Biology subjects

Kleman, J.-P.

Publications and source records attributed to Kleman, J.-P..

11 recordsLinked to original sources

bioRxiv2026

Dimeric Alix nucleates ESCRT-III CHMP4 polymerization

Alix is a key adaptor protein of the endosomal sorting complex required for transport (ESCRT) membrane remodeling machinery. Although Alix-mediated recruitment of the ESCRT-III subunit CHMP4 is well established, the molecular mechanisms underlying Alix activation and ESCRT-III polymerization remain poorly understood. Here, we present the crystal structure of the dimeric Alix V-domain (Alix-V) in complex with a llama nanobody. Dimerization is mediated by domain swapping, generating an X-shaped flexible conformation. We demonstrate that Alix forms dimers in vivo and provide evidence for the recruitment of dimeric Alix to plasma membrane repair sites and the cytokinetic midbody. Notably, a mutation disrupting Alix dimerization impairs plasma membrane repair. Furthermore, high-speed AFM experiments reveal that dimeric Alix, but not the monomeric form, nucleates CHMP4B filament polymerization. Our data establish that dimeric Alix is the active form responsible for nucleating two ESCRT-III CHMP4 filaments, whose geometry creates a platform for the recruitment of downstream ESCRT-III components required to assemble the active membrane remodeling and fission machinery.

biochemistry↗

bioRxiv2025

Filamentation-driven peripheral clustering of the inducible lysine decarboxylase is crucial for E. coli acid stress response

Bacteria have evolved numerous sophisticated acid stress response strategies to withstand fluctuating environmental pH, with enterobacterial inducible amino acid decarboxylases playing a major role. The lysine decarboxylase LdcI catalyses lysine-to-cadaverine conversion coupled to proton consumption and carbon dioxide release, thereby buffering cytoplasmic and extracellular pH. Our previous study showed that Escherichia coli LdcI forms intracellular patches under mild acid stress, and that at the corresponding acidic pH, purified LdcI polymerises into filaments, suggesting a potential role for polymerisation in its function. Here, we investigate the physiological relevance of LdcI filamentation in E. coli using 3D super-resolution fluorescence microscopy and a structure-based LdcI polymerisation-deficient E. coli mutant strain. We establish a semi-automated workflow for detection of clusters within a bacterial cell as well as quantitative analysis of their volume and localisation. We demonstrate that LdcI predominantly clusters at the cell periphery. We then show that disrupting LdcI polymerisation markedly reduces cluster size without significantly affecting localisation, suggesting that acid-stress induced LdcI clustering in vivo is driven by the enzymes filamentation. Growth and pH measurements reveal that the mutant strain exhibits reduced fitness and impaired extracellular buffering compared to the wild type, indicating that LdcI polymerisation enhances E. coli acid stress response. Our findings provide strong evidence that LdcI filamentation is a key regulatory feature of the E. coli capacity to counteract acid stress, optimising enzymatic activity by spatially organising LdcI within the cytoplasm. More broadly, this work adds to a growing body of evidence supporting the functional significance of metabolic enzyme self-assembly and its implications for bacterial stress adaptation. Author summaryAmino acid decarboxylases confer enterobacteria the ability to survive under acidic conditions. Kirchner et al. show that polymerisation of the acid-stress inducible lysine decarboxylase LdcI within E. coli cells leads to its peripheral intracellular clustering and fine-tunes its enzymatic activity in vivo.

microbiology↗

bioRxiv2024

Combining live cell fluorescence imaging with in situ cryo electron tomography sheds light on the septation process in Deinococcus radiodurans

Cell division is a fundamental biological process that allows a single mother cell to produce two daughter cells. In bacteria, different modes of cell division have been reported that are notably associated with distinctive cell shapes, but in all cases, division involves a step of septation, corresponding to the growth of a new dividing cell wall, followed by splitting of the two daughter cells. The radiation-resistant Deinococcus radiodurans is a spherical bacterium protected by a thick and unusual cell envelope. It has been reported to divide using a distinctive mode of septation in which two septa originating from opposite sides of the cell progress with a flat leading edge until meeting and fusing at mid-cell. In the present study, we have combined conventional and super-resolution fluorescence microscopy of live bacteria with in situ cryogenic electron tomography of bacterial lamellae to investigate the septation process in D. radiodurans. This work provides important insight into (i) the complex architecture of the cell envelope of this bacterium, (ii) the sliding doors septation process and (iii) the molecular mechanisms underlying septal growth and closure.

microbiology↗

bioRxiv2024

Phototropin connects blue light perception to starch metabolism in green algae

In photosynthetic organisms light acts as an environmental signal to control their development and physiology, and as energy source to drive the conversion of CO2 into carbohydrates used for growth or storage. The main storage carbohydrate in green algae is starch, which accumulates during the day and is broken down at night to meet cellular energy demands. The signalling role of light quality in the regulation of starch accumulation remains unexplored. Here, we identify PHOTOTROPIN-MEDIATED SIGNALLING KINASE 1 (PMSK1) as a key regulator of starch metabolism in Chlamydomonas reinhardtii. In its phosphorylated form (PMSK1-P), it activates GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAP1), promoting starch biosynthesis. We show that blue light, perceived by PHOTOTROPIN, induces PMSK1 dephosphorylation that in turn represses GAP1 mRNA levels and reduces starch accumulation. These findings reveal a novel blue light-mediated signaling pathway that advances our understanding of photoreceptor-controlled carbon metabolism in microalgae. One-Sentence SummaryBlue light perception by PHOTOTROPIN triggers kinase-mediated signaling to inhibit starch accumulation in the green alga Chlamydomonas.

plant biology↗

bioRxiv2023

Stress-induced nucleoid remodeling in Deinococcus radiodurans is associated with major changes in HU abundance and dynamics

Bacteria have developed a wide range of strategies to respond to stress, one of which is the rapid large-scale reorganization of their nucleoid, which is often associated with a major reprogramming of the gene expression profile. Nucleoid associated proteins (NAPs) are believed to be major actors in this process, but the molecular mechanisms underlying stress-induced nucleoid remodeling remain poorly understood. Here, using the radiation resistant bacterium, D. radiodurans, as a model, and advanced fluorescence microscopy approaches, we examined the changes in nucleoid morphology and compaction induced by either entry into stationary phase or exposure to UV-C light, and characterized the associated changes in abundance and dynamics of the major NAP in D. radiodurans, the heat-unstable (HU) protein. While both types of stress induced a similar macroscopic rearrangement of the nucleoid into a more compact structure, HU diffusion was significantly reduced in stationary phase cells, but was instead dramatically increased following exposure to UV-C, suggesting that the underlying mechanisms of remodeling are distinct. Furthermore, a detailed comparison of the cellular response to sublethal and lethal doses of UV-C light revealed that UV-induced nucleoid remodeling involves a rapid nucleoid condensation step associated with increased HU diffusion and abundance, followed by a slower decompaction phase to restore normal nucleoid morphology and HU dynamics, before cell growth and division can resume. Together, these findings shed light on the diversity and complexity of stressed-induced nucleoid remodeling processes in bacteria.

microbiology↗

bioRxiv2023

An inducible ESCRT-III inhibition tool to control HIV-1 budding

HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated auto-cleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins with variable modification of Gag VLP budding upon drug administration. Notably, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted a minor effect and synergized with CHMP2A-NS3. Localization studies demonstrated the re-localization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.

microbiology↗

bioRxiv2022

Biophysical ordering transitions underlie genome 3D re-organization during cricket spermiogenesis.

Spermiogenesis is a radical process of differentiation whereby sperm cells acquire a compact and specialized morphology to cope with the constraints of sexual reproduction while preserving their main cargo, an intact copy of the paternal genome. In animals, this often involves the replacement of most histones by sperm-specific nuclear basic proteins (SNBPs). Yet, how the SNBP-structured genome achieves compaction and accommodates shaping remain largely unknown. Here, we exploited confocal, electron and super-resolution microscopy observations, coupled with polymer modeling simulations to identify the higher-order architecture of sperm chromatin in the needle-shaped nucleus of the emerging model cricket Gryllus bimaculatus. Accompanying spermatid differentiation and shaping, the SNBP-based genome was strikingly reorganized as ~25nm-thick fibers orderly coiled along the elongated nucleus axis. This chromatin spool was further found to achieve large-scale helical twisting in the final stages of spermiogenesis, favoring its ultracompaction. Through a combination of microscopy observations and polymer simulations, we revealed that these dramatic transitions may be recapitulated by a surprisingly simple biophysical principle based on a nucleated rigidification of chromatin linked to the histone-to-SNBP transition within a confined nuclear space. Our work highlights a unique, liquid crystal-like mode of higher-order genome organization in ultracompact cricket sperm completely distinct from nucleosomal chromatin, and establishes a multidisciplinary methodological framework to explore the diversity of non-canonical modes of DNA organization. SIGNIFICANCE STATEMENTAnimal sperm cells are highly compact and atypically shaped compared to other cell types. How DNA is packaged and organized in the 3D space of sperm cell nuclei to cope with these constraints is poorly understood. In this work, we identified an original and elegant solution to this problem in crickets, whereby DNA fibers orderly spool and twist to fit into ultracompact, needle-shaped sperm cells. To understand this reorganization, we modeled DNA fibers in the nucleus as polymers and found that a relatively simple mechanism through which fibers become more rigid bit by bit can largely recapitulate our observations. Our multidisciplinary work highlights a simple solution to compact DNA to extreme levels in specialized nuclei.

cell biology↗

bioRxiv2022

Structural basis of CHMP2A-CHMP3 ESCRT-III polymer assembly and membrane cleavage

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo electron microscopy structures of membrane-coated CHMP2A-CHMP3 filaments of two different diameters at 3.3 and 3.6 [A] resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence towards the tube interior. Inter-filament interactions are electrostatic, which facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high speed atomic force microscopy imaging corroborate that CHMP2A-CHMP3 polymers and VPS4 can constrict and cleave narrow membrane tubes, thus acting as a minimal membrane fission machinery.

cell biology↗

bioRxiv2022

The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to antibiotics under anaerobiosis and has a major impact on cell membrane regulation.

microbiology↗

bioRxiv2020

Supramolecular assembly of the E. coli LdcI upon acid stress

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statement Bacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2019

Cell morphology and nucleoid dynamics in dividing D. radiodurans

Our knowledge of bacterial nucleoids originates mostly from studies of rod- or crescent-shaped bacteria. Here, we reveal that Deinococcus radiodurans, a relatively large, spherical bacterium, possessing a multipartite genome, and well-known for its radioresistance, constitutes a valuable system for the study of nucleoids in cocci. Using advanced microscopy, we show that as D. radiodurans progresses through its cell cycle, it undergoes coordinated morphological changes at both the cellular and nucleoid level. D. radiodurans nucleoids were found to be highly condensed, but also surprisingly dynamic, adopting multiple distinct configurations and presenting a novel chromosomal arrangement in which oriC loci are radially distributed around clustered ter sites maintained at the centre of cells. Single-molecule and ensemble studies of the abundant histone-like HU protein suggest that its loose binding to DNA may contribute to this remarkable plasticity. These findings clearly demonstrate that nucleoid organization is complex and tightly coupled to cell cycle progression.

microbiology↗