Biology subjects

Gonzalez-Jimenez, P.

Publications and source records attributed to Gonzalez-Jimenez, P..

4 recordsLinked to original sources

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↗

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↗