Cell & Tissue Imaging

Publications

Year of publication 2019

Jamecna D, Polidori DJ, Mesmin B, Dezi M, Lévy D, Bigay J, Antonny B (2019 Mar 22)

An intrinsically disordered region in OSBP acts as an entropic barrier to control protein dynamics and orientation at membrane contact sites

Developmental cell : DOI : doi.org/10.1016/j.devcel.2019.02.021 Learn more
Summary

Lipid transfer proteins (LTPs) acting at membrane contact sites (MCS) between the ER and other organelles contain domains involved in heterotypic (e.g. ER to Golgi) membrane tethering as well as domains involved in lipid transfer. Here, we show that a long ≈ 90 aa intrinsically unfolded sequence at the N-terminus of oxysterol binding protein (OSBP) controls OSBP orientation and dynamics at MCS. This Gly-Pro-Ala-rich sequence, whose hydrodynamic radius is twice as that of folded domains, prevents the two PH domains of the OSBP dimer from homotypically tethering two Golgi-like membranes and considerably facilitates OSBP in-plane diffusion and recycling at MCS. Although quite distant in sequence, the N-terminus of OSBP-related protein-4 (ORP4) has similar effects. We propose that N-terminal sequences of low complexity in ORPs form an entropic barrier that restrains protein orientation, limits protein density and facilitates protein mobility in the narrow and crowded MCS environment.

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Beber A, Taveneau C, Nania M, Tsai FC, Di Cicco A, Bassereau P, Lévy D, Cabral JT, Isambert H, Mangenot S*, Bertin A* (2019 Jan 24)

Membrane reshaping by micrometric curvature sensitive septin filaments

Nature communications : DOI : 10.1038/s41467-019-08344-5 Learn more
Summary

Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane.They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of Septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.

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Year of publication 2018

Verweij Frederik J, Revenu Celine, Arras Guillaume, Dingli Florent, Loew Damarys, Follain Gautier, Allio Guillaume, Goetz Jacky G., Herbomel Philippe, Del Bene Filippo, Raposo Graça, van Niel Guillaume (2018 Jul 30)

Live tracking of inter-organ communication by endogenous exosomes in vivo

BioRxiv : DOI : 10.1101/380311 Learn more
Summary

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Anna M Lilja, Veronica Rodilla, Mathilde Huyghe, Edouard Hannezo, Camille Landragin, Olivier Renaud, Olivier Leroy, Steffen Rulands, Benjamin D Simons, Silvia Fre (2018 May 23)

Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

Nature cell biology : DOI : 10.1038/s41556-018-0108-1 Learn more
Summary

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

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Year of publication 2017

Perrine Paul-Gilloteaux, Xavier Heiligenstein, Martin Belle, Marie-Charlotte Domart, Banafshe Larijani, Lucy Collinson, Graça Raposo, Jean Salamero (2017 Feb 1)

eC-CLEM: flexible multidimensional registration software for correlative microscopies.

Nature methods : 102-103 : DOI : 10.1038/nmeth.4170 Learn more
Summary

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Claire François-Martin, James E Rothman, Frederic Pincet (2017 Jan 25)

Low energy cost for optimal speed and control of membrane fusion.

Proceedings of the National Academy of Sciences of the United States of America : 1238-1241 : DOI : 10.1073/pnas.1621309114 Learn more
Summary

Membrane fusion is the cell’s delivery process, enabling its many compartments to receive cargo and machinery for cell growth and intercellular communication. The overall activation energy of the process must be large enough to prevent frequent and nonspecific spontaneous fusion events, yet must be low enough to allow it to be overcome upon demand by specific fusion proteins [such as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)]. Remarkably, to the best of our knowledge, the activation energy for spontaneous bilayer fusion has never been measured. Multiple models have been developed and refined to estimate the overall activation energy and its component parts, and they span a very broad range from 20 kBT to 150 kBT, depending on the assumptions. In this study, using a bulk lipid-mixing assay at various temperatures, we report that the activation energy of complete membrane fusion is at the lowest range of these theoretical values. Typical lipid vesicles were found to slowly and spontaneously fully fuse with activation energies of ∼30 kBT Our data demonstrate that the merging of membranes is not nearly as energy consuming as anticipated by many models and is ideally positioned to minimize spontaneous fusion while enabling rapid, SNARE-dependent fusion upon demand.

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Year of publication 2016

Elodie Gazquez, Yuli Watanabe, Florence Broders-Bondon, Perrine Paul-Gilloteaux, Julie Heysch, Viviane Baral, Nadège Bondurand, Sylvie Dufour (2016 Dec 2)

Endothelin-3 stimulates cell adhesion and cooperates with β1-integrins during enteric nervous system ontogenesis.

Scientific reports : 37877 : DOI : 10.1038/srep37877 Learn more
Summary

Endothelin-3 (EDN3) and β1-integrins are required for the colonization of the embryonic gut by enteric neural crest cells (ENCCs) to form the enteric nervous system (ENS). β1-integrin-null ENCCs exhibit migratory defects in a region of the gut enriched in EDN3 and in specific extracellular matrix (ECM) proteins. We investigated the putative role of EDN3 on ENCC adhesion properties and its functional interaction with β1-integrins during ENS development. We show that EDN3 stimulates ENCC adhesion to various ECM components in vitro. It induces rapid changes in ENCC shape and protrusion dynamics favouring sustained growth and stabilization of lamellipodia, a process coincident with the increase in the number of focal adhesions and activated β1-integrins. In vivo studies and ex-vivo live imaging revealed that double mutants for Itgb1 and Edn3 displayed a more severe enteric phenotype than either of the single mutants demonstrated by alteration of the ENS network due to severe migratory defects of mutant ENCCs taking place early during the ENS development. Altogether, our results highlight the interplay between the EDN3 and β1-integrin signalling pathways during ENS ontogenesis and the role of EDN3 in ENCC adhesion.

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Salomé Adam, Juliette Dabin, Odile Chevallier, Olivier Leroy, Céline Baldeyron, Armelle Corpet, Patrick Lomonte, Olivier Renaud, Geneviève Almouzni, Sophie E Polo (2016 Sep 20)

Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

Molecular cell : DOI : S1097-2765(16)30461-0 Learn more
Summary

Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.

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Marco Biondini, Amel Sadou-Dubourgnoux, Perrine Paul-Gilloteaux, Giulia Zago, Melis D Arslanhan, François Waharte, Etienne Formstecher, Maud Hertzog, Jinchao Yu, Raphael Guerois, Alexis Gautreau, Giorgio Scita, Jacques Camonis, Maria Carla Parrini (2016 Sep 4)

Direct interaction between Exocyst and Wave complexes promotes cell protrusions and motility.

Journal of cell science : DOI : jcs.187336 Learn more
Summary

Coordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave Regulatory Complex (WRC). The Exocyst complex, which functions in polarized exocytosis, has been involved in regulation of cell motility. Here we show a physical and functional connection between Exocyst and WRC. Purified components of Exocyst and WRC complexes directly associate in vitro and interactions interfaces are identified. The Exocyst/WRC interaction is confirmed in cells by co-immunoprecipitation and is shown to occur independently of the Arp2/3 complex. Disruption of the Exocyst/WRC interaction leads to impaired migration. By time-lapse microscopy coupled to image correlation analysis, we visualize the traffic of WRC toward the front in nascent protrusions. Exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between Exocyst and WRC complexes provides a novel mechanistic insight into the spatio-temporal regulation of cell migration.

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Marina Casiraghi, Marjorie Damian, Ewen Lescop, Elodie Point, Karine Moncoq, Nelly Morellet, Daniel Levy, Jacky Marie, Eric Guittet, Jean-Louis Banères, Laurent J Catoire (2016 Aug 5)

Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer.

Journal of the American Chemical Society : 11170-5 : DOI : 10.1021/jacs.6b04432 Learn more
Summary

Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.

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Megan K Dennis, Cédric Delevoye, Amanda Acosta-Ruiz, Ilse Hurbain, Maryse Romao, Geoffrey G Hesketh, Philip S Goff, Elena V Sviderskaya, Dorothy C Bennett, J Paul Luzio, Thierry Galli, David J Owen, Graça Raposo, Michael S Marks (2016 Aug 3)

BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers.

The Journal of cell biology : 293-308 : DOI : 10.1083/jcb.201605090 Learn more
Summary

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1-dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3-dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky-Pudlak syndrome variants.

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Katarzyna S Kopanska, Yara Alcheikh, Ralitza Staneva, Danijela Vignjevic, Timo Betz (2016 Jun 9)

Tensile Forces Originating from Cancer Spheroids Facilitate Tumor Invasion.

PloS one : e0156442 : DOI : 10.1371/journal.pone.0156442 Learn more
Summary

The mechanical properties of tumors and the tumor environment provide important information for the progression and characterization of cancer. Tumors are surrounded by an extracellular matrix (ECM) dominated by collagen I. The geometrical and mechanical properties of the ECM play an important role for the initial step in the formation of metastasis, presented by the migration of malignant cells towards new settlements as well as the vascular and lymphatic system. The extent of this cell invasion into the ECM is a key medical marker for cancer prognosis. In vivo studies reveal an increased stiffness and different architecture of tumor tissue when compared to its healthy counterparts. The observed parallel collagen organization on the tumor border and radial arrangement at the invasion zone has raised the question about the mechanisms organizing these structures. Here we study the effect of contractile forces originated from model tumor spheroids embedded in a biomimetic collagen I matrix. We show that contractile forces act immediately after seeding and deform the ECM, thus leading to tensile radial forces within the matrix. Relaxation of this tension via cutting the collagen does reduce invasion, showing a mechanical relation between the tensile state of the ECM and invasion. In turn, these results suggest that tensile forces in the ECM facilitate invasion. Furthermore, simultaneous contraction of the ECM and tumor growth leads to the condensation and reorientation of the collagen at the spheroid’s surface. We propose a tension-based model to explain the collagen organization and the onset of invasion by forces originating from the tumor.

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Léa Ripoll, Xavier Heiligenstein, Graça Raposo, Cédric Delevoye (2016 May 18)

Illuminating the dark side of recycling endosomes.

Cell cycle (Georgetown, Tex.) : 1309-10 : DOI : 10.1080/15384101.2016.1160682 Learn more
Summary

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Selma Maacha, Océane Anezo, Malika Foy, Géraldine Liot, Laurence Mery, Cécile Laurent, Xavier Sastre-Garau, Sophie Piperno-Neumann, Nathalie Cassoux, Nathalie Planque, Simon Saule (2016 Apr 21)

Protein Tyrosine Phosphatase 4A3 (PTP4A3) Promotes Human Uveal Melanoma Aggressiveness Through Membrane Accumulation of Matrix Metalloproteinase 14 (MMP14).

Investigative ophthalmology & visual science : 1982-90 : DOI : 10.1167/iovs.15-18780 Learn more
Summary

To study PTP4A3 phosphatase and MMP14 metalloprotease synergy in uveal melanoma aggressiveness.

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Florence Broders-Bondon, Perrine Paul-Gilloteaux, Elodie Gazquez, Julie Heysch, Matthieu Piel, Roberto Mayor, John D Lambris, Sylvie Dufour (2016 Apr 5)

Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin.

Developmental biology : 85-99 : DOI : 10.1016/j.ydbio.2016.03.022 Learn more
Summary

We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cadherin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cadherin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders.

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