In just 48 hours, the abstract has been viewed 800 times and downloaded 160 times! The findings produced by Raphaël Rodriguez’s team at Institut Curie have the international science community on tenterhooks.
And that was exactly what the researcher had hoped for by publishing this extensive study on a platform for biologists before publishing it in a science journal: “The findings in themselves apply to a number of different fields, from biochemistry to epigenetics (see below), and the impact they have will be manifold in a range of areas, from cancerology to the cosmetics industry.This makes the findings difficult to publish in journals which are often too specialized – yet we wanted to make them available to researchers, to ensure they were visible and can be used as soon as possible,” he explained.
The young team leader has long focused on a glycoprotein (a protein with sugars attached to it) called CD44. The protein has been the subject of numerous studies, and has been detected in a great many organs, from ovaries and the liver to the prostate and pancreas. CD44 also plays a role in many different biological processes, such as embryonic development, inflammation, immune response and cancer. In the latter in particular, the protein has been linked to metastases and recurrences! But how? Raphaël Rodriguez and his colleagues have finally pinpointed the answer.
CD44 plays a role in iron endocytosis, meaning the process by which iron penetrates cells. This finding is made all the more unexpected by the fact that until now, there was only one known iron endocytosis mechanism, in which a specific protein, transferrin, transports iron, and its corresponding receptor, TfR1, deposits iron on the surface of cells.
The researchers demonstrated that when these cancerous cells become metastatic, this new method of transporting iron, linked to CD44, may well even be predominant. Once inside the cells, this iron acts as a key catalyst, essential to unlocking some gene expressions. In normal cells, chemically-modified and DNA-associated proteins called histones muffle some genes, and due to the iron, these “epigenetic” markers disappear. The researchers are already developing molecules capable of blocking iron being transported within the cells. New possibilities linked to these basic findings in the fight against cancer are therefore now within reach.
This research is the culmination of Institut Curie’s long-standing expertise, and in particular that of Jean-Paul Thiery, the former Director of the Translational Research department, Edith Heard, Research Unit Director and Collège de France Professor, and Geneviève Almouzni, Honorary Director of the Research Center, both epigenetics specialists.
Raphaël Rodriguez hopes that these findings will help him to pursue his research and encourage other teams to contact him with a view to exploring other aspects of this work, such as in the field of immunity, as CD44 plays a role in this process, or potentially in the field of cosmetics, as the process of iron endocytosis via CD44 also involves hyaluronic acid, already feted for its “rejuvenating” properties in skincare, and therefore better understood in terms of its actions.
NB : this study has not been published yet. Preprint available here
Sebastian Müller, Fabien Sindikubwabo, Tatiana Cañeque, Anne Lafon, Antoine Versini, Bérangère Lombard, Damarys Loew, Adeline Durand, Céline Vallot, Sylvain Baulande, Nicolas Servant, Raphaël Rodriguez
Proteins: Exit This Way
Cells secrete numerous proteins, but these proteins do not exit the cell surface from just anywhere, according to a study conducted jointly by two Institut Curie Research Center teams and published in the Journal of Cell Biology.
Stéphanie Miserey-Lenkei is a member of Bruno Goud’s Molecular Mechanisms of Intracellular Transport team and is interested in the role of the protein RAB6. RAB6 is found in the Golgi apparatus and regulates the protein secretion process within cells. Protein secretion is essential to the life of both cells and organisms. Proteins secreted by a given cell act as messengers in long-distance communication with other cells. They can also remain on the cell surface and provide anchoring points with the cell environment or points for interaction with other cells within the tissue.
Gaelle Boncompain, a member of Franck Perez’s Dynamics of Intracellular Organization team, which focuses on intracellular transport mechanisms, has developed tools to not only synchronize protein secretion (the RUSH system) but to block proteins at the point at which they leave the cell (the SPI system). These tools are now used by numerous other laboratories worldwide.
Working with their postgraduate students, the two researchers combined their expertise to show how secreted proteins follow very specific pathways within vesicles that guide them—as though on rails—from the Golgi apparatus to preferential secretion sites on the cell surface. Furthermore, they found that these sites are located exactly at the points at which cells attach to their environment. “We also observed that, regardless of the type of protein secreted, it is always the transport system involving RAB6 that is used. And the molecular motors involved and the microtubules, the “rails”, are the same,” explains Stéphanie Miserey-Lenkei.
In cancer, many studies have found that secreted proteins are transported abnormally. Cell surface proteins are involved in the migration and adhesion of cells within their environment. “It will be important to figure out the role played by the forces exerted by and on the cell, as well as to test the existence of these preferential secretion sites in three-dimensional cultures,” points out Gaelle Boncompain. The researchers still need to work out why these preferential secretion sites exist. They already have several hypotheses and their upcoming work should be able to tell us more.
RAB6 and Microtubules Restrict Protein Secretion to Focal Adhesions
Lou Fourriere, Amal Kasri, Nelly Gareil, Sabine Bardin, Hugo Bousquet, David Pereira, Franck Perez, Bruno Goud, Gaelle Boncompain, Stéphanie Miserey-Lenkei
The European Commission is supporting three doctoral training networks involving Institut Curie
Of the 128 innovative training networks chosen by the European Commission as part of the Marie Sklodowska-Curie Actions, three involve teams from the Research Center. The projects will offer “high-level research and training opportunities” to doctoral candidates.
The chosen doctoral training programs “will contribute to improving the overall quality of doctoral training in Europe and beyond, adding to its innovative nature”, according to the European Commission when the results of its call for projects were announced in May 2019. The grant agreements are spread over four years.
Three of these Marie Sklodowska-Curie – International Training Networks (MSC-ITN) projects were obtained by teams at the Research Center at Institut Curie, including one as coordinator. The Marie Curie ITN Actions enable joint training for doctoral candidates, as part of a network of European partners and in a chosen scientific field.
The project leaders at Institut Curie are:
– Marie Dutreix, head of the Repair, Recombination and Cancer Team, Charles Fouillade and Pierre-Marie Girard from her team, alongside Célio Pouponnot, co-lead of the Signaling and Tumor Progression Team, which is participating in the Theradnet network, coordinated by the University of Zürich (Switzerland), studying therapeutic radiation. This network includes the same teams as the Radiate-ITN network, which studied radiation, was just successfully completed, and obtained the same level of support from Europe. Their participation in the project is supported by the European Commission to the value of approximately €0.5 million.
– Prof. Anne-Hélène Monsoro, head of the Signaling and Neural Crest Development Team, who is coordinating the NEUcrest network, involving 31 facilities and focusing on neural crest development. The overall budget of the NEUcrest project is €4.1 million. Institut Curie will directly manage approximately €0.5 million, not including the equivalent budget for management.
The total amount of support allocated to the Research Center as part of the MSC-ITN thus amounts to €1.37 million.
A new microfluidic method of studying single-cell epigenetics
Researchers at Institut Curie are combining their expertise with that of a team from the ESPCI and HiFiBiO to identify, cell by cell, the epigenetic signature of cells that are resistant to anticancer treatments. This new breakthrough has earned them a publication in Nature Genetics.
Microfluidics is a technique that allows single-cell analysis, i.e. to talk of cells individually, rather than observing the average behavior of a large cell population. Using this method, isolating individualized cells in microdroplets, researchers gradually discovered heterogeneity within the cells that make up a tumor. After revealing the genetic differences between different cells, they are now moving to the next level: observing epigenetic differences, no longer looking at DNA composition, but the way in which it is organized.
This study is more complicated than that of RNA (this DNA derivative enables the study of gene expression). This is why researchers at Institut Curie have collaborated with Andrew Griffiths and his team at the ESPCI, the École supérieure de physique et de chimie industrielle de Paris, and Annabelle Gérard from HiFiBio, a specialist microfluidics company. These microfluidics experts are thus associated with the team of Céline Vallot, data analysis specialist at Institut Curie.
Microfluidics is already a leading technique in molecular biology. Together, these researchers are taking it a step further. Keven Grosselin, a PhD student at ESPCI and HiFiBio, developed the technique that applies to epigenetics: “a three-step technique, different to and more complex than usual microfluidics, which examines gene expression,” comments Céline Vallot. For her part, she contributed her data analysis expertise: “The data collected using this technique is very scattered, there are lots of “gaps”, genes about which we have little information. Data processing thus allows the relevant information to be extracted, thereby answering our biological questions”. Through a collaboration with Elisabetta Marangoni and her preclinical investigation team at Institut Curie, researchers were able to examine the epigenetic map in cases of resistance to chemotherapy and hormone therapy.
This is how they revealed epigenetic subclones, the group of cells among the cells of a tumor that have identical characteristics to each other but are different to the rest in terms of epigenetics. “We have observed heterogeneous epigenetic maps within sensitive tumors”, explains Céline Vallot. This may explain how some tumors are resistant to treatment. Thanks to this collaboration, Institut Curie now has this new microfluidics technique, which could enable us to identify a subpopulation of cells, which may only be 16% of the tumor cells, but which could determine the acquisition of resistance.”
Six of the Research Center’s teams awarded the FRM label
The FRM (Fondation pour la Recherche Médicale, medical research foundation) label has been awarded to six of the Institut Curie Research Center’s teams. These three-year grants help fund research projects for amounts ranging from €200,000 to €400,000 per team (Valérie Borde, Deborah Bourc’his, Emmanuel Farge, Silvia Fre, Franck Perez, Graça Raposo).
Six of the Research Center’s teams have been selected to be awarded the FRM label, for a total of €2.2 million in funding. 53 teams were allocated this funding across France, meaning Institut Curie represents a total of 11% of the country’s beneficiaries.
Recipients’ research projects:
► Valérie Borde: “DNA synthesis control and consequences on fertility and genome stability during double-strand break repair”
► Deborah Bourc’his: “Epigenetic control of fertility by DNA methylation”
► Emmanuel Farge: “Stimulation by mechanotransduction of the Ret/β-cat tumorigenic pathway in heightening the number of stem cells and preclinical therapeutic application in colon cancer in mice”
► Silivia Fre: “Study of the mechanisms involved in controlling differentiation and plasticity in mammals’ stem cells”
► Franck Perez: “Study of the dynamics and mechanisms involved in anterograde and retrograde transport pathways”
► Graça Raposo: “Cellular and molecular bases for pigmentation in humans: physiopathology of intercellular communication towards melanosomes”
Research project summaries:
► Valérie Borde, Chromosome dynamics and recombination team
Project: “DNA synthesis control and consequences on fertility and genome stability during double-strand break repair”
Double-strand breaks in DNA are the most dangerous form of lesions for genomes contained in our cells’ chromosomes. These breaks occur accidentally following exposure to genotoxic agents, or are programmed by cells, such as during the meiosis that produces gametes (ovules and spermatozoids) as part of sexual reproduction. As these breaks are repaired, the stage in which the damaged DNA is rebuilt is risky, as it can engender mutations that are potentially hereditarily harmful. Our project aims to characterize the factors that occur during the new DNA synthesis stage, how these factors are regulated, what the consequences of their deregulation are on fertility, and how cells react to DNA-damaging agents such as those that occur during cancer chemotherapy. For this project, we will be drawing on multiple different experimental systems, from using Saccharomyces cerevisiae yeast to mouse models, and innovative approaches to monitor how DNA is repaired and what the contributing factors are.
► Deborah Bourc’his, Epigenetic decisions and reproduction in mammals team
Project: “Epigenetic control of fertility by DNA methylation”
Reproduction is a fundamental property in all living beings, designed to ensure genetic information is passed down over the generations, thus guaranteeing the continuing existence of species. Reproductive cells, meaning ovules and spermatozoids, have the dual task of acquiring the attributes needed to trigger fertilization, and protecting the DNA they carry from mutations to ensure the ‘right’ information is passed on to the next generation. Any form of interference with one of these functions leads to sterility or developmental abnormalities in the next generation, in turn leading to miscarriages and serious conditions. One of the most dangerous sources of genetic information mutations is transposons, genetic elements that have formed a natural, integral part of our DNA since the beginning of humanity, with the ability to cluster and multiply when not finely controlled. We have millions of copies of transposons in our bodies, with transposable elements making up over half of our DNA, where genes account for just 2% of it. In mammals, a specific epigenetic marker that involves methyl groups attaching to DNA plays a key role in protecting reproductive cells’ identity and integrity against transposons. Our research program focuses on the role played by DNA methylation in reproduction, and more specifically its influence on reproductive cell emergence during development, their specialization in reproducing and protecting the genetic information they carry from transposon activity. This program aims to better understand the root causes of infertility, with most cases unfortunately remaining all too often unexplained, despite an alarming drop in industrialized nations’ fertility rates.
► Emmanuel Farge, Mechanics and genetics of embryo and tumor development team
Project: “Stimulation by mechanotransduction of the Ret/β-cat tumorigenic pathway in heightening the number of stem cells and preclinical therapeutic application in colon cancer in mice”
To date, a certain number of cases of cancer have been found to be caused by mutations of genomes in some cells that lead to uncontrolled growth. The team’s data shows that the mechanical pressures in growing tumors are also likely to trigger deregulations leading to uncontrolled growth in the surrounding healthy tissue. This project aims to test the impact of this mechanical tumor growth in the development of the illness, and to develop new approaches to treatment that inhibit the effects of this mechanical tumorigenic induction in order to make significant progress in fighting the illness. Being awarded the FRM label in 2015 helped us understand which molecules (mechanical molecular sensors) and which colon cells are particularly sensitive to tumor growth pressure and result in tumorigenesis in response to pressure from surrounding tumors. In addition, it allowed us to inhibit the tumorigenesis’ mechanical induction using chemotherapeutic treatment to inhibit these molecular sensors in mice. Finally, it allowed us to demonstrate that the molecular sensors in question are found universally in all animal and human organs, restoring our faith in treatments based on inhibiting tumoral mechanical sensitivity in cancers, which could therefore be universally effective in a wide range of different cancers and organs. The project submitted for renewal of the label in 2019 involves finalizing this work to offer such a therapeutic approach, targeting the mechanically sensitive molecular elements revealed to be the most sensitive and significant in the work carried out for the 2015 label.
► Silivia Fre, Notch signaling in stem cells and tumours team:
Project: “Study of the mechanisms involved in controlling differentiation and plasticity in mammals’ stem cells”
Breast cancer is the most common form of cancer among women in developed countries. One of the mechanisms by which cancer develops involves the programs that maintain stem cells being deregulated. As a result, understanding the mechanisms by which normal stem cells develop will be crucial to understanding how and why these programs can be deregulated and lead to cancer. Mammals’ pluripotent stem cells are cells that can differentiate into different types of cells and are responsible for embryonic development in mammals. However, over the course of adulthood, unipotent progenitors that can only differentiate into a single type of cell replace the pluripotent stem cells. Although little is known on how stem cells in mammals evolve from pluripotent to unipotent, our laboratory recently discovered that mammals’ stem cells become unipotent very early on in mammogenesis. The aim of the project presented here is to determine how normal embryonic (pluripotent) and adult (unipotent) stem cells develop in mammals, in order to pinpoint the moment at which the stem cells become cells or go on to another specific differentiation. Cutting-edge technology will be used to explore the mechanisms that regulate how the cells shift from pluripotent to unipotent. Understanding these mechanisms will help us understand how normal unipotent cells can be restored to a pluripotent state similar to embryonic stem cells – the mechanism that leads to cancer. These findings will help in identifying early-stage breast cancer, as well as in discovering new treatment targets.
► Franck Perez,Dynamics of intracellular organization team
Project: “Study of the dynamics and mechanisms involved in anterograde and retrograde transport pathways”
To ensure their survival and specific functions, eukaryotic cells must continuously transport proteins to specific compartments: plasma membrane or internal compartments. The Golgi apparatus is the central organelle that modifies and sorts proteins in the secretory pathways (anterograde pathway) and proteins in retrograde transport. Defects in these pathways are linked to various pathologies, from developmental disorders or neuronal degeneration to some types of cancer. How the Golgi apparatus is able to control these two-way pathways and what the ensuing impact of these routes is on how the Golgi apparatus is structured, remains little-known. We have developed a system called RUSH, which allows us to analyze and quantify anterograde transport in a huge range of proteins. In this project, we will be developing a quantitative study system into retrograde transport pathways (retroRUSH). Using these two systems will enable us to analyze the sorting process and the effect membrane pathways have on the Golgi apparatus as well as interferences between the anterograde and retrograde pathways. In the project’s second phase, we will be studying the role proteins linked to the Golgi apparatus play in controlling these pathways. Some of these factors are directly responsible for the development of human pathologies. However, many studies suggest that long-term inactivation of these factors allow the cells to adapt and mask the fundamental effects of these deficiencies. Furthermore, some of these factors have essential functions that prevent the impact of their inactivation from being directly studied. For all these reasons, we will be implementing methods that allow these factors to be acutely inactivated, thus allowing us to study the roles they play in real time.
► Graça Raposo, Structure and membrane compartments team
Project: “Cellular and molecular bases for pigmentation in humans: physiopathology of intercellular communication towards melanosomes”
In skin melanocytes, melanin pigment is synthesized in structures called melanosomes, which are transferred to neighboring keratinocytes to be colored and protect skin from UV rays. Our project pinpoints the mechanisms involved in communication between melanocytes and keratinocytes, both of which are affected in genetic pigmentary disorders and types of skin cancer such as melanoma. We will characterize the messengers or extra-cellular vesicles involved in communication between melanocytes and keratinocytes as well as their role(s) in skin homeostasis. We will study how pigment in keratinocytes evolves depending on skin type, in particular how it is formed and maintained in melanosome reservoirs via the role played by extra-cellular vesicles and their content. We will study the mechanisms underpinning the differences in how melanosomes are distributed in skin types with high and low pigmentation. Through this project, we will be identifying new mechanisms necessary to normal skin pigmentation, as well as pathological processes that will pave the way for new avenues of treatment.
Scientific Publishing: Where we are and where we need to go
Mai 15th, 2019 – Ron Vale, Investigator at the Howard Hughes Medical Institute, SAN FRANCISCO, University of California, Professor & Vice-Chair, Dept. of Cellular & Molecular Pharmacology and member of Institut Curie ISAB gave a seminar about ‘’Where we are and where we need to go’’.
Watch the video of the talk:
New findings into the X-inactivation center
The bipartite structure of the X-inactivation center is responsible for regulating the Tsix and Xist genes during development, according to the work conducted by Dr. Joke van Bemmel and Dr. Rafael Galupa in the team of Prof. Edith Heard in Nature Genetics.
The X-inactivation center locus in mice is a powerful model for understanding the links between genome architecture and gene regulation, with the Xist and Tsix non-coding genes showing opposing expression patterns throughout development, despite being organized into an overlapping sense-antisense pair.
The X-inactivation center (Xic) is organized as two topologically associating domains (TADs), but the role of this organization is unclear. To further investigate this, a team of researchers from Institut Curie, overseen by Edith Heard, created inversions in the genome to permute the Xist/Tsix transcriptional unit and put their promoters in the TAD of each other. In doing so, they discovered that this led to an inversion in their expression patterns: Xist became positively regulated early on in male and female pluripotent cells alike, while Tsix aberrantly remains expressed upon differentiation.
This means that topological partitioning of the Xic is key to ensure proper X-inactivation. Conducted in collaboration with laboratories in Rotterdam, Oxford, Pasadena, Basel and Amsterdam, and with the support of Institut Curie’s platforms, this study shows how gene architecture in cis-regulatory sites can impact on regulating growth in mammals.
A team of biologists from Institut Curie’s Research Center is making discoveries on the normal functioning and the pathological malfunctions of caveolae, cellular structures with a number of implications.
Discovered in the 1950s, caveolae are small structures that form invaginations in cell membranes, and their function has long been a mystery. It was not until 2011 that biologists and physicists from Institut Curie uncovered their role, findings that were published in the journal Cell (1). These rafts of folds and back-folds are designed to absorb the mechanical impacts that cells are subject to; they fold and unfold like an accordion, so that the cell walls can bend without breaking.
Today, the same team, in collaboration with the Institut de myologie, has revealed new findings in the journal Nature Communications (2). Christophe Lamaze, head of the Membrane dynamics and mechanics of intracellular signaling team (Cellular and Chemical Biology Unit, CNRS / Inserm / Institut Curie and affiliated to the LabEx CelTisPhyBio and PSL), Mélissa Dewulf and Cedric Blouin, researchers in his team, studied the muscle cells of patients suffering from a myopathy in which the caveolin-3 gene, a muscle-specific caveolae, is muted. The result is that these cells are not very resistant to mechanical stress. But, more surprisingly, they found that caveolae play a role in the transmission of certain messages inside cells. Specifically, they found that in diseased cells, the response to interleukin-6, an inflammation molecule, was permanently activated, whereas in normal conditions this response is regulated by the caveolae. They have also discovered similar mechanisms in breast cancer cells that will be published soon. Notably, last year in the Journal of Cell Biology (3), they had shown that some components of the caveolae play a role in breast cancer cell invasion.
And from May 12 to 16 in La Baule, France, our caveolae specialists will bring together around one hundred researchers from around the world (including Europe, the United States, Chile, Japan and Australia) to discuss this burgeoning topic as part of a meeting sponsored by the European Molecular Biology Organization (EMBO), an association of Europe’s best researchers in life sciences.
Cells respond to mechanical stress by rapid disassembly of caveolae. Sinha B, Köster D, Ruez R, Gonnord P, Bastiani M, Abankwa D, Stan RV, Butler-Browne G, Vedie B, Johannes L, Morone N, Parton RG, Raposo G, Sens P, Lamaze C, Nassoy P. 2011 Feb 4;144(3):402-13. doi: 10.1016/j.cell.2010.12.031. PMID: 21295700
EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription. Torrino S, Shen WW, Blouin CM, Mani SK, Viaris de Lesegno C, Bost P, Grassart A, Köster D, Valades-Cruz CA, Chambon V, Johannes L, Pierobon P, Soumelis V, Coirault C, Vassilopoulos S, Lamaze C. J Cell Biol. 2018 Dec 3;217(12):4092-4105. doi: 10.1083/jcb.201801122. Epub 2018 Oct 22 PMID: 30348749
Large and reversible myosin-dependent forces in rigidity sensing
A new study by researchers from Columbia University, National University Singapore and Institut Curie suggests that cells are capable of generating unexpectedly large forces when sensing the mechanical properties of their environment.
In order to modulate processes such as growth, differentiation, and cell migration, cells constantly collect and transduce information about the molecular and mechanical properties of their environment. To probe the rigidity of their surroundings, for instance, myosin molecular motors temporarily generate small contractions within actin filaments anchoring the cell to neighboring tissues. Until now, it was largely unknown how much force single myosin molecules are able to generate during these contractions in living cells.
To measure those forces, the researchers study mouse embryonic fibroblasts on microfabricated dual-stiffness pillars. Using high-resolution microscopy to measure cell-induced pillar deflection, they show that during rigidity sensing, myosin motors generate forces that are about an order of magnitude larger than previously measured in single molecule in-vitro experiments. Furthermore, the authors observe that contractions and relaxations occur at the same rate, and in both cases, displacements occur in relatively slow discrete steps.
To better understand how large forces and slow stepwise displacements might be linked, Lohner et al. adapted a mathematical model of collective acto-myosin contractility developed at the Curie Institute in 1995. In the cell, acto-myosin contractions are powered by ATP hydrolysis. In order to explain the large force measurements in the model, the researchers had to assume that ATP hydrolysis rate greatly exceeds the step rate of myosin contractions and that the energy gained by a single ATP hydrolysis event is not sufficient for a single step. In this regime, what appears as a step is better described as an avalanche process, by which motors collectively move by half the periodicity of actin filaments. The large energy released during these events explains the resultant large forces found experimentally. The low probability of the events explains the relatively low frequency of discrete displacement steps.
These results provide new insights into the mechanisms of in-vivo cellular force generation and how cells achieve efficient and accurate mechano-sensitivity over a wide range of extracellular environments.
Cell membrane deformations are crucial for proper cell function. Specialized protein assemblies initiate inward or outward membrane deformations that turn into, for example, endocytic intermediates or filopodia. Actin assembly and dynamics are involved in this process, although their detailed role remains controversial. We show here that a dynamic, branched actin network is sufficient to initiate both inward and outward membrane deformation. With actin polymerization triggered at the membrane of liposomes, we produce inward filopodia-like structures at low tension, while outward endocytosis-like structures are robustly generated regardless of tension. Our results shed light on the mechanism of endocytosis, both in mammalian cells, where actin polymerization forces are required when membrane tension is increased, and in yeast, where those forces are necessary to overcome the opposing turgor pressure. By combining experimental observations with physical modeling, we propose a mechanism for actin-driven endocytosis controlled by membrane tension and the architecture of the actin network.