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HBEC-3 cells were transfected with non-silencing shRNA (Control), shRASSF1A-777 and shRASSF1A-779. A) Changes in RASSF1A and Yap expression assessed by immunofluorescence (Red=RASSF1A, Green=Yap, Blue=DAPI) experiments. Scale bar represents 50 µm. B) The migration speed (µm/h) of transfected cells was determined by wound assay on collagen IV. Mitomycin C (1 µg/ml) was added to medium 12h before wounding. The invasion capacity was assessed using a BioCoat Matrigel Invasion Chamber. The relative invasion was normalized to that of the cells transfected with Control. C) Representative images of colony formation of transfected cells in soft agar. Scale bar represents 20 µm. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

HBEC-3 2D-migration induced by RASS1A-knockdown showed features evoking both collective migration and multicellular streaming

Plasmids used in this study.

The cells were transiently transfected with siNeg, siRASSF1A or siRASSF1AC. The experiences were performed 48 hours after transfection A. The trans-endothelial invasion capacity of transfected cells was assessed using a cell culture insert (BioCoat Matrigel Invasion Chamber) on which a monolayer of endothelial cells has been grown previously. The total number of cells that invaded to the lower side of the filter after 48h was counted under the microscope (10X). The relative invasion was normalized to that of the cells transfected with siNeg. B. The migration speed (µm/h) of transfected HBEC-3 RasV12, H1975 and BEAS-2B cells was determined by wound healing assay on Fibronectin (HBEC-3) or collagen IV (H1975 and BEAS-2B). Mitomycin C (1 µg/ml) was added to medium 12h before wounding. C. The invasion capacity of transfected HBEC-3RasV12, H1975, BEAS-2B, BEAS-2B RasV12 cells was assessed using BioCoat Matrigel Invasion Chambers. The total number of cells that invaded to the lower side of the filter after 48h was counted under the microscope (10X). The relative invasion was normalized to that of the cells transfected with siNeg. D. The capacity of growth without adherence in soft agar of HBEC-3 RasV12 and BEAS-2B cells transfected by an inactive siRNA or SiRASSF1A was evaluated by quantification of colony formation. The number of colonies was scored after 7 days. One representative experiment is shown in a right panel. Scale bar represents 80 µm. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

Three independent air-liquid cultures, each requiring 21 days of growth before fixation and observation, were analyzed. For each culture, 2 slides through the porous membrane and the cells have been performed, those slides being processed at the center of the membrane to avoid artifacts linked to a peripheral location. For siNeg condition (only one stratum of cells) 50 to 70 cells were observed, whereas for siRASSF1A conditions, 50 to 250 cells (several strata of cells) were analyzed. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

The HBEC-3 cells were transiently transfected with siNeg, si RASSF1A or siRASSF1AC. The experiences were performed 48 hours after transfection. A. Expression of focal adhesion complex proteins was examined by RT-PCR analysis. B & C. Adhesion strength of the HBEC-3 cells on fibronectin (B) and laminin (C). For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

HBEC-3 cells were transiently transfected with siNeg or siRASSF1A in association with either pcDNA3-RASSF1A or pcDNA3-RASSF1C. The experiences were performed 48 hours after transfection. A. Representative images for RASSF1A expression changes by immunofluorescence and confocal microscopy. RASSF1A primary antibody (Red) followed by Alexa 555 conjugated secondary antibody along with DAPI for DNA (Blue) was used as a control along with DAPI for DNA (Blue). Scale bar represents 50 µm. B. Representative blots for quantification of Twist and Vimentin proteins levels by western blotting in HBEC-3 cells. Actin was used as an internal control. (n=2) C. The migration speed (µm/h) of transfected HBEC-3 cells was determined by wound healing assay on collagen I & IV. Mitomycin C (1 µg/ml) was added to medium 12h before scratch in order to limit cell proliferation. D. Representative images for nuclear localization of Yap in HBEC-3 cell by immunofluorescence and confocal microscopy. The cells stained by Yap primary antibody (Green) followed by Alexa488 conjugated secondary antibody, along with DAPI for DNA (Blue). Scale bar represents 50 µm. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

Representative images are shown for YAP localization changes, by immunofluorescence and confocal microscopy. HBEC-3 cells were transfected by inactive siNeg (upper lane), siRASS1A, siYAP or siRASSF1A plus siYAP (lower lane) and monitored 12, 24, 36 and 48h after transfection for YAP. The cells were stained by Yap primary antibody (Green) followed by Alexa488 conjugated secondary antibody, along with DAPI for DNA (Blue).

A) GST pull-down assay in HBEC-3 cells. Quantification of active GTP-bound RhoA levels as determined by western blotting. One representative experiment is shown in a top panel of histogram. B & C) HBEC-3 cells were transiently transfected with non-silencing siRNA (siNeg), siRASSF1A-1 in combination or not with B) three plasmids coding for wt-RhoA, RhoA N19 (dominant negative form) & RhoAV14 (constitutively active form) C) Cells were assayed for their invasive capacities with Narciclasine treatment. The invasion capacity was assessed using a BioCoat Matrigel Invasion Chamber. The relative invasion was normalized to that of the cells transfected with siNeg. D) Nuclear intensity of YAP was assessed by immunofluorescence and confocal microscopy (right panel), upon depletion of RASSF1A, transfection of RhoAwt expressing plasmid, co-transfection of siRASSF1A and RhoAwt plasmid, transfection of dominant negative RhoAN19 plasmid alone, or upon RASSF1A depletion, transfection of constitutively active RhoAV14 plasmid alone or upon RASSF1A depletion. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

Inactivation of the tumor suppressor gene RASSF1A by promoter hypermethylation represents a key event underlying the initiation and progression of lung cancer. RASSF1A inactivation is also associated with poor prognosis and may promote metastatic spread. In this study, we investigated how RASSF1A inactivation conferred invasive phenotypes to human bronchial cells. RNAi-mediated silencing of RASSF1A induced epithelial-to-mesenchymal transition (EMT), fomenting a motile and invasive cellular phenotype in vitro and increased metastatic prowess in vivo. Mechanistic investigations revealed that RASSF1A blocked tumor growth by stimulating cofilin/PP2A–mediated dephosphorylation of the guanine nucleotide exchange factor GEF-H1, thereby stimulating its ability to activate the antimetastatic small GTPase RhoB. Furthermore, RASSF1A reduced nuclear accumulation of the Hippo pathway transcriptional cofactor Yes-associated protein (YAP), which was reinforced by RhoB activation. Collectively, our results indicated that RASSF1 acts to restrict EMT and invasion by indirectly controlling YAP nuclear shuttling and activation through a RhoB-regulated cytoskeletal remodeling process, with potential implications to delay the progression of RASSF1-hypermethylated lung tumors. Cancer Res; 76(6); 1627–40. ©2016 AACR.

RASSF1 eight coding exon regions are represented along with the eight different transcripts arising from alternative splicing with the use of two different promoters. Only siRASSF1A and shRASSF1A-777, by annealing to 1alpha exon are able to deplete the mRNA species transcribed from the first promoter proceeding 1alpha(RASSF1A, D, E , F, G, H), whereas siRASSF1AC and shRASSF1A-779, by annealing to exon 4 or 4-5 respectively, are able to deplete all species, since they all contain exon 4. The 737 bp CpG island methylated in human cancers spans the first promoter for RASSF1A, D, E, F, G, H.

Oxalis features: independent control of pO2, pH and the liquid flowrate. pO2 equilibration time in the medium: 3 minutes. pO2 accuracy: 3 mmHg. Flowrate as low as 1 μL min−1 to avoid shear stress.

We describe a novel method to achieve a universal, massive, and fully automated analysis of cell motility behaviours, starting from time-lapse microscopy images. The approach was inspired by the recent successes in application of machine learning for style recognition in paintings and artistic style transfer. The originality of the method relies i) on the generation of atlas from the collection of single-cell trajectories in order to visually encode the multiple descriptors of cell motility, and ii) on the application of pre-trained Deep Learning Convolutional Neural Network architecture in order to extract relevant features to be used for classification tasks from this visual atlas. Validation tests were conducted on two different cell motility scenarios: 1) a 3D biomimetic gels of immune cells, co-cultured with breast cancer cells in organ-on-chip devices, upon treatment with an immunotherapy drug; 2) Petri dishes of clustered prostate cancer cells, upon treatment with a chemotherapy drug. For each scenario, single-cell trajectories are very accurately classified according to the presence or not of the drugs. This original approach demonstrates the existence of universal features in cell motility (a so called “motility style”) which are identified by the DL approach in the rationale of discovering the unknown message in cell trajectories.

Organ-on-chip and tumor-on-chip microfluidic cell cultures represent a fast-growing research field for modelling organ functions and diseases, for drug development, and for promising applications in personalized medicine. Still, one of the bottlenecks of this technology is the analysis of the huge amount of bio-images acquired in these dynamic 3D microenvironments, a task that we propose to achieve by exploiting the interdisciplinary contributions of computer science and electronic engineering. In this work, we apply this strategy to the study of oncolytic vaccinia virus (OVV), an emerging agent in cancer immunotherapy. Infection and killing of cancer cells by OVV were recapitulated and directly imaged in tumor-on-chip. By developing and applying appropriate image analysis strategies and advanced automatic algorithms, we uncovered synergistic cooperation of OVV and immune cells to kill cancer cells. Moreover, we observed that the kinetics of immune cells were modified in presence of OVV and that these immune modulations varied during the course of infection. A correlation between cancer cell infection and cancer-immune interaction time was pointed out, strongly supporting a cause-effect relationship between infection of cancer cells and their recognition by the immune cells. These results shed new light on the mode of action of OVV, and suggest new clinical avenues for immunotherapy developments.

International audience; Autophagy is a physiological degradation process that removes unnecessary or dysfunctional components of cells. It is important for normal cellular homeostasis and as a response to a variety of stresses, such as nutrient deprivation. Defects in autophagy have been linked to numerous human diseases, including cancers. Cancer cells require autophagy to migrate and to invade. Here, we study the intracellular topology of this interplay between autophagy and cell migration by an interdisciplinary live imaging approach which combines micro-patterning techniques and an autophagy reporter (RFP-GFP-LC3) to monitor over time, during directed migration, the back–front spatial distribution of LC3-positive compartments (autophagosomes and autolysosomes). Moreover, by exploiting a genetically controlled cell model, we assessed the impact of transformation by the Ras oncogene, one of the most frequently mutated genes in human cancers, which is known to increase both cell motility and basal autophagy. Static cells displayed an isotropic distribution of autophagy LC3-positive compartments. Directed migration globally increased autophagy and polarized both autophagosomes and autolysosomes at the front of the nucleus of migrating cells. In Ras-transformed cells, the front polarization of LC3 compartments was much less organized, spatially and temporally, as compared to normal cells. This might be a consequence of altered lysosome positioning. In conclusion, this work reveals that autophagy organelles are polarized toward the cell front during migration and that their spatial-temporal dynamics are altered in motile cancer cells that express an oncogenic Ras protein.

Cell-cell interactions are an observable manifestation of underlying complex biological processes occurring in response to diversified biochemical stimuli. Recent experiments with microfluidic devices and live cell imaging show that it is possible to characterize cell kinematics via computerized algorithms and unravel the effects of targeted therapies. We study the influence of spatial and temporal resolutions of time-lapse videos on motility and interaction descriptors with computational models that mimic the interaction dynamics among cells. We show that the experimental set-up of time-lapse microscopy has a direct impact on the cell tracking algorithm and on the derived numerical descriptors. We also show that, when comparing kinematic descriptors in two diverse experimental conditions, too low resolutions may alter the descriptors’ discriminative power, and so the statistical significance of the difference between the two compared distributions. The conclusions derived from the computational models were experimentally confirmed by a series of video-microscopy acquisitions of co-cultures of unlabelled human cancer and immune cells embedded in 3D collagen gels within microfluidic devices. We argue that the experimental protocol of acquisition should be adapted to the specific kind of analysis involved and to the chosen descriptors in order to derive reliable conclusions and avoid biasing the interpretation of results.

International audience; Bone is the most frequent metastasis site for breast cancer. As well as dramatically increasing disease burden, bone metastases are also an indicator of poor prognosis. One of the main challenges in investigating bone metastasis in breast cancer is engineering in vitro models that replicate the features of in vivo bone environments. Such in vitro models ideally enable the biology of the metastatic cells to mimic their in vivo behavior as closely as possible. Here, taking benefit of cutting-edge technologies both in microfabrication and cancer cell biology, we have developed an in vitro breast cancer bone-metastasis model. To do so we first 3D printed a bone scaffold that reproduces the trabecular architecture and that can be conditioned with osteoblast-like cells, a collagen matrix, and mineralized calcium. We thus demonstrated that this device offers an adequate soil to seed primary breast cancer bone metastatic cells. In particular, patient-derived xenografts being considered as a better approach than cell lines to achieve clinically relevant results, we demonstrate the ability of this biomimetic bone niche model to host patient-derived xenografted metastatic breast cancer cells. These patient-derived xenograft cells show a long-term survival in the bone model and maintain their cycling propensity, and exhibit the same modulated drug response as in vivo. This experimental system enables access to the idiosyncratic features of the bone microenvironment and cancer bone metastasis, which has implications for drug testing.

The monomeric GTPase RalB controls crucial physiological processes, including autophagy and invasion, but it still remains unclear how this multi-functionality is achieved. Previously, we reported that the RalGEF (Guanine nucleotide Exchange Factor) RGL2 binds and activates RalB to promote invasion. Here we show that RGL2, a major activator of RalB, is also required for autophagy. Using a novel automated image analysis method, Endomapper, we quantified the endogenous localization of the RGL2 activator and its substrate RalB at different endomembrane compartments, in an isogenic normal and Ras-transformed cell model. In both normal and Ras-transformed cells, we observed that RGL2 and RalB substantially localize at early and recycling endosomes, and to lesser extent at autophagosomes, but not at trans-Golgi. Interestingly the use of a FRET-based RalB biosensor indicated that RalB signaling is active at these endomembrane compartments at basal level in rich medium. Furthermore, induction of autophagy by nutrient starvation led to a considerable reduction of early and recycling endosomes, in contrast to the expected increase of autophagosomes, in both normal and Ras-transformed cells. However, autophagy mildly affected relative abundances of both RGL2 and RalB at early and recycling endosomes, and at autophagosomes. Interestingly, RalB activity increased at autophagosomes upon starvation in normal cells. These results suggest that the contribution of endosome membranes (carrying RGL2 and RalB molecules) increases total pool of RGL2-RalB at autophagosome forming compartments and might contribute to amplify RalB signaling to support autophagy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

STK38 (also known as NDR1) is a Hippo pathway serine/threonine protein kinase with multifarious functions in normal and cancer cells. Using a context-dependent proximity-labeling assay, we identify more than 250 partners of STK38 and find that STK38 modulates its partnership depending on the cellular context by increasing its association with cytoplasmic proteins upon nutrient starvation-induced autophagy and with nuclear ones during ECM detachment. We show that STK38 shuttles between the nucleus and the cytoplasm and that its nuclear exit depends on both XPO1 (aka exportin-1, CRM1) and STK38 kinase activity. We further uncover that STK38 modulates XPO1 export activity by phosphorylating XPO1 on serine 1055, thus regulating its own nuclear exit. We expand our model to other cellular contexts by discovering that XPO1 phosphorylation by STK38 regulates also the nuclear exit of Beclin1 and YAP1, key regulator of autophagy and transcriptional effector, respectively. Collectively, our results reveal STK38 as an activator of XPO1, behaving as a gatekeeper of nuclear export. These observations establish a novel mechanism of XPO1-dependent cargo export regulation by phosphorylation of XPO1's C-terminal auto-inhibitory domain.

The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anticancer strategies., eLife digest Cancers develop when cells in the body divide rapidly in an uncontroled manner. It is generally possible to cure cancers that remain contained within a small area. However, if the tumor cells start to move, the cancer may spread in the body and become life threatening. Currently, most of the anti-cancer treatments act to reduce the multiplication of these cells, but not their ability to migrate. A signal protein called Ras stimulates human cells to grow and move around. In healthy cells, the activity of Ras is tightly controled to ensure cells only divide and migrate at particular times, but in roughly 30% of all human cancers, Ras is abnormally active. Ras switches on another protein, named RalB, which is also involved in inappropriate cell migration. Yet, it is not clear how RalB is capable to help Ras trigger the migration of cells. Zago et al. used an approach called optogenetics to specifically activate the RalB protein in human cells using a laser that produces blue light. When activated, the light-controlled RalB started abnormal cell migration; this was used to dissect which molecules and mechanisms were involved in the process. Taken together, the experiments showed that, first, Ras ‘turns on’ RalB by changing the location of two proteins that control RalB. Then, the activated RalB regulates the exocyst, a group of proteins that travel within the cell. In turn, the exocyst recruits another group of proteins, named the Wave complex, which is part of the molecular motor required for cells to migrate. Zago et al. also found that, in patients, the RalB protein was present at abnormally high levels in samples of breast cancer cells that had migrated to another part of the body. Overall, these findings indicate that the role of RalB protein in human cancers is larger than previously thought, and they highlight a new pathway that could be a target for new anti-cancer drugs.

During mesenchymal cell motility, various actin regulators are recruited to the leading edge with exquisite precision in time and space to generate protrusion and retraction cycles. We present here an automated method, named CorRecD (from Correlation Recruitment Dynamics), which quantifies cell edge dynamics, protein recruitment and analyze their cross-correlation. The Wave Regulatory Complex (WRC), a master driver of protrusions, is used as a case-of-study. This biologist-friendly method relies on free software tools and can be applied to any fluorescently tagged protein of interest.

A major challenge in cancer research is the complexity of the tumor microenvironment and the necessity to take into account the host immunological setting. An innovative way to address these problems is to exploit the emerging technology of organ-on-chip to achieve co-cultures in microfluidic devises that recapitulate ex vivo the tumor ecosystem. We generated tumors-on-chip integrating four cell populations (cancer, immune, endothelial cells and fibroblasts) in a 3D collagen matrix, reconstituting HER2 breast cancer ecosystem. By time-lapse microscopy, differential live staining and a novel automated analysis method, we visualized, and quantified the complex dynamics of this ecosystem, in absence or in 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 (≳ 60 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) on the contrary inhibited cancer-immune interactions, antagonizing the action of the trastuzumab. These observations constitute a proof-of-concept that tumors-on-chip are novel powerful platforms that can be exploited to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level responses to anti-cancer drugs, and to dissect the roles of the various cellular components of the stroma.

During mesenchymal cell motility, various actin regulators are recruited to the leading edge with exquisite precision in time and space to generate protrusion and retraction cycles. We present here an automated method, named CorRecD (from Correlation Recruitment Dynamics), which quantifies cell edge dynamics, protein recruitment and analyze their cross-correlation. The Wave Regulatory Complex (WRC), a master driver of protrusions, is used as a case-of-study. This biologist-friendly method relies on free software tools and can be applied to any fluorescently tagged protein of interest.

Comment inBreast cancer: Fibroblast subtypes alter the microenvironment. [Nat Rev Clin Oncol. 2018]; International audience; Carcinoma-associated fibroblasts (CAF) are key players in the tumor microenvironment. Here, we characterize four CAF subsets in breast cancer with distinct properties and levels of activation. Two myofibroblastic subsets (CAF-S1, CAF-S4) accumulate differentially in triple-negative breast cancers (TNBC). CAF-S1 fibroblasts promote an immunosuppressive environment through a multi-step mechanism. By secreting CXCL12, CAF-S1 attracts CD4+CD25+ T lymphocytes and retains them by OX40L, PD-L2, and JAM2. Moreover, CAF-S1 increases T lymphocyte survival and promotes their differentiation into CD25HighFOXP3High, through B7H3, CD73, and DPP4. Finally, in contrast to CAF-S4, CAF-S1 enhances the regulatory T cell capacity to inhibit T effector proliferation. These data are consistent with FOXP3+ T lymphocyte accumulation in CAF-S1-enriched TNBC and show how a CAF subset contributes to immunosuppression.

Cell migration is central to many developmental, physiologic and pathological processes, including cancer progression. The Ral GTPases (RalA and RalB) which act down-stream the Ras oncogenes, are key players in the coordination between membrane trafficking and actin polymerization. A major direct effector of Ral, the exocyst complex, works in polarized exocytosis and is at the center of multiple protein-protein interactions that support cell migration by promoting protrusion formation, front-rear polarization, and extra-cellular matrix degradation. In this review we describe the recent advancements in deciphering the molecular mechanisms underlying this role of Ral via exocyst on cell migration. Among others, we will discuss the recently identified cross-talk between Ral and Rac1 pathways: exocyst binds to a negative regulator (the RacGAP SH3BP1) and to the major effector (the Wave Regulatory Complex, WRC) of Rac1, the master regulator of protrusions. Next challenge will be to better characterize the dynamics in space and in time of these molecular interplays, to better understand the pleiotropic functions of Ral in both normal and cancer cells.

International audience; The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

The dynamics of the cytoskeleton and cell shape relies on the coordinated activation of RhoGTPase molecular switches. Among them, Rac1 participates to the orchestration in space and time of actin branching and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single molecule imaging and super-resolution microscopy, we reveal an additional supramolecular organization of Rac1. We find that, similarly to H-Ras, Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble due to the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs, GAPs, downstream effectors, and possibly other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Using optogenetics and micropatterning tools, we find that activation of Rac1 leads to its immobilization in nanoclusters and that the local level of Rac1 activity matches the local density of nanoclusters. Altogether, our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms. This finding implies that graded distributions of nanoclusters might encode spatial information.Significance statementThe plasma membrane of eukaryotic cells is a highly organized surface where hundreds of incoming signals are transduced to the intracellular space. How cells encode faithfully this myriad of signals is a fundamental question. Here we show that Rac1, a critical membrane-bound protein involved in the regulation of cytoskeletal dynamics, forms small aggregates together with other regulating proteins. These supramolecular assemblies, called nanoclusters, are the “quantal” units of signaling. By increasing the local concentration, nanoclusters set thresholds for downstream signaling and ensure the fidelity of information transduction. We found that Rac1 nanoclusters are distributed as spatial gradients matching the patterns of Rac1 activity. We propose that cells can encode positional information through distributed signaling quanta, hereby ensuring spatial fidelity.

International audience; 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 the regulation of cell motility. Here, we show a physical and functional connection between exocyst and WRC. Purified components of exocyst and WRC 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 using time-lapse microscopy coupled to image correlation analysis, we visualized the trafficking of the WRC towards the front of the cell in nascent protrusions. The exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between the exocyst and WRC provides a new mechanistic insight into the spatio-temporal regulation of cell migration.

Summary The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss of function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 downregulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.

Migration collective : un partage des tâches entre cellules leaders et coordination supracellulaire Myriam Reffay1,2, Maria-Carla Parrini3, Olivier Cochet-Escartin1, Benoit Ladoux2,4, Axel Buguin1, Sylvie Coscoy1, Francois Amblard1, Jacques Camonis3, Pascal Silberzan1 1 Laboratoire physico-chimie Curie, UMR 168, Institut Curie, centre de recherche, CNRS, UPMC, Paris, F-75248, France ; 2 Laboratoire matiere et systemes complexes UMR 7057, Universite Paris Diderot, CNRS, Paris, F-75251, France ; 3 groupe analyse des reseaux de transduction, U830, Institut Curie, centre de recherche, Inserm, Paris, F-75248, France. 4 Adresses actuelles : Institut Jacques Monod (IJM), CNRS UMR 7592 et Universite Paris Diderot, Paris F75205, France ; Mechanobiology Institute, National University of Singapore 117411, Singapore. myriam.reffay@univ-paris-diderot.fr

Oncogenic Ras signalling occurs frequently in many human cancers. However, no effective targeted therapies are currently available to treat patients suffering from Ras-driven tumours. Therefore, it is imperative to identify downstream effectors of Ras signalling that potentially represent promising new therapeutic options. Particularly, considering that autophagy inhibition can impair the survival of Ras-transformed cells in tissue culture and mouse models, an understanding of factors regulating the balance between autophagy and apoptosis in Ras-transformed human cells is needed. Here, we report critical roles of the STK38 protein kinase in oncogenic Ras transformation. STK38 knockdown impaired anoikis resistance, anchorage-independent soft agar growth, and in vivo xenograft growth of Ras-transformed human cells. Mechanistically, STK38 supports Ras-driven transformation through promoting detachment-induced autophagy. Even more importantly, upon cell detachment STK38 is required to sustain the removal of damaged mitochondria by mitophagy, a selective autophagic process, to prevent excessive mitochondrial reactive oxygen species production that can negatively affect cancer cell survival. Significantly, knockdown of PINK1 or Parkin, two positive regulators of mitophagy, also impaired anoikis resistance and anchorage-independent growth of Ras-transformed human cells, while knockdown of USP30, a negative regulator of PINK1/Parkin-mediated mitophagy, restored anchorage-independent growth of STK38-depleted Ras-transformed human cells. Therefore, our findings collectively reveal novel molecular players that determine whether Ras-transformed human cells die or survive upon cell detachment, which potentially could be exploited for the development of novel strategies to target Ras-transformed cells.

The p21-activated kinase (PAK) 1 kinase, an effector of the Cdc42 and Rac1 GTPases, regulates cell protrusions and motility by controlling actin and adhesion dynamics. Its deregulation has been linked to human cancer. We show here that activation of PAK1 is necessary for protrusive activity during cell spreading. To investigate PAK1 activation dynamics at live protrusions, we developed a conformational biosensor, based on fluorescence resonance energy transfer. This novel PAK1 biosensor allowed the spatiotemporal visualization of PAK1 activation during spreading of COS-7 cells and during motility of normal rat kidney cells. By using this imaging approach in COS-7 cells, the following new insights on PAK1 regulation were unveiled. First, PAK1 acquires an intermediate semi-open conformational state upon recruitment to the plasma membrane. This semi-open PAK1 species is selectively autophosphorylated on serines in the N-terminal regulatory region but not on the critical threonine 423 in the catalytic site. Second, this intermediate PAK1 state is hypersensitive to stimulation by Cdc42 and Rac1. Third, interaction with PIX proteins contributes to PAK1 stimulation at membrane protrusions, in a GTPase-independent way. Finally, trans-phosphorylation events occur between PAK1 molecules at the membrane possibly playing a relevant role for its activation. This study leads to a model for the complex and accurate regulation of PAK1 kinase in vivo at cell protrusions.

International audience; We report here a simple yet robust transient compartmentalization system for microfluidic platforms. Cylindrical microfilaments made of commercially available fishing lines are embedded in a microfluidic chamber and employed as removable walls, dividing the chamber into several compartments. These partitions allow tight sealing for hours, and can be removed at any time by longitudinal sliding with minimal hydrodynamic perturbation. This allows the easy implementation of various functions, previously impossible or requiring more complex instrumentation. In this study, we demonstrate the applications of our strategy, firstly to trigger chemical diffusion, then to make surface co-coating or cell co-culture on a two-dimensional substrate, and finally to form multiple cell-laden hydrogel compartments for three-dimensional cell co-culture in a microfluidic device. This technology provides easy and low-cost solutions, without the use of pneumatic valves or external equipment, for constructing well-controlled microenvironments for biochemical and cellular assays.

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.

Characterizing the migration of a population of cells remains laborious and somewhat subjective. Advances in genetics and robotics allow researchers to perform many experiments in parallel, but analyzing the large sets of data remains a bottleneck. Here we describe a rapid, fully automated correlation-based method for cell migration analysis, compatible with standard video microscopy. This method allows for the computation of quantitative migration parameters via an extensive dynamic mapping of cell displacements.

The Ras proto-oncogenic proteins, prototypes of the small GTPases, work as molecular switches: they are active when bound to GTP and inactive when bound to GDP. A variety of evidence suggested that the Ras paradigm is not fully valid for the Rho-family of small GTPases. Indeed, permanent activation is not sufficient but it is rather the continuous oscillation between the GDP-bound and GTP-bound conformations (namely the GDP/GTP cycling or GTPase flux), that is required for Rho-GTPases to perform their biological functions and properly coordinate actin cytoskeleton reorganization. In our recent study, we show that Rac1 needs to cycle between the GDP and GTP states in order to efficiently control cell motility. Similarly, it was previously reported that GDP/GTP cycling is required by RhoA for cytokinesis and by Cdc42 for cell polarization. The future challenge is to understand why the GTPase flux is so important for the biological actions of Rho GTPases.