RESUMO
Neutrophils are critical to innate immunity, including host defense against bacterial and fungal infections. They achieve their host defense role by phagocytosing pathogens, secreting their granules full of cytotoxic enzymes, or expelling neutrophil extracellular traps (NETs) during the process of NETosis. NETs are weblike DNA structures decorated with histones and antimicrobial proteins released by activated neutrophils. Initially described as a means for neutrophils to neutralize pathogens, NET release also occurs in sterile inflammation, promotes thrombosis, and can mediate tissue damage. To effectively manipulate this double-edged sword to fight a particular disease, researchers must work toward understanding the mechanisms driving NETosis. Such understanding would allow the generation of new drugs to promote or prevent NETosis as needed. While knowledge regarding the (patho)physiological roles of NETosis is accumulating, little is known about the cellular and biophysical bases of this process. In this review, we describe and discuss our current knowledge of the molecular, cellular, and biophysical mechanisms mediating NET release as well as open questions in the field.
Assuntos
Armadilhas Extracelulares/metabolismo , Animais , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Citosol/metabolismo , DNA/metabolismo , HumanosRESUMO
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
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
INTRODUCTION: Although an essential frontline service in the prevention of child morbidity and mortality, there are indications that routine vaccinations have been disrupted during the COVID-19 pandemic. The present study aimed to compare vaccination coverage before COVID-19 in Mali in 2019 and during COVID-19 in 2020. OBJECTIVE: To compare vaccination coverages before COVID-19 in Mali in 2019 and during COVID-19 in 2020. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: We collected routine immunization data from 2019 to 2020 of children under one year in the health district of Commune V in Bamako which includes twelve community health centers (CSCom). RESULTS: Considering all vaccines together, coverage in 2019 was higher than in 2020 (88.7% vs. 71,6%) (p < 10- 3, Fig. 1). In 2020, low proportions of children vaccinated were observed in May (51.1%) two months after the first COVID-19 case in Mali on March 25, 2020. For all vaccines, the mean number of children vaccinated was significantly higher in 2019 (before COVID-19) as compared to 2020 (during COVID-19) (p < 0.05). However, in September and October 2019 BCG vaccine coverage was lower in 2019 as compared to 2020 (p < 10- 3). CONCLUSION: COVID-19 pandemic has affected routine childhood vaccine coverage in Commune V of Bamako, particularly in May 2020. Therefore, new strategies are needed to improve vaccine coverage in young children below 1.
Assuntos
COVID-19 , Cobertura Vacinal , Humanos , Criança , Lactente , Pré-Escolar , Mali/epidemiologia , Estudos Transversais , Pandemias , COVID-19/epidemiologia , COVID-19/prevenção & controle , Vacinação , Vacina BCGRESUMO
Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high-resolution time-lapse microscopy of mouse and human neutrophils and differentiated HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers of organelles and stimulated with bacterial toxins or Candida albicans to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin decondensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a stepwise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.
Assuntos
Armadilhas Extracelulares/imunologia , Neutrófilos/imunologia , Proteína-Arginina Desiminase do Tipo 4/imunologia , Animais , Cromatina/imunologia , Citoesqueleto/imunologia , DNA/imunologia , DNA/metabolismo , Armadilhas Extracelulares/metabolismo , Células HL-60 , Histonas/imunologia , Humanos , Imunidade Inata , Inflamação/imunologia , Camundongos , Microtúbulos/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/metabolismo , Membrana Nuclear/imunologiaRESUMO
During cell migration in confinement, the nucleus has to deform for a cell to pass through small constrictions. Such nuclear deformations require significant forces. A direct experimental measure of the deformation force field is extremely challenging. However, experimental images of nuclear shape are relatively easy to obtain. Therefore, here we present a method to calculate predictions of the deformation force field based purely on analysis of experimental images of nuclei before and after deformation. Such an inverse calculation is technically non-trivial and relies on a mechanical model for the nucleus. Here we compare two simple continuum elastic models of a cell nucleus undergoing deformation. In the first, we treat the nucleus as a homogeneous elastic solid and, in the second, as an elastic shell. For each of these models we calculate the force field required to produce the deformation given by experimental images of nuclei in dendritic cells migrating in microchannels with constrictions of controlled dimensions. These microfabricated channels provide a simplified confined environment mimicking that experienced by cells in tissues. Our calculations predict the forces felt by a deforming nucleus as a migrating cell encounters a constriction. Since a direct experimental measure of the deformation force field is very challenging and has not yet been achieved, our numerical approaches can make important predictions motivating further experiments, even though all the parameters are not yet available. We demonstrate the power of our method by showing how it predicts lateral forces corresponding to actin polymerisation around the nucleus, providing evidence for actin generated forces squeezing the sides of the nucleus as it enters a constriction. In addition, the algorithm we have developed could be adapted to analyse experimental images of deformation in other situations.
Assuntos
Movimento Celular/fisiologia , Núcleo Celular/fisiologia , Modelos Biológicos , Actinas/metabolismo , Algoritmos , Animais , Fenômenos Biomecânicos , Núcleo Celular/ultraestrutura , Forma Celular/fisiologia , Biologia Computacional , Simulação por Computador , Células Dendríticas/citologia , Células Dendríticas/fisiologia , Elasticidade/fisiologia , Camundongos , Microtecnologia , Imagem com Lapso de TempoRESUMO
Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear. Using osteosarcoma cells, we found that in addition to its known localizations on microtubules and the plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and focal adhesions. Cells depleted of MARK proteins demonstrated that MARK2 promotes phosphorylation of both myosin II and the myosin phosphatase targeting subunit MYPT1 to synergistically drive myosin II contractility and stress fiber formation in cells. Studies with isolated proteins showed that MARK2 directly phosphorylates myosin II regulatory light chain, while its effects on MYPT1 phosphorylation are indirect. Using a mutant lacking the membrane-binding domain, we found that membrane association is required for focal adhesion targeting of MARK2, where it specifically enhances cell protrusion by promoting FAK phosphorylation and formation of focal adhesions oriented in the direction of migration to mediate directionally persistent cell motility. Together, our results define MARK2 as a master regulator of the actomyosin and microtubule cytoskeletal systems and focal adhesions to mediate directional cancer cell migration.
Assuntos
Actomiosina , Adesões Focais , Actomiosina/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Adesões Focais/metabolismo , Mamíferos , Cadeias Leves de Miosina/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , FosforilaçãoRESUMO
In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.
Assuntos
Movimento Celular/fisiologia , Colágeno/metabolismo , Leucócitos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Quimiocina CCL21/metabolismo , Quimiotaxia/fisiologia , Células Dendríticas/citologia , Humanos , Transdução de Sinais/fisiologiaRESUMO
Cell migration is a hallmark of dendritic cells (DCs) function. It is needed for DCs to scan their environment in search for antigens as well as to reach lymphatic organs in order to trigger T lymphocyte's activation. Such interaction leads to tolerance in the case of DCs migrating under homeostatic conditions or to immunity in the case of DCs migrating upon encounter with pathogen-associated molecular patterns. Cell migration is therefore essential for DCs to transfer information from peripheral tissues to lymphoid organs, thereby linking innate to adaptive immunity. This stresses the need to unravel the molecular mechanisms involved. However, the tremendous complexity of the tissue microenvironment as well as the limited spatio-temporal resolution of in vivo imaging techniques has made this task difficult. To bypass this problem, we have developed microfabrication-based experimental tools that are compatible with high-resolution imaging. Here, we will discuss how such devices can be used to study DC migration under controlled conditions that mimic their physiological environment in a robust quantitative manner.
Assuntos
Microambiente Celular , Quimiotaxia , Células Dendríticas/imunologia , Microscopia , Microtecnologia , Imunidade Adaptativa , Animais , Polaridade Celular , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Desenho de Equipamento , Humanos , Imunidade Inata , Microscopia/instrumentação , Microscopia/métodos , Microtecnologia/instrumentação , Microtecnologia/métodos , Miniaturização , Fenótipo , Transdução de Sinais , Fatores de TempoRESUMO
Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Movimento Celular , Núcleo Celular/metabolismo , Células Dendríticas , Neutrófilos , Lâmina Nuclear/metabolismo , Animais , Immunoblotting , Lamina Tipo A/metabolismo , Camundongos , PolimerizaçãoRESUMO
Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA-mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42-Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4-MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.