by oxidative stress.

cell biology↗

bioRxiv2024

Key role of vimentin in the organization of the primary cilium

We previously reported the presence of the intermediate filament vimentin at the primary cilium of lung cancer epithelial cells. In this study we further demonstrate that vimentin is intimately intertwined with acetylated tubulin at this structure. Interestingly, although vimentin can be detected along the whole length of the primary cilium, phospho-serine 56 vimentin is found particularly enriched at its basal region in A549 lung cancer cells. Vimentin appears to play a pivotal role in ciliogenesis, since its depletion in MEF or in A549 cells results in a lower proportion of cells displaying primary cilia and recognizable basal bodies. Furthermore, the detectable cilia in vimentin depleted cells are shorter. In addition, the centriolar structure appears disrupted in vimentin deficient cells, as indicated by an abnormal distribution of {gamma}- and acetylated tubulin. Moreover, these cells display a defective organization of the pericentriolar material, characterized by a marked decrease in the levels of pericentrin and a diffuse distribution of Rab11. Taken together, our results show that vimentin is present at the primary cilium and suggest that it plays an important role in cilium structure and biogenesis, since its depletion leads to marked morphological defects and altered organization of key elements of this structure.

cell biology↗

bioRxiv2023

Intracellular pH modulates vimentin remodeling in response to oxidants

Vimentin plays key roles in cell mechanosensing, cytoskeletal crosstalk and stress responses, and is finely tuned by posttranslational modifications. The vimentin single cysteine residue, C328, is a hotspot for modification and essential for filament remodeling by oxidants and electrophiles. With a pKa near physiological pH, C328 reactivity could be sensitive to cellular pH fluctuations. Here, we show that C328 modifications and vimentin reorganization by various reactive agents are modulated in response to pH variations. Lowering intracellular pH prevents, whereas intracellular alkalinization potentiates vimentin network disruption by oxidative and electrophilic species, including diamide, hydrogen peroxide and hydroxynonenal. The protective effect associated with low pH is selective for vimentin since it does not preclude oxidant-elicited disruption of actin or tubulin structures. Vimentin C328A and C328H mutants are resistant to disruption under all pH conditions, which highlights the importance of the thiol group at this position for the sensitizing effect of alkaline pH. Chemogenetic and optogenetic modulation of cellular pH allow spatiotemporal tuning of vimentin susceptibility to oxidants, suggesting the potential role of pH in the regulation of vimentin organization at precise locations. Alkalinization and generation of reactive oxygen species cooperate at the cell front during migration. We show that vimentin disassembly at cell edges of migrating fibroblasts, and lamellipodia formation, are affected by pH changes and the presence of C328. We propose that vimentin C328 could behave as a coincidental pH and redox responsive element, contributing to the precise regulation of vimentin assembly by the concert of these factors, illustrating the pH dependence of cysteine-mediated redox signaling.

cell biology↗

bioRxiv2023

Vimentin cysteine 328 modifications finely tune network organization and influence actin remodeling under oxidative and electrophilic stress

Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a "sensor" transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin.

cell biology↗

bioRxiv2021

Immunolocalization studies of vimentin and ACE2 on the surface of cells exposed to SARS-CoV-2 Spike proteins

The Spike protein from SARS-CoV-2 mediates docking of the virus onto cells and contributes to viral invasion. Several cellular receptors are involved in SARS-CoV-2 Spike docking at the cell surface, including ACE2 and neuropilin. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Here we have explored the binding of Spike protein constructs to several cell types using low-temperature immunofluorescence approaches in live cells, to minimize internalization. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which showed scarce colocalization with a lipid raft marker, but consistent coincidence with ACE2. Under our conditions, vimentin immunoreactivity appeared as spots or patches unevenly distributed at the surface of diverse cell types. Remarkably, several observations including potential antibody internalization and adherence to cells of vimentin-positive structures present in the extracellular medium exposed the complexity of vimentin cell surface immunoreactivity, which requires careful assessment. Notably, overall colocalization of Spike and vimentin signals markedly varied with the cell type and the immunodetection sequence. In turn, vimentin-positive spots moderately colocalized with ACE2; however, a particular enrichment was detected at elongated structures positive for acetylated tubulin, consistent with primary cilia, which also showed Spike binding. Thus, these results suggest that vimentin-ACE2 interaction could occur at selective locations near the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking.

cell biology↗

bioRxiv2021

Elucidating vimentin interaction with zinc ions and its interplay with oxidative modifications through crosslinking assays and molecular dynamics simulations

The intermediate filament protein vimentin is involved in essential cellular processes, including cell division and stress responses. Vimentin oxidative modifications impact network reorganization and its single cysteine residue, Cys328, acts as a redox sensor. Vimentin binds zinc, which influences its assembly by undefined mechanisms. Here, results from combined biochemical and molecular dynamics studies support that zinc ions interact with Cys328 in its thiolate form, whereas Glu329 and Asp331 stabilize zinc coordination. Vimentin oxidation can induce disulfide crosslinking, implying a close proximity of cysteine residues in certain vimentin associations, validated by our computational models. Notably, micromolar zinc concentrations selectively prevent Cys328 alkylation and crosslinking. These effects are not mimicked by magnesium, consistent with the fewer magnesium ions hosted at the cysteine region. Altogether, our results pinpoint the region surrounding Cys328, highly conserved in type III intermediate filaments, as a hot spot for zinc binding, which modulates Cys328 reactivity and vimentin assembly.

biochemistry↗

bioRxiv2021

Dynamic posttranslational modifications of cytoskeletal proteins unveil hot spots under nitroxidative stress

The cytoskeleton is a supramolecular structure consisting of interacting protein networks that support cell dynamics in essential processes such as migration and division, as well as in responses to stress. Fast cytoskeletal remodeling is achieved with the participation of regulatory proteins and posttranslational modifications (PTMs). Redox-related PTMs are emerging as critical players in cytoskeletal regulation. Here we used a cellular model of mild nitroxidative stress in which a peroxynitrite donor induced transient changes in the organization of three key cytoskeletal proteins, i.e., vimentin, actin and tubulin. Nitroxidative stress-induced reconfiguration of intermediate filaments, microtubules and actin structures were further correlated with their PTM profiles and dynamics of the PTM landscape. Using high-resolution mass spectrometry, 62 different PTMs were identified and relatively quantified in vimentin, actin and tubulin proteins, including 12 enzymatic, 13 oxidative and 2 nitric oxide-derived modifications as well as 35 modifications by carbonylated lipid peroxidation products, thus evidencing the occurrence of a chain reaction with formation of reactive species and the activation of multiple signaling pathways. Our results unveil the presence of certain modifications under basal conditions and their modulation in response to stress in a target-, residue- and reactive species-dependent manner. Moreover, we identified protein PTM "hot spots", such as the single cysteine residue of vimentin, supporting its role in PTM cross-talk and redox sensing. Finally, identification of novel PTMs in these proteins may pave the way for unveiling novel cytoskeleton regulatory mechanisms.

cell biology↗

bioRxiv2019

Amoxicillin inactivation by thiol-catalyzed cyclization reduces protein haptenation and antibacterial potency

Serum and cellular proteins are targets for the formation of adducts with the {beta}-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other {beta}-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.

pharmacology and toxicology↗

bioRxiv2018

Vimentin filaments interact with the mitotic cortex allowing normal cell division

The vimentin network displays remarkable plasticity to support basic cellular functions. Here, we show that in several cell types vimentin filaments redistribute to the cell periphery during mitosis, forming a robust scaffold interwoven with cortical actin and affecting the mitotic cortex properties. Importantly, the intrinsically disordered tail domain of vimentin is essential for this redistribution, which allows normal mitotic progression. A tailless vimentin mutant forms curly bundles, which remain entangled with dividing chromosomes leading to mitotic catastrophes or asymmetric partitions. Serial deletions of the tail domain induce increasing impairments of cortical association and mitosis progression. Disruption of actin, but not of microtubules, mimics the impact of tail deletion. Pathophysiological stimuli, including HIV-protease and lipoxidation, induce similar alterations. Interestingly, filament integrity is dispensable for cortical association, which also occurs in vimentin particles. These results unveil novel implications of vimentin dynamics in cell division by means of its interplay with the mitotic cortex.

cell biology↗