RESUMO
Immune cells experience large cell shape changes during environmental patrolling because of the physical constraints that they encounter while migrating through tissues. These cells can adapt to such deformation events using dedicated shape-sensing pathways. However, how shape sensing affects immune cell function is mostly unknown. Here, we identify a shape-sensing mechanism that increases the expression of the chemokine receptor CCR7 and guides dendritic cell migration from peripheral tissues to lymph nodes at steady state. This mechanism relies on the lipid metabolism enzyme cPLA2, requires nuclear envelope tensioning and is finely tuned by the ARP2/3 actin nucleation complex. We also show that this shape-sensing axis reprograms dendritic cell transcription by activating an IKKß-NF-κB-dependent pathway known to control their tolerogenic potential. These results indicate that cell shape changes experienced by immune cells can define their migratory behavior and immunoregulatory properties and reveal a contribution of the physical properties of tissues to adaptive immunity.
Assuntos
Movimento Celular , Células Dendríticas , Homeostase , Linfonodos , Camundongos Endogâmicos C57BL , Receptores CCR7 , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Linfonodos/imunologia , Linfonodos/citologia , Receptores CCR7/metabolismo , Camundongos , Movimento Celular/imunologia , Forma Celular , NF-kappa B/metabolismo , Camundongos Knockout , Transdução de Sinais/imunologia , Quinase I-kappa B/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismoRESUMO
Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Dano ao DNA , Exodesoxirribonucleases/metabolismo , Membrana Nuclear/metabolismo , Fosfoproteínas/metabolismo , Animais , Linhagem Celular , Senescência Celular , Colágeno/metabolismo , Progressão da Doença , Feminino , Humanos , Camundongos , Invasividade Neoplásica , Membrana Nuclear/ultraestrutura , Proteólise , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cell migration in confined 3D tissue microenvironments is critical for both normal physiological functions and dissemination of tumor cells. We discovered a cytoskeletal structure that prevents damage to the nucleus during migration in confined microenvironments. The formin-family actin filament nucleator FMN2 associates with and generates a perinuclear actin/focal adhesion (FA) system that is distinct from previously characterized actin/FA structures. This system controls nuclear shape and positioning in cells migrating on 2D surfaces. In confined 3D microenvironments, FMN2 promotes cell survival by limiting nuclear envelope damage and DNA double-strand breaks. We found that FMN2 is upregulated in human melanomas and showed that disruption of FMN2 in mouse melanoma cells inhibits their extravasation and metastasis to the lung. Our results indicate a critical role for FMN2 in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage to promote cell survival during confined migration and thus promote cancer metastasis.
Assuntos
Núcleo Celular/metabolismo , Adesões Focais , Neoplasias Pulmonares/secundário , Melanoma/patologia , Proteínas dos Microfilamentos/metabolismo , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Actinas/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos/citologia , Matriz Extracelular/metabolismo , Feminino , Forminas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido NervosoRESUMO
The integrity of the nuclear envelope (NE) is essential for maintaining the structural stability of the nucleus. Rupture of the NE has been frequently observed in cancer cells, especially in the context of mechanical challenges, such as physical confinement and migration. However, spontaneous NE rupture events, without any obvious physical challenges to the cell, have also been described. The molecular mechanism(s) of these spontaneous NE rupture events remain to be explored. Here, we show that DNA damage and subsequent ATR activation leads to NE rupture. Upon DNA damage, lamin A/C is phosphorylated in an ATR-dependent manner, leading to changes in lamina assembly and, ultimately, NE rupture. In addition, we show that cancer cells with intrinsic DNA repair defects undergo frequent events of DNA-damage-induced NE rupture, which renders them extremely sensitive to further NE perturbations. Exploiting this NE vulnerability could provide a new angle to complement traditional, DNA-damage-based chemotherapy.
Assuntos
Lamina Tipo A , Membrana Nuclear , Membrana Nuclear/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Fosforilação , Dano ao DNA , DNA/metabolismo , Núcleo Celular/metabolismoRESUMO
The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration--one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confinement, adhesion, and contractility.
Assuntos
Mesoderma/citologia , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Células Epiteliais/citologia , Fibroblastos/citologia , Adesões Focais , Células HeLa , Humanos , Pele/citologiaRESUMO
Cell movement has essential functions in development, immunity, and cancer. Various cell migration patterns have been reported, but no general rule has emerged so far. Here, we show on the basis of experimental data in vitro and in vivo that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to cell migration speed. We suggest that this universal coupling constitutes a generic law of cell migration, which originates in the advection of polarity cues by an actin cytoskeleton undergoing flows at the cellular scale. Our analysis relies on a theoretical model that we validate by measuring the persistence of cells upon modulation of actin flow speeds and upon optogenetic manipulation of the binding of an actin regulator to actin filaments. Beyond the quantitative prediction of the coupling, the model yields a generic phase diagram of cellular trajectories, which recapitulates the full range of observed migration patterns.
Assuntos
Actinas/metabolismo , Movimento Celular , Modelos Biológicos , Animais , Linhagem Celular , Polaridade Celular , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Camundongos Endogâmicos C57BL , OryziasRESUMO
The cellular cortex provides crucial mechanical support and plays critical roles during cell division and migration. The proteins of the ERM family, comprised of ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the contributions of the ERM proteins to leukocyte migration, we generated single and triple ERM knockout macrophages. Surprisingly, we found that even in the absence of ERM proteins, macrophages still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore, we discovered that, unlike every other cell type previously investigated, the single or triple knockout of ERM proteins does not affect macrophage migration in diverse contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanical properties of their cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their uniquely adaptive cortical plasticity.
RESUMO
Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers1-3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis4-8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis9. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.
Assuntos
Instabilidade Cromossômica , Dano ao DNA , Duplicação Gênica , Fase S , Tetraploidia , Instabilidade Cromossômica/genética , Replicação do DNA , Humanos , Cariótipo , Mitose , Fase S/genéticaRESUMO
Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force coupling is usually mediated by transmembrane adhesion receptors, especially those of the integrin family, amoeboid cells such as leukocytes can migrate extremely fast despite very low adhesive forces1. Here we show that leukocytes cannot only migrate under low adhesion but can also transmit forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographical features of the substrate to propel themselves. Here the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating retrograde shear forces that are sufficient to drive the cell body forwards. Notably, adhesion-dependent and adhesion-independent migration are not mutually exclusive, but rather are variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate interchangeably and simultaneously. As adhesion-free migration is independent of the chemical composition of the environment, it renders cells completely autonomous in their locomotive behaviour.
Assuntos
Citoesqueleto de Actina/metabolismo , Movimento Celular , Microambiente Celular , Linfócitos T/citologia , Actinas/metabolismo , Animais , Adesão Celular , Linhagem Celular , Humanos , Camundongos , Linfócitos T/metabolismo , Talina/deficiênciaRESUMO
Cells exist in an astonishing range of volumes across and within species. However, our understanding of cell size control remains limited, owing in large part to the challenges associated with accurate determination of cell volume. Much of our comprehension of size regulation derives from yeast models, but even for these morphologically stereotypical cells, assessment of cell volume has mostly relied on proxies and extrapolations from two-dimensional measurements. Recently, the fluorescence exclusion method (FXm) was developed to evaluate the size of mammalian cells, but whether it could be applied to smaller cells remained unknown. Using specifically designed microfluidic chips and an improved data analysis pipeline, we show here that FXm reliably detects subtle differences in the volume of fission yeast cells, even for those with altered shapes. Moreover, it allows for the monitoring of dynamic volume changes at the single-cell level with high time resolution. Collectively, our work highlights how the coupling of FXm with yeast genetics will bring new insights into the complex biology of cell growth.
Assuntos
Saccharomyces cerevisiae , Schizosaccharomyces , Animais , Ciclo Celular , Tamanho Celular , Mamíferos , Microfluídica , Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Citoesqueleto , Microtúbulos , Movimento Celular , Polímeros , Projetos de PesquisaRESUMO
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.
Assuntos
Actinas , Neoplasias , DNA , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Citosol/metabolismo , Transdução de SinaisRESUMO
Imbalance in the finely orchestrated system of chromatin-modifying enzymes is a hallmark of many pathologies such as cancers, since causing the affection of the epigenome and transcriptional reprogramming. Here, we demonstrate that a loss-of-function mutation (LOF) of the major histone lysine methyltransferase SETDB1 possessing oncogenic activity in lung cancer cells leads to broad changes in the overall architecture and mechanical properties of the nucleus through genome-wide redistribution of heterochromatin, which perturbs chromatin spatial compartmentalization. Together with the enforced activation of the epithelial expression program, cytoskeleton remodeling, reduced proliferation rate and restricted cellular migration, this leads to the reversed oncogenic potential of lung adenocarcinoma cells. These results emphasize an essential role of chromatin architecture in the determination of oncogenic programs and illustrate a relationship between gene expression, epigenome, 3D genome and nuclear mechanics.
Assuntos
Cromatina , Neoplasias Pulmonares , Humanos , Cromatina/genética , Epigenoma , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Heterocromatina , Fenótipo , Neoplasias Pulmonares/genéticaRESUMO
Blebs and pseudopods can both power cell migration, with blebs often favored in tissues, where cells encounter increased mechanical resistance. To investigate how migrating cells detect and respond to mechanical forces, we used a "cell squasher" to apply uniaxial pressure to Dictyostelium cells chemotaxing under soft agarose. As little as 100 Pa causes a rapid (<10 s), sustained shift to movement with blebs rather than pseudopods. Cells are flattened under load and lose volume; the actin cytoskeleton is reorganized, with myosin II recruited to the cortex, which may pressurize the cytoplasm for blebbing. The transition to bleb-driven motility requires extracellular calcium and is accompanied by increased cytosolic calcium. It is largely abrogated in cells lacking the Piezo stretch-operated channel; under load, these cells persist in using pseudopods and chemotax poorly. We propose that migrating cells sense pressure through Piezo, which mediates calcium influx, directing movement with blebs instead of pseudopods.
Assuntos
Dictyostelium/citologia , Dictyostelium/metabolismo , Canais Iônicos/metabolismo , Proteínas de Protozoários/metabolismo , Pseudópodes/metabolismo , Fenômenos Biomecânicos , Movimento Celular , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Dictyostelium/química , Dictyostelium/genética , Canais Iônicos/genética , Mecanotransdução Celular , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Pressão , Proteínas de Protozoários/genética , Pseudópodes/genéticaRESUMO
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.
Assuntos
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
Migrating cells exhibit various motility patterns, resulting from different migration mechanisms, cell properties, or cell-environment interactions. The complexity of cell dynamics is reflected, e.g., in the diversity of the observed forms of velocity autocorrelation function-which has been widely served as a measure of diffusivity and spreading. By analyzing the dynamics of migrating dendritic cells in vitro, we disentangle the contributions of direction θ and speed v to the velocity autocorrelation. We find that the ability of cells to maintain their speed or direction of motion is unequal, reflected in different temporal decays of speed and direction autocorrelation functions, ACv(t)â¼t-1.2 and ACθ(t)â¼t-0.5, respectively. The larger power-law exponent of ACv(t) indicates that the cells lose their speed memory considerably faster than the direction memory. Using numerical simulations, we investigate the influence of ACθ and ACv as well as the direction-speed cross correlation Cθ-v on the search time of a persistent random walker to find a randomly located target in confinement. Although ACθ and Cθ-v play the major roles, we find that the speed autocorrelation ACv can be also tuned to minimize the search time. Adopting an optimal ACv can reduce the search time even up to 10% compared with uncorrelated spontaneous speeds. Our results suggest that migrating cells can improve their search efficiency, especially in crowded environments, through the directional or speed persistence or the speed-direction correlation.
Assuntos
Células Dendríticas , Movimento (Física)RESUMO
Classic Hodgkin lymphoma (cHL) is usually characterized by a low tumour cell content, derived from crippled germinal centre B cells. Rare cases have been described in which the tumour cells show clonal T-cell receptor rearrangements. From a clinicopathological perspective, it is unclear if these cases should be classified as cHL or anaplastic large T-cell lymphoma (ALCL). Since we recently observed differences in the motility of ALCL and cHL tumour cells, here, we aimed to obtain a better understanding of T-cell-derived cHL by investigating their global proteomic profiles and their motility. In a proteomics analysis, when only motility-associated proteins were regarded, T-cell-derived cHL cell lines showed the highest similarity to ALK- ALCL cell lines. In contrast, T-cell-derived cHL cell lines presented a very low overall motility, similar to that observed in conventional cHL. Whereas all ALCL cell lines, as well as T-cell-derived cHL, predominantly presented an amoeboid migration pattern with uropod at the rear, conventional cHL never presented with uropods. The migration of ALCL cell lines was strongly impaired upon application of different inhibitors. This effect was less pronounced in cHL cell lines and almost invisible in T-cell-derived cHL. In summary, our cell line-derived data suggest that based on proteomics and migration behaviour, T-cell-derived cHL is a neoplasm that shares features with both cHL and ALCL and is not an ALCL with low tumour cell content. Complementary clinical studies on this lymphoma are warranted.
Assuntos
Doença de Hodgkin , Linfoma Anaplásico de Células Grandes , Doença de Hodgkin/genética , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Humanos , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Proteômica , Linfócitos T/metabolismoRESUMO
During trafficking through tissues, T cells fine-tune their motility to balance the extent and duration of cell-surface contacts versus the need to traverse an entire organ. Here we show that in vivo, myosin IIA-deficient T cells had a triad of defects, including overadherence to high-endothelial venules, less interstitial migration and inefficient completion of recirculation through lymph nodes. Spatiotemporal analysis of three-dimensional motility in microchannels showed that the degree of confinement and myosin IIA function, rather than integrin adhesion (as proposed by the haptokinetic model), optimized motility rate. This motility occurred via a myosin IIA-dependent rapid 'walking' mode with multiple small and simultaneous adhesions to the substrate, which prevented spurious and prolonged adhesions. Adhesion discrimination provided by myosin IIA is thus necessary for the optimization of motility through complex tissues.