Membrane repair and extracellular vesicles

The group’s activities focus on the pathophysiological study of membrane repair and extracellular vesicles. Dysregulation of membrane repair—a crucial process for cells subjected to mechanical stress—leads to numerous diseases such as muscular dystrophies and cancer. Extracellular vesicles are membrane-bound vesicles released by all cell types, involved in a wide range of pathophysiological processes such as cell communication, coagulation, and cancer, and they hold potential applications as biomarkers or therapeutic agents.

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BioFAP Team

Membrane Repair and Extracellular Vesicles Group

Membrane Repair and Muscular Dystrophies

Anthony Bouter

A defect in the membrane repair process leads to cell death and may contribute to the development of degenerative diseases, such as muscular dystrophies. The same primary mutation often results in variable disease progression due to the presence of secondary factors. However, most of these secondary factors remain to be identified. Our project aims to study the involvement of annexins, macrophages, and fibro-adipogenic progenitors in the development of muscular dystrophies, particularly Duchenne muscular dystrophy, dysferlinopathies, caveolinopathies, and facioscapulohumeral muscular dystrophy.

Sarcolemma microrupture is a physiological phenomenon induced by the mechanical stresses to which muscle fibers are subjected. Normal skeletal muscle cells are able to repair these ruptures within minutes, thanks to a calcium-dependent protein machinery. This machinery notably involves proteins from the annexin and ferlin families. With the support of AFM-Téléthon, we have shown that annexins (Croissant et al., Int. J. Mol. Sci., 2021; Croissant et al., Membranes, 2022), specifically ANXA5 (Carmeille et al., Biochim. Biophys. Acta, 2016) and ANXA6 (Croissant et al., Cells, 2020), play a key role in the membrane repair of human skeletal muscle cells.

Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, which impair membrane repair and lead to degeneration of skeletal muscle (Bansal et al., Nature 2003). It has been shown that disease severity is associated with increased expression of ANXA2 by muscle cells and its release into the extracellular environment, which promotes adipogenesis in skeletal striated muscle (Hogarth et al., Nat Commun 2019).

Our project aims to study the involvement of annexins, macrophages, and fibroadipogenic progenitors in the development of muscular dystrophies. Focusing on ANXA1 to ANXA6, we investigate human skeletal muscle cell lines as well as muscle biopsies obtained from control individuals or patients with various muscular dystrophies. A collection of muscle biopsies from control subjects and patients with muscular dystrophy has been established at the University Hospital of Bordeaux, in collaboration with Prof. Marie-Laure Martin-Négrier and Dr. Guilhem Solé, and has been approved for research use.

Techniques implemented:

Cell culture
Fluorescence microscopy
Immunohistochemistry/Immunocytochemistry (fluorescence)
Western blot
Laser ablation
Shear stress

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Extracellular Vesicles – Pathophysiological Roles

Alain Brisson

Extracellular vesicles (EVs) are membrane-bound vesicles released by all cells, representing a newly discovered class of cellular components. EVs have attracted major interest due to their involvement in a wide range of physiological and pathological processes, as well as their potential applications as biomarkers or therapeutic agents.

Our research focuses on EVs derived from blood cells, with the following objectives:

To provide a detailed characterization – including size, phenotype, and concentration – of the various EV populations present in the blood of healthy individuals, as well as in individuals with sickle cell disease or pre-eclampsia;
To identify markers of sickle cell disease or pre-eclampsia, with particular attention to EVs exhibiting pro-coagulant activity. Innovative approaches, including immuno-gold cryo-electron microscopy and flow cytometry, are applied and further developed to address these projects.

Membrane repair and preeclampsia

Anthony Bouter

Preeclampsia is a major hypertensive disorder affecting up to 8% of pregnancies. This condition is often associated with intrauterine growth restriction, which can lead to fetal death in extreme cases. It is also a major cause of maternal mortality in developing countries. The etiology of preeclampsia remains unknown. In collaboration with Daniel Vaiman’s team (Cochin Institute, Paris), we are characterizing the membrane repair and cell fusion machinery of trophoblasts, two processes crucial in placental biology. We hypothesize that failures in these cellular mechanisms lead to trophoblast death by necrosis and may be one of the causes of preeclampsia development.

In the event of trophoblastic cell necrosis, the cellular debris and extracellular vesicles released into the maternal blood carry signaling molecules responsible for inducing maternal hypertension and inflammation, which are characteristic features of preeclampsia.

The rupture of the plasma membrane is a physiological event that occurs in cells exposed to mechanical stress. These cells possess a molecular machinery that enables rapid membrane repair (McNeil & Steinhardt, Annu. Rev. Cell Dev. Biol. 2003). The absence of repair leads to cell death and may contribute to the development of degenerative diseases (McNeil & Steinhardt, Annu. Rev. Cell Dev. Biol. 2003). We have contributed to the current understanding of membrane repair, notably by revealing the central role played by Annexin-A5 (ANXA5) in murine perivascular cells, human placental cells, and skeletal muscle cells (Bouter et al., Nat. Commun. 2011; Carmeille et al., Biochim. Biophys. Acta 2015; Bouter et al., Placenta 2015; Carmeille et al., Biochim. Biophys. Acta 2016).

As part of a research project funded by the ANR, in collaboration with Daniel Vaiman’s team (Institut Cochin, Paris), we aim to demonstrate that one of the causes of preeclampsia is a defect in the membrane repair and/or fusion machinery of placental cells. The human placental trophoblast, an epithelium-like tissue covering the placenta, acts as a barrier separating maternal and fetal blood.

It is a two-layered structure, with the outer layer forming a syncytium, the syncytiotrophoblast (ST), which expands and regenerates through the fusion of the underlying mononucleated cytotrophoblasts (CT) throughout pregnancy.

We have shown that both CT and ST cells possess a membrane repair machinery (Carmeille et al., Biochim. Biophys. Acta, 2015). We also demonstrated that human placental cells, modified to mimic preeclamptic cells, exhibit defective membrane repair due to the absence of ANXA1 (Ducat et al., iScience, 2020). We are working to identify the molecular actors responsible for disease development in preeclamptic patients.

Techniques used:

Cell culture
Fluorescence microscopy
Immunocytofluorescence
Western blot
CRISPR/Cas9, shRNA
Laser ablation
Shear stress

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Réparation membranaire et cancer

Anthony Bouter

During tumor invasion and metastasis, cancer cells are exposed to significant mechanical stresses due to their migration through the extracellular matrix, dense cellular areas, and complex fluids (blood and lymph). These mechanical stresses can cause membrane damage.

The aim of this project is to slow tumor invasion and metastasis by inhibiting the membrane repair mechanism of cancer cells. Among the proteins essential for membrane repair in breast and pancreatic cancer cells, we have already identified Annexin-A2 (ANXA2), ANXA5, and ANXA6 as major players. We are currently working on designing inhibitors capable of preventing tumor invasion and metastasis.

Cells of many tissues, such as skeletal or cardiac muscles, the intestinal epithelium, or the vascular endothelium, are exposed to mechanical stress, which often induces the formation of tears in their plasma membrane (McNeil et al., Annu. Rev. Cell Dev. Biol. 2003). Normal cells are capable of repairing these ruptures within minutes, thanks to a Ca²⁺-dependent molecular machinery involving proteins from the annexin family (Croissant et al., Int. J. Mol. Sci. 2021).

During tumor invasion and metastasis, cancer cells are subjected to considerable shear forces due to their migration through the extracellular matrix, densely populated cell areas, and complex fluids (blood and lymph), which can result in numerous plasma membrane lesions (Wirtz et al., Nat. Rev. Cancer 2011). Few studies have formally addressed membrane repair in the context of cancer, but many works have highlighted a positive correlation between the key players of the membrane repair machinery and tumor invasion.

Building on the work of Jyoti Jaiswal’s group (Washington, USA) and Jesper Nylandsted’s group (Copenhagen, Denmark), we hypothesized that invasive cancer cells possess an enhanced membrane repair machinery that contributes to their survival during tumor progression (Bouvet et al., Sci. Rep. 2020; Gounou et al., Biol. Cell 2023; Gounou et al., Cell. Mol. Life Sci. 2024). We demonstrated that ANXA2, A5, and A6 participate in membrane repair in breast and pancreatic cancer cells (Bouvet et al., Sci. Rep. 2020; Gounou et al., Biol. Cell 2023; Gounou et al., Cell. Mol. Life Sci. 2024), and we observed that the overexpression of these annexins is a poor prognostic factor in many cancers, including stomach, lung, breast, and pancreatic cancers (Gounou et al., Cell. Mol. Life Sci. 2024).

The current project aims to develop pharmacological inhibitors capable of drastically reducing tumor invasion and cancer metastasis. The work is primarily conducted on breast and pancreatic cancers.

Techniques employed: