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.
The Influence of Pregnancy Varies by Timing in BRCA1 and BRCA2 Mutation Carriers
The lower risk of breast cancer from multiple pregnancies and from breast feeding seen in average risk women were confirmed to extend to those at the highest risk of breast cancer by the largest prospective study of BRCA1 and BRCA2 mutations carriers to date published today in the Journal of the National Cancer Institute Cancer Spectrum. Women with BRCA1 mutations who had two, three or four or more full-term pregnancies were at 21%, 30%, and 50% decrease risk of breast cancer compared to women with a single full-term pregnancy. Breastfeeding also reduced risk in BRCA1 mutation carriers. In contrast, women with BRCA2 mutations did not have a decrease in risk from multiple pregnancies except if they had four or more pregnancies. Women with BRCA1 mutations who had only one full-term pregnancy were at an increased of breast cancer as were women with BRCA2 mutations except if they had four or more pregnancies.
“What we have learned is that timing really matters for many risk factors and the dual effect of pregnancy we see in non-mutation carriers with a long term protection but short term increase following a pregnancy may not extend to women with BRCA1 and BRCA2 or at a longer term not yet observed in those cohorts,” said lead author Mary Beth Terry, PhD, Professor of Epidemiology and Environmental Health Sciences at the Mailman School of Public Health at Columbia University and the Herbert Irving Comprehensive Cancer Center. “Moreover, the hormonal upheaval that occurs during the first pregnancy may have a more or less important impact on the risk of breast cancer depending on whether the first pregnancy occurs during periods of life at higher risk of developing a breast cancer or at less high risk, periods shifted by about ten years between BRCA2 and BRCA1 mutation carriers, with a later peak for BRCA2 mutation carriers,“ said senior author Nadine Andrieu, PhD, Director of research at the Institut National de la Santé et de la Recherche Médicale and at the Institut Curie, Paris, France.
This study followed 5,707 BRCA1 and 3,535 BRCA2 mutation carriers using a retrospective cohort analysis and 2,276 BRCA1 and 1,610 BRCA2 mutation carrier. The cohort known as IBCCS (International BRCA1/2 Carrier Cohort Study) includes data from 21 national or center-based prospective follow-up studies whose the national EMBRACE cohort from UK, the national GENEPSO cohort from FR with the Groupe génétique et cancer of Unicancer and the Genetic group and the national HEBON cohort from NL, the Kathleen Cuningham Foundation Constortium for Research into Familial Breast Cancer Followup Study, and the Breast Cancer Family Registry.
The research was supported with funding from the National Cancer Institute (NCI), NIH, DHHS -USA, Cancer Research – UK, the National Health and Medical Research Council of Australia, the New South Wales Cancer Council, the Victorian Health Promotion Foundation, the Victorian Breast Cancer Research Consortium, Cancer Australia, the Australian National Breast Cancer Foundation, the National Health and Medical Research Council, the Queensland Cancer Fund, and the Cancer Foundation of Western Australia, Hungarian Research Grants ,the Norwegian EEA Financial Mechanism, the Swedish Cancer Society, Lund Hospital Funds, and European Research Council Advanced Grant, the Spanish Ministry of Economy and Competitiveness (MINECO), the Spanish Research Network on Rare diseases (CIBERER), the Canadian Institutes of Health Research, the Ministry of Economic Development, Innovation and Export Trade, the Ministère de l’Économie, de la Science et de l’ Innovation du Québec, and The Quebec Breast Cancer Foundation, the German Cancer Research Center (DKFZ), the German Cancer Aid, the Fondation de France, the Ligue Nationale Contre le Cancer and Institut National du Cancer –FR, European Regional Development FEDER –SP, the Dutch Cancer Society, the Netherlands Organisation of Scientific Research, the Pink Ribbon- NL and the European ERA-NET TRANSAN JTC 2012 Cancer 12-054.
The study, “The influence of Number and Timing of Pregnancies on Breast Cancer Risk for Women with BRCA1 or BRCA2 mutations,”
Atoh1 Controls Primary Cilia Formation to Allow for SHH-Triggered Granule Neuron Progenitor Proliferation
During cerebellar development, granule neuron progenitors (GNPs) proliferate by transducing Sonic Hedgehog (SHH) signaling via the primary cilium. Precise regulation of ciliogenesis, thus, ensures proper GNP pool expansion. Here, we report that Atoh1, a transcription factor required for GNPs formation, controls the presence of primary cilia, maintaining GNPs responsiveness to SHH. Loss of primary cilia abolishes the ability of Atoh1 to keep GNPs in a proliferative state. Mechanistically, Atoh1 promotes ciliogenesis by transcriptionally regulating Cep131, which facilitates centriolar satellite (CS) clustering to the basal body. Importantly, ectopic expression of Cep131 counteracts the effects of Atoh1 loss in GNPs by restoring proper localization of CS and ciliogenesis. This Atoh1-CS-primary cilium-SHH pro-proliferative pathway is also conserved in SHH-type medulloblastoma, a pediatric brain tumor arising from the GNPs. Together, our data reveal how Atoh1 modulates the primary cilium to regulate GNPs development.
As we kick off 2019, Stéphanie Descroix, Hélène Salmon Wolfgang Keil and Thomas Walter will be taking over their own research teams at Institut Curie. A new governance which signals a new boost for some promising research themes.
In 2019, there are four major team changes to report within the Research Center at Institut Curie.
First of all, Stéphanie Descroix also belongs to this UMR 168, and her team will also be hosted at the IPGG. She takes the reins from Jean-Louis Viovy – now researcher emeritus – to head two types of bio-physical projects. First she will develop new microfluidic tools. She will thus analyze certain circulating biomarkers, the molecules present in the blood that signal the presence of cancer, its development or the effectiveness of a treatment. In addition, her team will develop organs on chips. Thanks to these miniature systems that can be controlled as we wish, researchers can determine the influence of each parameter of the living conditions of the cells or tumors. The exceptional technical platform at IPGG will allow biologists, clinicians and biophysicists to find answers to a broad range of questions.
Hélène Salmon won another international call for bids to create a new team in the Immunity and cancer unit (U932). Immunity is a theme that’s dear to Institut Curie, and immunotherapies are currently changing the outlook for a large number of patients suffering from cancer, even in the advanced stages. Hélène Salmon and her colleagues will be working on the signal exchanges between the stroma cells, the tissue supporting organs and tumors, and the immune cells responsible for preserving the integrity of the organism. For this the team will benefit from 1.5 million euros in funding over 5 years, awarded by the ARC to the person that the association considers “a future leader in oncology.”
Biophysicist Wolfgang Keil responded to an international call to create a new team in the physical chemistry laboratory at Institut Curie (UMR168). Hosted by the Institut Pierre-Gilles de Gennes (IPGG), this group of researchers will be tasked with furthering knowledge on developmental biology. During these early stages in the life of a multi-cellular organism, the cascade divisions from a single cell lead to the creation of a huge variety of cells, specializing in the different functions needed for the adult organism. This young team leader has a conviction: “The mathematical and physical concepts are essential to understanding precisely how the joint action of gene networks and environmental factors leads to the emergence of biological forms.”
Lastly, Thomas Walter is taking over as interim leader of Jean-Philippe Vert’s team at the Bioinformatics center at the École des Mines, in partnership with Institut Curie. This large and experienced group of 13 people, already created, will thus continue to develop machine learning and artificial intelligence methods to assist biology and health. Researchers manipulate big data; these enormous quantities of diverse data, figures and information which are now produced by biological and genome examinations. Artificial intelligence – now and in the future – helps us discover biomarkers, assist physicians in their diagnoses and better prescribe personalized treatments.
Dissecting Effects of Anti-cancer Drugs and Cancer-Associated Fibroblasts by On-Chip Reconstitution of Immunocompetent Tumor Microenvironments
A major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2+ breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (>50 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) antagonized the effects of trastuzumab. These observations constitute a proof of concept that tumors-on-chip are powerful platforms to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level drug responses, and to dissect the roles of stromal components.
Reconstitution of an HER2+ Tumor Ecosystem On-Chip, Including CAFs and Immune Cells
We designed a microfluidic device for cell co-cultures (called a chip for short), based on published works (Chen et al., 2017, Lucarini et al., 2017, Parlato et al., 2017, Zervantonakis et al., 2012) (Figure 1A). The chips were microfabricated by soft lithography using PDMS (polydimethylsiloxane), a silicone rubber that is biocompatible, gas permeable, and transparent. The chip design consisted of 5 parallel microchambers (500- to 1000-μm wide, 150- to 200-μm high), separated by regularly spaced micropillars that allow the confinement of hydrogels by means of a balance between surface tension and capillary forces. The various cell types were positioned inside the chips, mimicking their original in vivo architecture in tumors.
Cancer cells (the HER2+ breast cancer BT474 cell line), cancer-associated fibroblasts (the breast CAF cell line Hs578T), and immune cells (PBMCs [peripheral blood mononuclear cells] from healthy donors) were embedded into 3D biomimetic hydrogels (made of collagen type I at 2.3 mg/mL, the major component of the extracellular matrix [ECM]), inside the 2 inner lateral chambers. To compare conditions with and without CAFs within the same chip, the left gel chamber was without CAFs, while the right gel chamber was with CAFs. The 2 outer lateral chambers were used as medium reservoirs. This work was focused on the effects of CAFs and immune cells; however, endothelial cells were always included since in vivo they contribute to shaping the biochemical environment by secreting a variety of cytokines and other soluble factors ( Buchanan et al., 2012, Lee et al., 2015 ). Endothelial cells (primary human umbilical vein endothelial cells [HUVECs]) were grown as 2D monolayers in the central chamber, as previously reported (Zervantonakis et al., 2012). For simplicity, the majority of co-cultures and observations were performed without adding perfusion in this vessel compartment. Considering that 3D cell co-cultures were achieved in microfluidics devices and that the literature extensively uses the organ-on-chip terminology even for systems without perfusion, we adopted the tumor-on-chip terminology for our approach.
These tumors-on-chip were visualized by high-content video-microscopy (multi-positioning, multi-colors, 2- to 120-min time intervals) for 4–5 days (Video S1). To discriminate cancer cells from CAFs, the fibroblasts were pre-stained in red with a live dye (CellTrace Yellow reagent) (Video S2). In addition, to quantitatively describe the tumor ecosystem, we implemented appropriate methods to measure on-chip proliferation, apoptotic death, and cell-cell interactions (Figures 1B and 1C). To monitor proliferation, nuclei were live stained using a far-red nuclear dye (SiR-DNA), which allows for the identification and counting of mitotic events (Video S3). To monitor cell death, apoptotic cells were identified using a green probe detecting caspase 3 and 7 activity (CellEvent caspase-3/7) (Video S4). Cells undergoing apoptosis, in addition to emitting green fluorescence, also showed a change in morphology. The overall observation of hundreds of videos revealed extremely rich and complex cell-cell interactions involving immune cells (Video S5). Using the ad hoc-designed CellHunter automated method (
Biselli et al., 2017, Parlato et al., 2017), we tracked the cell dynamics within the co-cultures and measured the interaction times with each immune cell for each cancer cell (Figure 1C), thus providing a high-content description of the cancer-immune cell interactions inside the reconstituted tumor ecosystem. The relative positioning and morphologies of the different cell populations were investigated by live snapshot confocal microscopy; cancer cells, immune cells, and fibroblasts are distributed along the z dimension of the collagen gel, and several cell-cell contacts occur in this true 3D ecosystem. The endothelial cells create vertical barriers, although not continuous ones, at the interfaces between the endothelium channel and the collagen gels (Figure 2).
Researchers from everywhere are gathered in San Diego from December 8th to 12th, for the world-renowned cell biology meeting organized by the American Society of Cell Biology (ASCB), where Institut Curie’s collaborators will have over 30 poster or oral presentations.
The ASCB annual meeting is an important event for cell biology researchers. This year, Institut Curie’s Research Center is particularly well represented, with 6 oral presentations and more than 20 posters.
Patricia Bassereau, Membrane and cellular functions team leader, CNRS Research Officer – Stretching cells with Patricia Bassereau
Patricia Bassereau has been invited to take to the stage at the ASCB meeting. She presented her team’s latest findings in cellular membrane physics alongside cell biologist Evelyne Coudrier. Find out more
Translational control of tumor immune escape via the eIF4F–STAT1–PD-L1 axis in melanoma
Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between programmed death ligand-1 (PD-L1) and programmed death-1 (PD-1) receptor, is a powerful anticancer approach. However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex mechanisms underlying its regulation are not completely understood. Here, we show that the eukaryotic translation initiation complex, eIF4F, which binds the 5′ cap of mRNAs, regulates the surface expression of interferon-γ-induced PD-L1 on cancer cells by regulating translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor. eIF4F complex formation correlates with response to immunotherapy in human melanoma. Pharmacological inhibition of eIF4A, the RNA helicase component of eIF4F, elicits powerful antitumor immune-mediated effects via PD-L1 downregulation. Thus, eIF4A inhibitors, in development as anticancer drugs, may also act as cancer immunotherapies.
13These authors jointly supervised this work: Stéphan Vagner, Caroline Robert
Microtubules – the critical highways of neurons – must stay “clear” for good traffic and neuronal survival
The excess of tubulin polyglutamylation perturbs transport in primary hippocampal neurons and causes extensive neurodegeneration in mice and humans.
Researchers from Carsten Janke’s group from Institut Curie have recently published two articles of important relevance in the field of Neurobiology.
Neurobiology is the branch of biology, which explores neurons, these tentacle cells also known as nerve cells, which are the primary components of our Nervous System.
A typical neuron1 consists of a cell body, dendrites and an axon2 (see images 1 and 3) 1,4. Most neurons receive signals via the dendrites and send out signals down the axon. However, to allow the spreading of those signals, materials have to be transported to the right place at the right time in the axons.
“Scientists are amazed that microscopic materials can be transported more than several feet along one neuron that goes from the spinal cord to the foot. This is equivalent scale to a person carrying a package walking along the wall of China” 3.
Neuronal cells have to transport mitochondria, vesicles and other materials from one side to the other for their correct functionality. This means from the neuronal cell body to the end of the axon (synaptic terminal) and vice versa. In order to do this, neurons use microtubules as highways along the vast length of their axon.
Image 1, 2 and 3. From top to bottom: 1A typical neuronal cell with its nucleus in pink and axon and dendrites in blue. 3Great Wall of China, represents the analogy with axons of some neurons. Those axons are so long, that the material travelling along them can be compared to the transport of a package along the Great Wall of China. 3Main parts of a neuron: cell body, dendrites and axon.
Many neurodegenerative diseases can be related to the dysfunction of microtubules. Therefore, studying the mechanisms that can influence and alter the properties and functions of microtubules is of important interest since they could play a role in neuronal disorders.
One of the mechanisms that alter the properties of microtubules, and how things move on them, are “tubulin posttranslational modifications”. But, what does this exactly mean? And what is tubulin? Let’s start answering the second question! In cells, there are big structures, polymers, built from small elements, or monomers, like a big structure built from small Lego bricks. Microtubules are one such polymer, and their Lego bricks are the tubulin proteins. Meaning one tubulin after another tubulin, after another tubulin, after another tubulin… form a microtubule! (see figure 4) 5.
Figure 4. The analogy between microtubules and a Lego structure. Lego blocks assembled as an example of how monomers (a single Lego blocks or tubulin) form a polymer (a Lego structure or a microtubule). Left image: a Lego structure, right image: a 3D model of a microtubule.
Regarding the first question – what are posttranslational modifications? – we need to imagine a factory chain. In this factory, the manufacturers produce tubulin, and, as in every factory, the products undergo several steps in the whole production chain. After the first step of protein production, tubulin assembles into microtubules, which then undergo some changes, meaning that at the end of its assembly line, microtubules are modified (see figure 5) 6.
Figure 5. Image adapted to show the different steps of the maturation of the protein tubulin. In this case the posttranslational modification that is common on neuronal microtubules is named polyglutamylation.
The scientists of this discovery, focused particularly in a modification named “polyglutamylation”, that consists in the addition of glutamate groups to the tubulins forming the microtubules.
In general, as many things in life, balance is good and excess of something can have bad consequences. In this study, scientists, using mouse models, have discovered how neurodegeneration occurs as a direct cause of an excess of polyglutamylation. In other words, when microtubules get too much of these glutamate groups (in red in the diagram), they change they properties in the axons of neurons, disturbing the transport and therefore leading to the degeneration of a variety of neurons in the central nervous system (see figure 6)7.
Figure 6. Image showing the analogy between transport of materials on microtubules in cells and traffic of cars on roads. Balanced microtubule polyglutamylation in neurons would be the case of good car traffic. However, the excess of polyglutamylation provokes a disruption in the traffic and leads to neurodegeneration in neurons.
Importantly in this research, the authors Magiera and collaborators were able to reverse the neurodegeneration by inhibiting the process of polyglutamylation, a discovery holding the promise that specific inhibitors could be used in the near future as potential therapeutic agents to treat disabling and often, fatal neurodegeneration, for which so far, no treatment is available.
Their second article – Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration – in which they analyzed 13 patients, makes the important discovery that dysregulated microtubule polyglutamylation is also detrimental to human neurons. This gives more value to their discovery in mice and entails more strongly this “posttranslational modification” as a potential target for drug development for human neurodegenerative disorders.
The dark side of centromeres: types, causes and consequences of structural abnormalities implicating centromeric DNA
Centromeres are the chromosomal domains required to ensure faithful transmission of the genome during cell division. They have a central role in preventing aneuploidy, by orchestrating the assembly of several components required for chromosome separation. However, centromeres also adopt a complex structure that makes them susceptible to being sites of chromosome rearrangements. Therefore, preservation of centromere integrity is a difficult, but important task for the cell. In this review, we discuss how centromeres could potentially be a source of genome instability and how centromere aberrations and rearrangements are linked with human diseases such as cancer.
There is not one but several types of HIV. Although HIV-1, which is the most common, wreaks havoc in infected populations, this is not the case for HIV-2 which less frequently leads to the development of AIDS. But why does the immune system do a better job of fighting this version of the virus? Researchers from Inserm and Institut Curie looked at this question, and identified the NONO protein, a detector which is more sensitive to HIV-2 and responsible for direct recognition of the virus by the immune system. This work, published in the journal Cell, provides a better understanding of the natural control of HIV and paves the way for new progress in the search for a vaccine for this virus.
AIDS develops when the immune system of an HIV-positive individual becomes unable to fight infection and becomes dramatically weak. The majority of people infected and not treated develop fatal AIDS. But in some cases AIDS does not develop in certain untreated HIV-positive individuals. This explains the existence of several forms of HIV.
Although HIV-1, which affects 25 million people, without treatment leads to AIDS in 99% of cases, this is not the case for HIV-2. This particular form of HIV is very close to HIV-1 but differs in terms of genetics. It is found mostly in West Africa and affects 1 million people. HIV-2 leads to the development of AIDS in fewer than 25% of cases, has no impact on the life expectancy of most people infected, and proves to be difficult to transmit to others. Furthermore, HIV-positive individuals with the HIV-2 form of the virus, and who also contract HIV-1, show improved resistance to the latter.
Researchers from Inserm and Institut Curie in Unit 932 Immunity and Cancer (Inserm/Institut Curie/PSL University/Paris Descartes University) studied the reasons for the immune system’s better control of HIV-2.
In 2010, this research team had already shown that dendritic cells – the immune system’s “sentinel” cells – were able to detect HIV-2 much more efficiently than HIV-1. In order for the immune response to be effective, there needs to be good immune recognition.
Based on this observation, the researchers sought to understand the molecular mechanisms involved in the dendritic cells’ recognition of HIV-2, and to find out why this recognition is effective in comparison to that of HIV-1.
They thus discovered that the NONO protein, located in the dendritic cells, acted as a detector able to recognize the internal casing (or capsid) of HIV-2 a lot better than that of HIV-1, and as a result to trigger an immune response to fight the virus. The capsid – which surrounds the genetic material of viruses – is made up of proteins, and NONO is apparently able to recognize a specific protein pattern of the capsid of HIV-2.
This study provides a better understanding of the natural mechanisms involved in infection control by HIV. According to Nicolas Manel, Inserm researcher in charge of the study: “the next step in this research project is to understand how this detection system works at the molecular level and how this detection triggers the immune response. We are developing innovative vaccine strategies in the lab, and this discovery paves the way for the new studies needed to develop a new generation of vaccines capable of “imitating” the HIV-2 capsid, and as a result triggering an immune response in people infected with the HIV-1 virus.”