Institute of Cell Biology

Emerging evidence suggests that Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) bind to the cytoskeleton or focal adhesions (FAs), controlling spatio-temporal Rho GTPase activity through feedback mechanisms. We explore such feedback in the Rho-specific GAP Deleted in Liver Cancer 1 (DLC1), which binds to FAs through mechanosensitive interactions. Using a FRET biosensor, we show that DLC1 loss of function leads to globally increased Rho activity and contractility in fibroblasts. Although Rho activity appears macroscopically steady, individual molecules undergo 'signaling flux'-a dynamic cycle of activation and deactivation. To measure this flux, we built a genetic circuit that enables both optogenetic activation of Rho and simultaneous readout of Rho activity. In cells at mechanical steady state, this reveals that DLC1 globally controls the rate of Rho deactivation, both at FAs and at the plasma membrane. Transient induction of local contractility, however, shows DLC1 associating with and dissociating from FAs during their reinforcement and relaxation, which might provide local positive feedback on Rho activity for robust FA disassembly. Together, our results indicate that DLC1 regulates Rho activity both globally at steady state and locally at FAs under tension, highlighting the complexity of spatio-temporal Rho GTPase signaling.

Kinetoplastids, such as Trypanosoma brucei , are eukaryotes that likely separated from the main lineage at an exceptionally early point in evolution. Consequently, many aspects of kinetoplastid biology differ significantly from other eukaryotic model systems, including yeasts, plants, worms, flies, and mammals. As in many eukaryotes, the T. brucei genome contains repetitive elements at various chromosomal locations, including centromere- and telomere-associated repeats and interspersed retrotransposon elements. T. brucei also contains intermediate-sized and mini-chromosomes that harbour abundant 177 bp repeat arrays and 70 bp repeat elements implicated in Variable Surface Glycoprotein (VSG) gene switching. In many eukaryotes, repetitive elements are assembled in specialised chromatin such as heterochromatin; however, apart from centromere- and telomere-associated repeats, little is known about chromatin-associated proteins that decorate these and other repetitive elements in kinetoplastids. Here, we utilise affinity selection of synthetic TALE DNA binding proteins designed to target specific repeat elements to identify enriched proteins by proteomics. Validating the approach, a telomere repeat binding TelR-TALE identifies many proteins previously implicated in telomere function. Furthermore, the 70R-TALE designed to bind 70 bp repeats indicates that proteins involved in DNA repair are enriched on these elements that reside adjacent to VSG genes. Interestingly, the 177 bp repeat binding 177R-TALE enriches for many kinetochore proteins, suggesting that intermediate-sized and mini-chromosomes assemble kinetochores related in composition to those located on the main megabase chromosomes. This provides a first insight into the chromatin landscape of repetitive regions of the trypanosome genome with relevance for their mechanisms of chromosome integrity, immune evasion, and cell replication.

Peroxisomes are dynamic organelles involved in multiple metabolic pathways that respond to cellular and environmental conditions. Yeasts are a useful model for peroxisome studies, as their growth in media containing peroxisome proliferators, such as methanol, induces peroxisome biogenesis. We analyzed Ogataea polymorpha strains defective in peroxisome biogenesis (pex3Δ) or peroxisomal matrix protein import (pex6Δ). The mutant strains differed in their ability to survive dehydration and rehydration after incubation in peroxisome-inducing conditions, but these differences were not related to resistance to oxidative, hyperosmotic, or heat stress. These results indicate that peroxisomes support efficient entry into anhydrobiosis and subsequent recovery through a mechanism that is independent of general stress tolerance. We hypothesized that this effect is mediated by autophagic processes required for the removal of damaged organelles during desiccation. To test this hypothesis, we compared cells with basal peroxisome levels to cells with increased peroxisome numbers following the induction of peroxisome division. Autophagy was inhibited indirectly by disrupting vacuolar acidification with ammonium chloride. This strategy enabled us to explore how the peroxisome abundance and autophagic activity affect the ability of cells to enter anhydrobiosis and survive recovery.

Yeasts are remarkably versatile microorganisms whose applications reach far beyond their traditional roles in fermentation. In recent years, they have also emerged as valuable tools in areas related to biosecurity and civilian protection. This paper explores how both conventional and non-conventional yeast can contribute to the detection, neutralisation, and prevention of biological and chemical threats. We review the use of recombinant yeast cells in biosensors for heavy metals, organic pollutants and endocrine-disrupting compounds, as well as their role in bioremediation and toxin removal. Special attention is given to the development of yeast-based vaccine platforms, including RNA and antigen-display systems using Saccharomyces cerevisiae and Komagataella phaffii. These technologies illustrate how yeast can bridge biotechnology and security, offering low-cost, scalable and sustainable solutions. However, practical deployment still faces challenges such as biosensor stability, regulatory barriers for genetically modified strains and the need for standardised calibration. Altogether, yeast biotechnology is positioned as a promising and resilient component of future biodefense and environmental protection strategies, strengthening preparedness in the face of hybrid biological and chemical threats.

Protectin DX (PDX) is a member of the superfamily of specialized proresolving mediators and exerts anti-inflammatory actions in animal models; however, its signaling mechanism remains unclear. Here, we demonstrate the analgesic actions of PDX in a mouse model of tibial fracture–induced postoperative pain (fPOP). Intravenous early- and late-phase treatment of PDX (100 ng/mouse) effectively alleviated fPOP. Compared with protectin D1 (PD1)/neuroprotectin D1, DHA, steroids, and meloxicam, PDX provided superior pain relief. While dexamethasone and meloxicam prolonged fPOP, PDX shortened the pain duration. The analgesic effects of PDX were abrogated in Gpr37−/− mice, which displayed deficits in fPOP resolution. PDX was shown to bind GPR37 and induce calcium responses in peritoneal macrophages. LC-MS/MS–based lipidomic analysis revealed that endogenous PDX levels were approximately 10-fold higher than those of PD1 in muscle at the fracture site. PDX promoted macrophage polarization via GPR37-dependent phagocytosis and efferocytosis through calcium signaling in vitro, and it further enhanced macrophage viability and efferocytosis in vivo via GPR37. Finally, PDX rapidly modulated nociceptor neuron responses by suppressing C-fiber–induced muscle reflex in vivo and calcium responses in DRG neurons ex vivo and by reducing TRPA1/TRPV1-induced acute pain and neurogenic inflammation in vivo. Our findings highlight multiple benefits of PDX to manage postoperative pain and promote perioperative recovery.

Upconverting nanoparticles (UCNPs) have attracted much attention in nanomedicine due to their ability to upconvert photons. However, their adverse effects hinder the biomedical applications. In this paper, bisphosphonate-modified poly(isobutylene-alt-maleic acid)-graft-poly(N,N-dimethylacrylamide)-coated NaYF4:Yb,Er,Pr UCNPs (UCNP@PIMAPDMA) nanoparticles were designed, which exhibited luminescence emission simultaneously in the visible and NIR-II regions. The developed UCNPs were characterized by a range of physicochemical methods, including transmission electron and energy dispersive microscopy (TEM and EDAX), dynamic light scattering (DLS), X-ray diffraction analysis (XRD), spectrofluorometry, X-ray photoelectron spectroscopy (XPS), and so forth. The UCNP@PIMAPDMA nanoparticles were also evaluated in cell cultures and experimental animals. The particles showed good biocompatibility with cultured human embryonic kidney HEK293 cells commonly used in toxicological studies. Neat UCNPs were cytotoxic towards these cells, which was confirmed by measuring their viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Blood serum proteins adhered to the surface of UCNP@PIMAPDMA particles, forming a protein corona that may contribute to particle biosafety. After intravenous injection of these particles into laboratory mice, there were no statistically significant changes in body mass of the treated animals. Also, no big adverse effects on blood cell profile, enzymatic and metabolic markers of hepatotoxicity and nephrotoxicity were observed. Finally, the application potential of UCNP@PIMAPDMA nanoparticles was confirmed by successfully imaging the cytoplasm of rat mesenchymal stem cells and rat C6 glioblastoma cells using laser scanning confocal microscopy.

A quarter of the human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop pulmonary TB. We developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: the development of necrotic lung granulomas and determined that the sst1-susceptible phenotype was driven by the aberrant macrophage activation. This study demonstrates that the aberrant response of the sst1-susceptible macrophages to prolonged stimulation with TNF is primarily driven by conflicting Myc and antioxidant response pathways leading to a coordinated failure (1) to properly sequester intracellular iron and (2) to activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled superinduction of Ifnβ and sustained the type I interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within the granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. The upregulation of the Myc pathway in peripheral blood cells of human TB patients was significantly associated with poor outcomes of TB treatment. Thus, Myc dysregulation in activated macrophages results in an aberrant macrophage activation and represents a novel target for host-directed TB therapies.

Abstract Microtubules, composed of α- and β-tubulin polymers, are essential components of the eukaryotic cytoskeleton. They maintain cellular shape and structural integrity and play critical roles in cell division and in intracellular vesicular transport. In Plasmodium, the parasite that causes malaria, nuclear replication during the liver stage is among the fastest known in eukaryotic cells and relies heavily on microtubules for DNA segregation and cytoskeletal organization. Despite their importance, the role of microtubules in liver stage development remains largely unexplored. Here, we investigated microtubule dynamics during liver stage development using a combination of cell and molecular biology techniques, expansion microscopy, and live-cell imaging. By employing antibodies specific for α-tubulin post-translational modifications (PTMs), we found that the Plasmodium sporozoites subpellicular microtubules (SSPM) persist during liver infection, giving rise to liver stage parasite microtubule bundles (LSPMB). These LSPMB form multimeric tubulin structures within hepatocytes and are redistributed to the hemi-spindle poles of parasite nuclei during schizogony. Deletion of the C-terminal region encompassing all known Plasmodium α-tubulin PTM sites prevented sporozoite migration from the mosquito midgut to the salivary glands, effectively blocking parasite transmission. Using Plasmodium microtubule-specific depolymerisation drugs, we found that while LSPMB are stable in sporozoites, they exhibit dynamic behavior during hepatocyte infection. Given the regulatory role of PTMs in microtubule dynamics, we generated parasite mutants by substituting and deleting key α-tubulin C-terminal residues involved in PTMs. Substitution of the polyglutamylation site with alanine and deletion of the C-terminal tyrosination/detyrosination motifs impaired parasite growth during liver infection. Together, our findings reveal extensive microtubule remodeling during liver stage development and establish α-tubulin C-terminal modifications as critical regulators of both intracellular development and parasite transmission of Plasmodium parasites.

Abstract Microtubules, composed of α- and β-tubulin polymers, are essential components of the eukaryotic cytoskeleton. They maintain cellular shape and structural integrity and play critical roles in cell division and in intracellular vesicular transport. In Plasmodium, the parasite that causes malaria, nuclear replication during the liver stage is among the fastest known in eukaryotic cells and relies heavily on microtubules for DNA segregation and cytoskeletal organization. Despite their importance, the role of microtubules in liver stage development remains largely unexplored. Here, we investigated microtubule dynamics during liver stage development using a combination of cell and molecular biology techniques, expansion microscopy, and live-cell imaging. By employing antibodies specific for α-tubulin post-translational modifications (PTMs), we found that the Plasmodium sporozoites subpellicular microtubules (SSPM) persist during liver infection, giving rise to liver stage parasite microtubule bundles (LSPMB). These LSPMB form multimeric tubulin structures within hepatocytes and are redistributed to the hemi-spindle poles of parasite nuclei during schizogony. Deletion of the C-terminal region encompassing all known Plasmodium α-tubulin PTM sites prevented sporozoite migration from the mosquito midgut to the salivary glands, effectively blocking parasite transmission. Using Plasmodium microtubule-specific depolymerisation drugs, we found that while LSPMB are stable in sporozoites, they exhibit dynamic behavior during hepatocyte infection. Given the regulatory role of PTMs in microtubule dynamics, we generated parasite mutants by substituting and deleting key α-tubulin C-terminal residues involved in PTMs. Substitution of the polyglutamylation site with alanine and deletion of the C-terminal tyrosination/detyrosination motifs impaired parasite growth during liver infection. Together, our findings reveal extensive microtubule remodeling during liver stage development and establish α-tubulin C-terminal modifications as critical regulators of both intracellular development and parasite transmission of Plasmodium parasites.

All living organisms have developed strategies to respond to chromosomal damage and preserve genome integrity. One such response is the repair of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA lesions. In Escherichia coli, DSBs are repaired via RecBCD-dependent homologous recombination. RecBCD is essential for accurate chromosome maintenance, but its over-expression can lead to reduced DNA repair ability. This apparent paradox suggests that RecBCD copy numbers may need to be tightly controlled within an optimal range. Using single-molecule fluorescence microscopy, we have established that RecB is present in very low abundance at mRNA and protein levels. RecB transcription shows high fluctuations, yet cell-to-cell protein variability remains remarkably low. Here, we show that the post-transcriptional regulator Hfq binds to recB mRNA and downregulates RecB protein translation in vivo. Furthermore, specific disruption of the Hfq-binding site leads to more efficient translation of recB mRNAs. In addition, we observe a less effective reduction of RecB protein fluctuations in the absence of Hfq. This fine-tuning Hfq-mediated mechanism might have the underlying physiological function of maintaining RecB protein levels within an optimal range.