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Cellular movement is essential for many vital biological functions where it plays a pivotal role both at the single cell level, such as during division or differentiation, and at the macroscopic level within tissues, where coordinated migration is crucial for proper morphogenesis. It also has an impact on various pathological processes, one for all, cancer spreading. Cell migration is a complex phenomenon and diverse experimental methods have been developed aimed at dissecting and analysing its distinct facets independently. In parallel, corresponding analytical procedures and tools have been devised to gain deep insight and interpret experimental results. Here we review established experimental techniques designed to investigate specific aspects of cell migration and present a broad collection of historical as well as cutting-edge computational tools used in quantitative analysis of cell motion.
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Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.
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Proteínas Adaptadoras de Transdução de Sinal , Proteínas do Tecido Nervoso , Proteínas Nucleares , Pseudópodes , Proteínas Supressoras de Tumor , Humanos , Animais , Células HeLa , Linhagem Celular , Actinas/metabolismo , Pseudópodes/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Membrana Celular/metabolismoRESUMO
Glioblastomas exhibit remarkable heterogeneity at various levels, including motility modes and mechanoproperties that contribute to tumor resistance and recurrence. In a recent study using gridded micropatterns mimicking the brain vasculature, glioblastoma cell motility modes, mechanical properties, formin content, and substrate chemistry are linked. Now is presented, SP2G (SPheroid SPreading on Grids), an analytic platform designed to identify the migratory modes of patient-derived glioblastoma cells and rapidly pinpoint the most invasive sub-populations. Tumorspheres are imaged as they spread on gridded micropatterns and analyzed by this semi-automated, open-source, Fiji macro suite that characterizes migration modes accurately. SP2G can reveal intra-patient motility heterogeneity with molecular correlations to specific integrins and EMT markers. This system presents a versatile and potentially pan-cancer workflow to detect diverse invasive tumor sub-populations in patient-derived specimens and offers a valuable tool for therapeutic evaluations at the individual patient level.
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Cancers feature substantial intratumoral heterogeneity of genetic and phenotypically distinct lineages. Although interactions between coexisting lineages are emerging as a potential contributor to tumor evolution, the extent and nature of these interactions remain largely unknown. We postulated that tumors develop ecological interactions that sustain diversity and facilitate metastasis. Using a combination of fluorescent barcoding, mathematical modeling, metabolic analysis, and in vivo models, we show that the Allee effect, i.e., growth dependency on population size, is a feature of tumor lineages and that cooperative ecological interactions between lineages alleviate the Allee barriers to growth in a model of triple-negative breast cancer. Soluble metabolite exchange formed the basis for these cooperative interactions and catalyzed the establishment of a polyclonal community that displayed enhanced metastatic dissemination and outgrowth in xenograft models. Our results highlight interclonal metabolite exchange as a key modulator of tumor ecology and a contributing factor to overcoming Allee effect-associated growth barriers to metastasis.
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Corantes , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Modelos Animais de Doenças , Densidade DemográficaRESUMO
The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.
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Mpox , Vaccinia virus , Humanos , Movimento Celular , Surtos de Doenças , Células EpiteliaisRESUMO
Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing, and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodium cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a MYO6-DOCK7 axis essential for spatially restricting RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative-mode motion in otherwise solid and static carcinoma cell collectives.
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Mama , Pseudópodes , Cicatrização , Movimento (Física)RESUMO
Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.
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Heterocromatina , Neoplasias , Humanos , 1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Núcleo Celular/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Neoplasias/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodia cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a novel MYO6-DOCK7 axis that is critical for spatially restriction of RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative mode motion in otherwise solid and static carcinoma cell collectives. Highlights: Collective motion of jammed epithelia requires myosin VI activityThe MYO6-DOCK7 axis is critical to restrict the activity of RAC1 in a planar polarized fashionMYO6-DOCK7-RAC1 activation ensures long-range coordination of movements by promoting orientation and persistence of cryptic lamellipodiaMyosin VI overexpression is exploited by infiltrating breast cancer cells.
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The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.
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Actinas , Neoplasias , DNA , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Citosol/metabolismo , Transdução de SinaisRESUMO
Circulating tumor cell (CTC) clusters are associated with increased metastatic potential and worse patient prognosis, but are rare, difficult to count, and poorly characterized biophysically. The PillarX device described here is a bimodular microfluidic device (Pillar-device and an X-magnetic device) to profile single CTCs and clusters from whole blood based on their size, deformability, and epithelial marker expression. Larger, less deformable clusters and large single cells are captured in the Pillar-device and sorted according to pillar gap sizes. Smaller, deformable clusters and single cells are subsequently captured in the X-device and separated based on epithelial marker expression using functionalized magnetic nanoparticles. Clusters of established and primary breast cancer cells with variable degrees of cohesion driven by different cell-cell adhesion protein expression are profiled in the device. Cohesive clusters exhibit a lower deformability as they travel through the pillar array, relative to less cohesive clusters, and have greater collective invasive behavior. The ability of the PillarX device to capture clusters is validated in mouse models and patients of metastatic breast cancer. Thus, this device effectively enumerates and profiles CTC clusters based on their unique geometrical, physical, and biochemical properties, and could form the basis of a novel prognostic clinical tool.
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Neoplasias da Mama , Células Neoplásicas Circulantes , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Separação Celular , Feminino , Humanos , Dispositivos Lab-On-A-Chip , Camundongos , Células Neoplásicas Circulantes/patologia , PrognósticoRESUMO
Tumor-to-stroma ratio (TSR) is a prognostic factor that expresses the relative amounts of tumor and intratumoral stroma. In this study, its clinical and molecular relevance was evaluated in prostate cancer (PCa). The feasibility of automated quantification was tested in digital scans of tissue microarrays containing 128 primary tumors from 72 PCa patients stained immunohistochemically for epithelial cell adhesion molecule (EpCAM), followed by validation in a cohort of 310 primary tumors from 209 PCa patients. In order to investigate the gene expression differences between tumors with low and high TSR, we applied multigene expression analysis (nCounter® PanCancer Progression Panel, NanoString) of 42 tissue samples. TSR scores were categorized into low (<1 TSR) and high (≥1 TSR). In the pilot cohort, 31 patients (43.1%) were categorized as low and 41 (56.9%) as high TSR score, whereas 48 (23.0%) patients from the validation cohort were classified as low TSR and 161 (77.0%) as high. In both cohorts, high TSR appeared to indicate the shorter time to biochemical recurrence in PCa patients (Log-rank test, p = 0.04 and p = 0.01 for the pilot and validation cohort, respectively). Additionally, in the multivariate analysis of the validation cohort, TSR predicted BR independent of other factors, i.e., pT, pN, and age (p = 0.04, HR 2.75, 95%CI 1.07-7.03). Our data revealed that tumors categorized into low and high TSR score show differential expression of various genes; the genes upregulated in tumors with low TSR score were mostly associated with extracellular matrix and cell adhesion regulation. Taken together, this study shows that high stroma content can play a protective role in PCa. Automatic EpCAM-based quantification of TSR might improve prognostication in personalized medicine for PCa.
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Glioblastoma are heterogeneous tumors composed of highly invasive and highly proliferative clones. Heterogeneity in invasiveness could emerge from discrete biophysical properties linked to specific molecular expression. We identified clones of patient-derived glioma propagating cells that were either highly proliferative or highly invasive and compared their cellular architecture, migratory, and biophysical properties. We discovered that invasiveness was linked to cellular fitness. The most invasive cells were stiffer, developed higher mechanical forces on the substrate, and moved stochastically. The mechano-chemical-induced expression of the formin FMN1 conferred invasive strength that was confirmed in patient samples. Moreover, FMN1 expression was also linked to motility in other cancer and normal cell lines, and its ectopic expression increased fitness parameters. Mechanistically, FMN1 acts from the microtubule lattice and promotes a robust mechanical cohesion, leading to highly invasive motility.
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Movimento Celular/fisiologia , Forminas/metabolismo , Glioblastoma/metabolismo , Invasividade Neoplásica/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proteínas Fetais/metabolismo , Glioblastoma/patologia , Humanos , Proteínas dos Microfilamentos/metabolismoRESUMO
ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.
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Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Núcleo Celular/metabolismo , Estresse Mecânico , Citoesqueleto de Actina , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Encéfalo , Cromatina , Citoplasma , Citoesqueleto/metabolismo , Dano ao DNA , Camundongos Knockout , Metástase Neoplásica , Neurogênese , Membrana Nuclear/metabolismoRESUMO
BACKGROUND: Prostate cancer (PCa) is among the most commonly diagnosed malignancies in men. Although 5-year survival in patients with localised disease reaches nearly 100%, metastatic disease still remains incurable. Therefore, there is a need for markers indicating metastatic dissemination. METHODS: EGFR overexpression (EGFRover) was tracked in 1039 primary tumours, circulating tumour cells from 39 d'Amico high-risk patients and metastatic samples from 21 castration-resistant PCa cases. EGFR status was compared to clinical parameters and multiple molecular factors were assessed using immunohistochemistry and gene ontology analysis. The functional aspect of EGFR was evaluated by plating PC-3 cells on soft and rigid matrices. RESULTS: EGFRover was found in 14% of primary tumours, where it was associated with shorter metastasis-free survival and was an independent indicator of worse overall survival. EGFRover correlated with a pro-migratory and pro-metastatic phenotype of tumour cells as well as rich collagen fibre content. All circulating tumour cells (detected in 13% of cases) were positive for EGFR, independent of their EMT-related phenotype. EGFRover was more prevalent in castration-resistant bone metastases (29% of patients) and supported growth of human PCa cells on rigid matrices mimicking bone stiffness. CONCLUSIONS: EGFRover is a stable, EMT-independent marker of PCa disseminating to rigid organs, preferentially bones.
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Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/secundário , Células Neoplásicas Circulantes/metabolismo , Neoplasias da Próstata/patologia , Biomarcadores Tumorais/genética , Neoplasias Ósseas/mortalidade , Movimento Celular , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Transição Epitelial-Mesenquimal , Receptores ErbB/genética , Receptores ErbB/metabolismo , Estudos de Viabilidade , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/mortalidade , Neoplasias de Próstata Resistentes à Castração/patologia , Vimentina/metabolismoRESUMO
Collective cellular behavior in confluent monolayers supports physiological and pathological processes of epithelial development, regeneration, and carcinogenesis. Here, the attainment of a mature and static tissue configuration or the local reactivation of cell motility involve a dynamic regulation of the junctions established between neighboring cells. Tricellular junctions (tTJs), established at vertexes where three cells meet, are ideally located to control cellular shape and coordinate multicellular movements. However, their function in epithelial tissue dynamic remains poorly defined. To investigate the role of tTJs establishment and maturation in the jamming and unjamming transitions of epithelial monolayers, a semi-automatic image-processing pipeline is developed and validated enabling the unbiased and spatially resolved determination of the tTJ maturity state based on the localization of fluorescent reporters. The software resolves the variation of tTJ maturity accompanying collective transitions during tissue maturation, wound healing, and upon the adaptation to osmolarity changes. Altogether, this work establishes junctional maturity at tricellular contacts as a novel biological descriptor of collective responses in epithelial monolayers.
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Dendritic cells "patrol" the human body to detect pathogens. In their search, dendritic cells perform a random walk by amoeboid migration. The efficiency of pathogen detection depends on the properties of the random walk. It is not known how the dendritic cells control these properties. Here, we quantify dendritic cell migration under well-defined 2-dimensional confinement and in a 3-dimensional collagen matrix through recording their long-term trajectories. We find 2 different migration states: persistent migration, during which the dendritic cells move along curved paths, and diffusive migration, which is characterized by successive sharp turns. These states exhibit differences in the actin distributions. Our theoretical and experimental analyses indicate that this kind of motion can be generated by spontaneous actin polymerization waves that contribute to dendritic cell polarization and migration. The relative distributions of persistent and diffusive migration can be changed by modification of the molecular actin filament nucleation and assembly rates. Thus, dendritic cells can control their migration patterns and adapt to specific environments. Our study offers an additional perspective on how dendritic cells tune their searches for pathogens.
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Actinas/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Sinais (Psicologia) , Células Dendríticas/fisiologia , Actinas/ultraestrutura , Medula Óssea , Membrana Celular , Forma Celular , Colágeno , Células Dendríticas/citologia , Géis , Humanos , PolimerizaçãoRESUMO
Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.
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Actinas/metabolismo , Adesão Celular/fisiologia , Miosina Tipo I/metabolismo , Miosinas/metabolismo , Fagocitose/fisiologia , Actinas/ultraestrutura , Animais , Células da Medula Óssea , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Feminino , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Miosina Tipo I/genética , Miosinas/genética , Cultura Primária de Células , Células RAW 264.7 , Receptores Fc/metabolismo , Receptores Fc/ultraestrutura , Imagem com Lapso de TempoRESUMO
Cancer cells' ability to migrate through constricting pores in the tissue matrix is limited by nuclear stiffness. MT1-MMP contributes to metastasis by widening matrix pores, facilitating confined migration. Here, we show that modulation of matrix pore size or of lamin A expression known to modulate nuclear stiffness directly impinges on levels of MT1-MMP-mediated pericellular collagenolysis by cancer cells. A component of this adaptive response is the centrosome-centered distribution of MT1-MMP intracellular storage compartments ahead of the nucleus. We further show that this response, including invadopodia formation in association with confining matrix fibrils, requires an intact connection between the nucleus and the centrosome via the linker of nucleoskeleton and cytoskeleton (LINC) complex protein nesprin-2 and dynein adaptor Lis1. Our results uncover a digest-on-demand strategy for nuclear translocation through constricted spaces whereby confined migration triggers polarization of MT1-MMP storage compartments and matrix proteolysis in front of the nucleus depending on nucleus-microtubule linkage.