Biology subjects

Cantaloube, S.

Publications and source records attributed to Cantaloube, S..

7 recordsLinked to original sources

bioRxiv2026

Light-dependent cell fixing with DNA-targeting fluorophores

Light has become of paramount importance for the spatiotemporal control of cell labeling, activity and death. We found that palmatine, a DNA-binding alkaloid, mediates the fixation of live cells upon visible light irradiation. Fixation takes a few seconds or minutes depending on the power used, is accompanied by nuclear fluorescent labeling in target cells, exhibits a high degree of control from cell islets to single cells, yielding cells with intact nucleus and membrane boundaries. 'Optofixation' relies on palmatine-DNA interaction, singlet oxygen photogeneration and lipid peroxidation, producing fixing levels of endogenous aldehydes. We also establish that many commonly used nuclear dyes exhibit optofixation properties across the UV-far red spectrum. Comprising the straightforward inactivation and labeling of native target cells by small fluorophores, this unprecedented, comparatively simple technology has the potential for major applications in biological research.

cell biology↗

bioRxiv2025

PacL-organized membrane-associated effluxosomes coordinate multi-metal resistance in Mycobacterium tuberculosis

Metal ion homeostasis is crucial for bacterial pathogens to withstand metal-induced stress during infection. However, the mechanisms underlying bacterial resistance to metal stress remain incompletely understood, particularly how bacteria coordinate responses to simultaneous exposure to multiple metals. Here, we uncover a previously unrecognized mechanism by which Mycobacterium tuberculosis, the causative agent of tuberculosis, orchestrates a coordinated response to multi-metal stress. We demonstrate that M. tuberculosis assembles dynamic, membrane-associated platforms, organized by PacL proteins, that confer resistance to multiple metals simultaneously. PacL proteins function as scaffolds, clustering multiple P-type ATPase (P-ATPase) pumps, CtpC, CtpG, and CtpV, into functional complexes we term "effluxosomes". Our findings show that PacL proteins are critical for stabilizing CtpG within membrane-associated clusters, conferring cadmium tolerance, while CtpC serves as a backup, promoting cross-resistance to both zinc and cadmium. Using super-resolution microscopy and single-particle tracking, we elucidate the 3D structure and dynamics of effluxosomes in the mycobacterial membrane. We further demonstrate that conserved residues within the transmembrane domain of PacL proteins are crucial for the assembly of dynamic effluxosomes, which are essential for P-ATPase activity. Additionally, we reveal that PacL1 exhibits metallochaperone activity, binding zinc, cadmium, and copper via a conserved C-terminal motif. Proximity labeling further identifies an extensive PacL1 interaction network, encompassing multiple proteins involved in stress adaptation. Our findings introduce effluxosomes as dynamic, membrane-associated efflux machineries that mediate coordinated multi-metal resistance in M. tuberculosis, providing new insights into bacterial metal homeostasis and unveiling potential antimicrobial targets.

microbiology↗

bioRxiv2025

Broad application of plant protein tagging with ALFA tag for nanobody-based imaging and biochemical approaches

Epitope tags are broadly used for detecting, modifying or purifying proteins of interest, but their breadth of application is often rather poor. Recently, the rationally designed ALFA tag and its ALFA Nanobody complements the repertoire of epitope tags and emerged as a highly versatile epitope tag characterized in various animal models and that outperforms existing tags. Here we evaluated the ALFA tag/ALFA Nanobody technology in plants and showcase its application for in planta protein detection across multiple compartments or cellular structures, protein:protein interaction studies, protein purification, induced-proximity approaches, and super-resolution microcopy. Most importantly, we shed light on the edge of ALFA tagging technology for proteins difficult to tag for topological or functionals reasons. We provide the proof of concept of ALFA tag technology for the detection and functional analysis of the Arabidopsis IRT1 iron transporter. Overall, such versatile and validated toolbox of ALFA tag and ALFA Nanobody applications will serve as a resource of great interest for functional studies in plants.

plant biology↗

bioRxiv2025

New Insights into nuclear import and nucleolar localization of yeast RNA exosome subunits

The RNA exosome is a multiprotein complex essential for RNA maturation and degradation. In budding yeast, a nine-subunit protein complex (Exo9) associated with Rrp44 forms Exo10 in the cytoplasm and, in complex with Rrp6, Exo11 in the nucleus. Depending on its subcellular localization, the exosome interacts with different cofactors and RNA substrates. In the cytoplasm, Exo10 associates with the SKI complex via Ski7, while in the nucleus, Exo11 interacts with the TRAMP complex. Within the nucleolus, the exosome participates in ribosomal RNA (rRNA) processing, facilitated by Mtr4-dependent adaptors Utp18 and Nop53. In this manuscript, we have performed a comprehensive study that addresses the targeting mechanism and precise subcellular localization of all members of the Exo11 complex. We observed a high concentration of all Exo11 subunits in the nucleolus and identified the importins Srp1 () and Kap95 ({beta}) as responsible for the nuclear import of Exo9 subunits. Notably, Exo9 subunits localization was not significantly disrupted in the simultaneous absence of NLS-containing subunits Rrp6 and Rrp44, suggesting redundant nuclear import pathways for Exo9. Additionally, we show evidence that Ski7 may play a role in the Exo9 retention in the cytoplasm. To explore the exosome sub-nucleolar localization, we compared Rrp43 with nuclear exosome cofactors and show that it is enriched in the same nucleolar region as Mtr4 and Nop53. In conclusion, our findings provide a detailed characterization of Exo11 distribution, highlight the primary nuclear import mechanisms for Exo9, and reveal the specific localization of the exosome within the granular component (GC) of the yeast nucleolus, suggesting a spatial regulation of the RNA processing pathway. Highlights- Comprehensive study of the localization of all exosome subunits in yeast. - Identification of karyopherins involved in exosome nuclear import. - Ski7 dependent cytoplasmic retention of exosome. - Yeast sub-nucleolar organization of exosome and its cofactors.

molecular biology↗

bioRxiv2023

Enhancer-driven local 3D chromatin domain folding modulates transcription in human mammary tumor cells

The genome is organized in functional compartments and structural domains at the sub-megabase scale. How within these domains interactions between numerous cis-acting enhancers and promoters regulate transcription remains an open question. Here, we determined chromatin folding and composition over several hundred kb around estrogen responsive genes in human breast cancer cell lines following hormone stimulation. Modeling of 5C data at 1.8 kb resolution was combined with quantitative 3D analysis of multicolor FISH measurements at 100 nm resolution and integrated with ChIP-seq data on transcription factor binding and histone modifications. We found that rapid estradiol induction of the progesterone gene (PGR) expression occurs in the context of pre-existing, cell type specific chromosomal architectures encompassing the 90 kb PGR coding region and an enhancer-spiked 5 300 kb upstream genomic region. In response to estradiol, interactions between estrogen-receptor (ER) bound regulatory elements are re-enforced. While initial enhancer - gene contacts coincide with RNA Pol 2 binding and transcription initiation, sustained hormone stimulation promotes ER accumulation creating a regulatory hub stimulating transcript synthesis. In addition to implications for estrogen receptor signaling, we uncover that preestablished chromatin architectures efficiently regulate gene expression upon stimulation without the need for de-novo extensive rewiring of long-range chromatin interactions.

cell biology↗

bioRxiv2020

RIOK2 phosphorylation by RSK promotes synthesis of the human small ribosomal subunit

Ribosome biogenesis lies at the nexus of various signaling pathways coordinating protein synthesis with cell growth and proliferation. This process is regulated by well-described transcriptional mechanisms, but a growing body of evidence indicates that other levels of regulation exist. Here we show that the Ras/mitogen-activated protein kinase (MAPK) pathway stimulates post-transcriptional stages of human ribosome synthesis. We identify RIOK2, a pre-40S particle assembly factor, as a new target of the MAPK-activated kinase RSK. RIOK2 phosphorylation by RSK promotes cytoplasmic maturation of late pre-40S particles, which is required for optimal protein synthesis and cell proliferation. RIOK2 phosphorylation facilitates its release from pre-40S particles and its nuclear re-import, prior to completion of small ribosomal subunits. Our results bring a detailed mechanistic link between the Ras/MAPK pathway and the maturation of human pre-40S particles, which open a hitherto poorly explored area of ribosome biogenesis.

molecular biology↗

bioRxiv2020

Super-resolved live-cell imaging using Random Illumination Microscopy

Super-resolution fluorescence microscopy has been instrumental to progress in biology. Yet, the photo-induced toxicity, the loss of resolution into scattering samples or the complexity of the experimental setups curtail its general use for functional cell imaging. Here, we describe a new technology for tissue imaging reaching a 114nm/8Hz resolution at 30 {micro}m depth. Random Illumination Microscopy (RIM) consists in shining the sample with uncontrolled speckles and extracting a high-fidelity super-resolved image from the variance of the data using a reconstruction scheme accounting for the spatial correlation of the illuminations. Super-resolution unaffected by optical aberrations, undetectable phototoxicity, fast image acquisition rate and ease of use, altogether, make RIM ideally suited for functional live cell imaging in situ. RIM ability to image molecular and cellular processes in three dimensions and at high resolution is demonstrated in a wide range of biological situations such as the motion of Myosin II minifilaments in Drosophila.

bioengineering↗