Compressive forces stabilize microtubules in living cells.

Microtubules are cytoskeleton components with unique mechanical and dynamic properties. They are rigid polymers that alternate phases of growth and shrinkage. Nonetheless, the cells can display a subset of stable microtubules, but it is unclear whether microtubule dynamics and mechanical properties are related. Recent in vitro studies suggest that microtubules have mechano-responsive properties, being able to stabilize their lattice by self-repair on physical damage. Here we study how microtubules respond to cycles of compressive forces in living cells and find that microtubules become distorted, less dynamic and more stable. This mechano-stabilization depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. This process seems to be instrumental for cell migration in confined spaces. Overall, these results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected, being a central mediator of cellular mechano-responses.

HIF2α is a direct regulator of neutrophil motility.

Orchestrated recruitment of neutrophils to inflamed tissue is essential during the initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it remains unclear how these factors influence the migration of neutrophils to and at the site of inflammation during their transmigration through the blood-endothelial cell barrier, as well as their motility in the interstitial space. Here, we reveal that activation of hypoxia-inducible factor 2 (HIF2α) as a result of a deficiency in HIF prolyl hydroxylase domain protein 2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNA sequencing analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2α-dependent neutrophil motility. Thus, we propose that the novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation because it promotes neutrophil movement through highly confined tissue landscapes.

Reconstitution of cell migration at a glance.

Single cells migrate in a myriad of physiological contexts, such as tissue patrolling by immune cells, and during neurogenesis and tissue remodeling, as well as in metastasis, the spread of cancer cells. To understand the basic principles of single-cell migration, a reductionist approach can be taken. This aims to control and deconstruct the complexity of different cellular microenvironments into simpler elementary constrains that can be recombined together. This approach is the cell microenvironment equivalent of in vitro reconstituted systems that combine elementary molecular players to understand cellular functions. In this Cell Science at a Glance article and accompanying poster, we present selected experimental setups that mimic different events that cells undergo during migration in vivo These include polydimethylsiloxane (PDMS) devices to deform whole cells or organelles, micro patterning, nano-fabricated structures like grooves, and compartmentalized collagen chambers with chemical gradients. We also outline the main contribution of each technique to the understanding of different aspects of single-cell migration.

TIF1γ interferes with TGFß1/SMAD4 signaling to promote poor outcome in operable breast cancer patients.

BACKGROUND: The Transforming growth factor ß (TGFß) signaling has a paradoxical role in cancer development and outcome. Besides, the prognostic significance of the TGFß1, SMAD4 in breast cancer patients is an area of many contradictions. The transcriptional intermediary factor 1γ (TIF1γ) is thought to interact with the TGFß/SMAD signaling through different mechanisms. Our study aims to define the prognostic significance of TGFß1, SMAD4 and TIF1γ expression in breast cancer patients and to detect possible interactions among those markers that might affect the outcome. METHODS: Immunohistochemistry was performed on tissue microarray (TMA) blocks prepared from samples of 248 operable breast cancer patients who presented at Centre Léon Bérard (CLB) between 1998 and 2001. The intensity and the percentage of stained tumor cells were integrated into a single score (0-6) and a cutoff was defined for high or low expression for each marker. Correlation was done between TGFß1, SMAD4 and TIF1γ expression with the clinico-pathologic parameters using Pearson's chi-square test. Kaplan-Meier method was used to estimate distant metastasis free survival (DMFS), disease free survival (DFS) and overall survival (OS) and the difference between the groups was evaluated with log-rank test. RESULTS: 223 cases were assessable for TIF1γ, 204 for TGFß1 and 173 for SMAD4. Median age at diagnosis was 55.8 years (range: 27 to 89 years). Tumors were larger than 20 mm in 49.2% and 45.2% had axillary lymph node (LN) metastasis (N1a to N3). 19.4% of the patients had SBR grade I tumors, 46.8% grade II tumors and 33.9% grade III tumors. ER was positive in 85.4%, PR in 75.5% and Her2-neu was over-expressed in 10% of the cases. Nuclear TIF1γ, cytoplasmic TGFß1, nuclear and cytoplasmic SMAD4 stainings were high in 35.9%, 30.4%, 27.7% and 52.6% respectively. TIF1γ expression was associated with younger age (p=0.006), higher SBR grade (p<0.001), more ER negativity (p=0.035), and tumors larger than 2 cm (p=0.081), while TGFß1 was not associated with any of the traditional prognostic factors. TGFß1 expression in tumor cells was a marker of poor prognosis regarding DMFS (HR=2.28; 95% CI: 1.4 to 3.8; p=0.002), DFS (HR=2.00; 95% CI: 1.25 to 3.5; p=0.005) and OS (HR=1.89; 95 % CI: 1.04 to 3.43; p=0.037). TIF1γ expression carried a tendency towards poorer DMFS (p=0.091), DFS (p=0.143) and OS (p=0.091). In the multivariate analysis TGFß1 remained an independent predictor of shorter DMFS, DFS and OS. Moreover, the prognostic significance of TGFß1 was more obvious in the TIF1γ high patient subgroup than in the patients with TIF1γ low expression. The subgroup expressing both markers had the worst DMFS (HR=3.2; 95% CI: 1.7 to 5.9; p<0.0001), DFS (HR=3.02; 95 % CI: 1.6 to 5.6; p<0.0001) and OS (HR=2.7; 95 % CI: 1.4 to 5.4; p=0.005). CONCLUSION: There is a crosstalk between the TIF1γ and the TGFß1/SMAD4 signaling that deteriorates the outcome of operable breast cancer patients and when combined together they can serve as an effective prognostic tool for those patients.

Near-infrared co-illumination of fluorescent proteins reduces photobleaching and phototoxicity.

Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5-9.2-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins.

EGFR-dependent aerotaxis is a common trait of breast tumour cells.

BACKGROUND: Aerotaxis, the chemotactism to oxygen, is well documented in prokaryotes. We previously reported for the first time that non-tumorigenic breast epithelial cells also display unequivocal directional migration towards oxygen. This process is independent of the hypoxia-inducible factor (HIF)/prolyl hydroxylase domain (PHD) pathway but controlled by the redox regulation of epidermal growth factor receptor (EGFR), with a reactive oxygen species (ROS) gradient overlapping the oxygen gradient at low oxygen concentration. Since hypoxia is an acknowledged hallmark of cancers, we addressed the putative contribution of aerotaxis to cancer metastasis by studying the directed migration of cancer cells from an hypoxic environment towards nearby oxygen sources, modelling the in vivo migration of cancer cells towards blood capillaries. METHODS: We subjected to the aerotactic test described in our previous papers cells isolated from fresh breast tumours analysed by the Pathology Department of the Saint-Etienne University Hospital (France) over a year. The main selection criterion, aside from patient consent, was the size of the tumour, which had to be large enough to perform the aerotactic tests without compromising routine diagnostic tests. Finally, we compared the aerotactic properties of these primary cells with those of commonly available breast cancer cell lines. RESULTS: We show that cells freshly isolated from sixteen human breast tumour biopsies, representative of various histological characteristics and grades, are endowed with strong aerotactic properties similar to normal mammary epithelial cell lines. Strikingly, aerotaxis of these primary cancerous cells is also strongly dependent on both EGFR activation and ROS. In addition, we demonstrate that aerotaxis can trigger directional invasion of tumour cells within the extracellular matrix contrary to normal mammary epithelial cells. This contrasts with results obtained with breast cancer cell lines, in which aerotactic properties were either retained or impaired, and in some cases, even lost during the establishment of these cell lines. CONCLUSIONS: Altogether, our results support that aerotaxis may play an important role in breast tumour metastasis. In view of these findings, we discuss the prospects for combating metastatic spread. TRIAL REGISTRATION: IRBN1462021/CHUSTE.

Atypical CXCL12 signaling enhances neutrophil migration by modulating nuclear deformability.

To reach inflamed tissues from the circulation, neutrophils must overcome physical constraints imposed by the tissue architecture, such as the endothelial barrier or the three-dimensional (3D) interstitial space. In these microenvironments, neutrophils are forced to migrate through spaces smaller than their own diameter. One of the main challenges for cell passage through narrow gaps is the deformation of the nucleus, the largest and stiffest organelle in cells. Here, we showed that chemokines, the extracellular signals that guide cell migration in vivo, modulated nuclear plasticity to support neutrophil migration in restricted microenvironments. Exploiting microfabricated devices, we found that the CXC chemokine CXCL12 enhanced the nuclear pliability of mouse bone marrow-derived neutrophils to sustain their migration in 3D landscapes. This previously uncharacterized function of CXCL12 was mediated by the atypical chemokine receptor ACKR3 (also known as CXCR7), required protein kinase A (PKA) activity, and induced chromatin compaction, which resulted in enhanced cell migration in 3D. Thus, we propose that chemical cues regulate the nuclear plasticity of migrating leukocytes to optimize their motility in restricted microenvironments.

The apoptosis inhibitor Bcl-xL controls breast cancer cell migration through mitochondria-dependent reactive oxygen species production.

The Bcl-xL apoptosis inhibitor plays a major role in vertebrate development. In addition to its effect on apoptosis, Bcl-xL is also involved in cell migration and mitochondrial metabolism. These effects may favour the onset and dissemination of metastasis. However, the underlying molecular mechanisms remain to be fully understood. Here we focus on the control of cell migration by Bcl-xL in the context of breast cancer cells. We show that Bcl-xL silencing led to migration defects in Hs578T and MDA-MB231 cells. These defects were rescued by re-expressing mitochondria-addressed, but not endoplasmic reticulum-addressed, Bcl-xL. The use of BH3 mimetics, such as ABT-737 and WEHI-539 confirmed that the effect of Bcl-xL on migration did not depend on interactions with BH3-containing death accelerators such as Bax or BH3-only proteins. In contrast, the use of a BH4 peptide that disrupts the Bcl-xL/VDAC1 complex supports that Bcl-xL by acting on VDAC1 permeability contributes to cell migration through the promotion of reactive oxygen species production by the electron transport chain. Collectively our data highlight the key role of Bcl-xL at the interface between cell metabolism, cell death, and cell migration, thus exposing the VDAC1/Bcl-xL interaction as a promising target for anti-tumour therapy in the context of metastatic breast cancer.

Redox regulation of EGFR steers migration of hypoxic mammary cells towards oxygen.

Aerotaxis or chemotaxis to oxygen was described in bacteria 130 years ago. In eukaryotes, the main adaptation to hypoxia currently described relies on HIF transcription factors. To investigate whether aerotaxis is conserved in higher eukaryotes, an approach based on the self-generation of hypoxia after cell confinement was developed. We show that epithelial cells from various tissues migrate with an extreme directionality towards oxygen to escape hypoxia, independently of the HIF pathway. We provide evidence that, concomitant to the oxygen gradient, a gradient of reactive oxygen species (ROS) develops under confinement and that antioxidants dampen aerotaxis. Finally, we establish that in mammary cells, EGF receptor, the activity of which is potentiated by ROS and inhibited by hypoxia, represents the molecular target that guides hypoxic cells to oxygen. Our results reveals that aerotaxis is a property of higher eukaryotic cells and proceeds from the conversion of oxygen into ROS.

Mitochondrial Ca2+ uptake controls actin cytoskeleton dynamics during cell migration.

Intracellular Ca2+ signaling regulates cell migration by acting on cytoskeleton architecture, cell directionality and focal adhesions dynamics. In migrating cells, cytosolic Ca2+ pool and Ca2+ pulses are described as key components of these effects. Whereas the role of the mitochondrial calcium homeostasis and the Mitochondria Cacium Uniporter (MCU) in cell migration were recently highlighted in vivo using the zebrafish model, their implication in actin cystokeleton dynamics and cell migration in mammals is not totally characterized. Here, we show that mcu silencing in two human cell lines compromises their migration capacities. This phenotype is characterized by actin cytoskeleton stiffness, a cell polarization loss and an impairment of the focal adhesion proteins dynamics. At the molecular level, these effects appear to be mediated by the reduction of the ER and cytosolic Ca2+ pools, which leads to a decrease in Rho-GTPases, RhoA and Rac1, and Ca2+-dependent Calpain activites, but seem to be independent of intracellular ATP levels. Together, this study highlights the fundamental and evolutionary conserved role of the mitochondrial Ca2+ homeostasis in cytoskeleton dynamics and cell migration.

Combination of a discovery LC-MS/MS analysis and a label-free quantification for the characterization of an epithelial-mesenchymal transition signature.

Disease phenotype reorganizations are the consequences of signaling pathway perturbations and protein abundance modulations. Characterizing the protein signature of a biological event allows the identification of new candidate biomarkers, new targets for treatments and selective patient therapy. The combination of discovery LC-MS/MS analyses and targeted mass spectrometry using selected reaction monitoring (SRM) mode has emerged as a powerful technology for biomarker identification and quantification owing to faster development time and multiplexing capability. The epithelial-mesenchymal transition (EMT) is a process that controls local invasion and metastasis generation by stimulating changes in adhesion and migration of cells but also in metabolic pathways. In this study, the non-transformed human breast epithelial cell line MCF10A, treated by TGFß or overexpressing mutant K-Ras(v12), two EMT inducers frequently involved in cancer progression, was used to characterize protein abundance changes during an EMT event. The LC-MS/MS analysis and label-free quantification revealed that TGFß and K-Ras(v12) induce a similar pattern of protein regulation and that besides the expected cytoskeletal changes, a strong increase in the anabolism and energy production machinery was observed. BIOLOGICAL SIGNIFICANCE: To our knowledge, this is the first proteomic analysis combining a label-free quantification with an SRM validation of proteins regulated by TGFß and K-Rasv12. This study reveals new insights in the characterization of the changes occurring during an epithelial-mesenchymal transition (EMT) event. Notably, a strong increase in the anabolism and energy production machinery was observed upon both EMT inducers.

RNA helicases DDX5 and DDX17 dynamically orchestrate transcription, miRNA, and splicing programs in cell differentiation.

The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation that dynamically orchestrate several layers of gene expression.