RESUMO
Molecular interactions are key to all cellular processes, and particularly interesting to investigate in the context of gene regulation. Protein-protein interactions are challenging to examine in vivo as they are dynamic, and require spatially and temporally resolved studies to interrogate them. Foerster Resonance Energy Transfer (FRET) is a highly sensitive imaging method, which can interrogate molecular interactions. FRET can be detected by Fluorescence Lifetime Imaging Microscopy (FLIM-FRET), which is more robust to concentration variations and photobleaching than intensity-based FRET but typically needs long acquisition times to achieve high photon counts. New variants of non-fitting lifetime-based FRET perform well in samples with lower signal and require less intensive instrument calibration and analysis, making these methods ideal for probing protein-protein interactions in more complex live 3D samples. Here we show that a non-fitting FLIM-FRET variant, based on the Average Arrival Time of photons per pixel (AAT- FRET), is a sensitive and simple way to detect and measure protein-protein interactions in live early stage zebrafish embryos.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Peixe-Zebra , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Fenômenos Biofísicos , FagocitoseRESUMO
DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP-AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.
Assuntos
Instabilidade Genômica/imunologia , Imunidade Inata/genética , Micronúcleos com Defeito Cromossômico , Nucleotidiltransferases/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromotripsia , Citoplasma/enzimologia , Citoplasma/genética , DNA/metabolismo , Dano ao DNA , Feminino , Instabilidade Genômica/genética , Humanos , Inflamação/enzimologia , Inflamação/genética , Lasers , Masculino , Camundongos , Microdissecção , Mitose , Membrana Nuclear/metabolismo , Nucleotidiltransferases/genética , Análise de Célula Única , TranscriptomaRESUMO
During apoptosis, pro-apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP Such caspase-independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS-STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS-STING signalling pathway. Using super-resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP In a temporal manner, we find that following MOMP, BAX/BAK-mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK-dependent MOMP Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase-independent cell death.
Assuntos
Apoptose , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Linhagem Celular Tumoral , DNA Mitocondrial/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , PermeabilidadeRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1005673.].
RESUMO
Human ß-defensin 3 (hBD3) is a cationic host defence peptide and is part of the innate immune response. HBD3 is present on a highly copy number variable block of six ß-defensin genes, and increased copy number is associated with the autoimmune disease psoriasis. It is not known how this increase influences disease development, but psoriasis is a T cell-mediated disease and activation of the innate immune system is required for the initial trigger that leads to the amplification stage. We investigated the effect of hBD3 on the response of primary macrophages to various TLR agonists. HBD3 exacerbated the production of type I Interferon-ß in response to the viral ligand mimic polyinosinic:polycytidylic acid (polyI:C) in both human and mouse primary cells, although production of the chemokine CXCL10 was suppressed. Compared to polyI:C alone, mice injected with both hBD3 peptide and polyI:C also showed an enhanced increase in Interferon-ß. Mice expressing a transgene encoding hBD3 had elevated basal levels of Interferon-ß, and challenge with polyI:C further increased this response. HBD3 peptide increased uptake of polyI:C by macrophages, however the cellular response and localisation of polyI:C in cells treated contemporaneously with hBD3 or cationic liposome differed. Immunohistochemistry showed that hBD3 and polyI:C do not co-localise, but in the presence of hBD3 less polyI:C localises to the early endosome. Using bone marrow derived macrophages from knockout mice we demonstrate that hBD3 suppresses the polyI:C-induced TLR3 response mediated by TICAM1 (TRIF), while exacerbating the cytoplasmic response through MDA5 (IFIH1) and MAVS (IPS1/CARDIF). Thus, hBD3, a highly copy number variable gene in human, influences cellular responses to the viral mimic polyI:C implying that copy number may have a significant phenotypic effect on the response to viral infection and development of autoimmunity in humans.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , RNA Helicases DEAD-box/genética , Psoríase/genética , Receptor 3 Toll-Like/genética , beta-Defensinas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Medula Óssea , Quimiocina CXCL10/genética , RNA Helicases DEAD-box/metabolismo , Humanos , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon , Lipossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Poli I-C/administração & dosagem , Psoríase/patologia , Receptor 3 Toll-Like/antagonistas & inibidores , beta-Defensinas/metabolismoRESUMO
We demonstrate a spectroscopic imaging based super-resolution approach by separating the overlapping diffraction spots into several detectors during a single scanning period and taking advantage of the size-dependent emission wavelength in nanoparticles. This approach has been tested using off-the-shelf quantum dots (Invitrogen Qdot) and in-house novel ultra-small (~3 nm) Ge QDs. Furthermore, we developed a method-specific Gaussian fitting and maximum likelihood estimation based on a Matlab algorithm for fast QD localisation. This methodology results in a three-fold improvement in the number of localised QDs compared to non-spectroscopic images. With the addition of advanced ultra-small Ge probes, the number can be improved even further, giving at least 1.5 times improvement when compared to Qdots. Using a standard scanning confocal microscope we achieved a data acquisition rate of 200 ms per image frame. This is an improvement on single molecule localisation super-resolution microscopy where repeated image capture limits the imaging speed, and the size of fluorescence probes limits the possible theoretical localisation resolution. We show that our spectral deconvolution approach has a potential to deliver data acquisition rates on the ms scale thus providing super-resolution in live systems.
Assuntos
Fluorescência , Pontos Quânticos , Espectrometria de Fluorescência , Funções VerossimilhançaRESUMO
Toll-like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro-inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium-transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca(2+) handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non-immune cells--including cardiomyocytes--using the same ligand-receptor system.
Assuntos
Trifosfato de Adenosina/biossíntese , Fibroblastos/fisiologia , Miócitos Cardíacos/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Receptor Toll-Like 9/fisiologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Retículo Endoplasmático/metabolismo , Camundongos , Mitocôndrias/metabolismo , Ligação Proteica , Estresse FisiológicoRESUMO
The resolution limit of conventional light microscopy has proven to be limiting for many biological structures such as viruses including Human immunodeficiency virus (HIV). Individual HIV virions are impossible to study using confocal microscopy as they are well below the 200 nm resolution limit of conventional light microscopes. Structured illumination microscopy (SIM) allows a twofold enhancement in image resolution compared to standard widefield illumination and so provides an excellent tool for study of HIV. Viral capsids (CAs) vary between 110 and 146 nm so this study challenges the performance of SIM microscopes. SIM microscopy was first developed in 2000, commercialised in 2007 and rapidly developed. Here we present the changes in capabilities of the SIM microscopes for study of HIV localisation as the instrumentation for structured illumination microscopy has evolved over the past 8 years.
Assuntos
HIV/ultraestrutura , Aumento da Imagem , Microscopia/métodos , Células HeLa , Humanos , Microscopia/instrumentação , Sensibilidade e EspecificidadeRESUMO
3-Phosphoinositide-dependent protein kinase-1 (PDK1) and phospholipase C (PLC)γ1 are two key enzymes in signal transduction that control several intracellular processes. Despite the fact that PLCγ1 has been investigated for several years, the mechanisms of activation of this enzyme are still not completely clear. Similarly, although PDK1 has been mostly investigated for its role in activation of Akt, a crucial enzyme in regulation of several cellular processes, it has become evident recently that the role of PDK1 in physiological and pathological conditions is not limited to Akt activation. Here we demonstrate that PDK1 regulates PLCγ1 activation in a mechanism involving association of the two enzymes and modulation of PLCγ1 tyrosine phosphorylation. We further show that this novel PDK1-PLCγ1 pathway is important for cancer cell invasion. The identification of a PDK1-PLCγ1 pathway reveals the existence of a previously undetected link between two of the most important enzymes in signal transduction. This is likely to have profound consequences for our understanding of several cellular functions that are dependent on phosphoinositides and controlled by PDK1 and PLCγ1.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fosfolipase C gama/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Cálcio/metabolismo , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/metabolismo , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Indazóis/farmacologia , Invasividade Neoplásica/genética , Fosfolipase C gama/genética , Fosforilação , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
BACKGROUND INFORMATION: Cell-cell adhesion and contraction play an essential role in the maintenance of geometric shape and polarisation of epithelial cells. However, the molecular regulation of contraction during cell elongation leading to epithelial polarisation and acquisition of geometric cell shape is not clear. RESULTS: Upon induction of cell-cell adhesion, we find that human keratinocytes acquire specific geometric shapes favouring hexagons, by re-modelling junction length/orientation and thus neighbour allocation. Acquisition of geometric shape correlates temporally with epithelial polarisation, as shown by an increase in lateral height. ROCK1 and ROCK2 are important regulators of myosin II contraction, but their specific role in epithelial cell shape has not been addressed. Depletion of ROCK proteins interferes with the correct proportion of hexagonal cell shapes and full elongation of lateral domain. Interestingly, ROCK proteins are not essential for maintenance of circumferential thin bundles, the main contractile epithelial F-actin pool. Instead, ROCK1 or ROCK2 regulates thin bundle contraction and positioning along the lateral domain, an important event for the stabilisation of the elongating lateral domain. Mechanistically, E-cadherin clustering specifically leads to ROCK1/ROCK2-dependent inactivation of myosin phosphatase and phosphorylation of myosin regulatory light chain. These events correlate temporally with the increase in lateral height and thin bundle compaction towards junctions. CONCLUSION: We conclude that ROCK proteins are necessary for acquisition of elongated and geometric cell shape, two key events for epithelial differentiation.
Assuntos
Diferenciação Celular , Células Epiteliais , Quinases Associadas a rho/metabolismo , Actinas/metabolismo , Caderinas/metabolismo , Adesão Celular/fisiologia , Polaridade Celular/fisiologia , Forma Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Morfogênese , Cadeias Leves de Miosina/metabolismo , Miosina Tipo II/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismoRESUMO
Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes, play key roles in fundamental cellular processes, including the sensing and transduction of mechanical cues. Here, we investigate systems-level changes in the integrin adhesome in patient-derived cutaneous squamous cell carcinoma cells and identify the actin regulatory protein Mena as a key node in the adhesion complex network. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We uncover an unexpected role for Mena at the nuclear membrane, where it controls nuclear architecture, chromatin repositioning and gene expression. Our findings identify an adhesion protein that regulates gene transcription via direct signalling across the nuclear envelope.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Cutâneas , Humanos , Actinas/genética , Actinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Núcleo Celular/metabolismo , Expressão Gênica , Integrinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Membrana Nuclear/metabolismo , Lâmina Nuclear/metabolismo , Neoplasias Cutâneas/metabolismoRESUMO
Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.
Assuntos
Diferenciação Celular/genética , RNA Helicases DEAD-box/metabolismo , Melanócitos/citologia , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Mutação , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatases/genética , Células-Tronco/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Inappropriate stimulation or defective negative regulation of the type I interferon response can lead to autoinflammation. In genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome, we identified biallelic mutations in LSM11 and RNU7-1, which encode components of the replication-dependent histone pre-mRNA-processing complex. Mutations were associated with the misprocessing of canonical histone transcripts and a disturbance of linker histone stoichiometry. Additionally, we observed an altered distribution of nuclear cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and enhanced interferon signaling mediated by the cGAS-stimulator of interferon genes (STING) pathway in patient-derived fibroblasts. Finally, we established that chromatin without linker histone stimulates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) production in vitro more efficiently. We conclude that nuclear histones, as key constituents of chromatin, are essential in suppressing the immunogenicity of self-DNA.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Interferon Tipo I/biossíntese , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U7/genética , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Linhagem Celular , DNA/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Células HCT116 , Células HEK293 , Doenças Hereditárias Autoinflamatórias/genética , Doenças Hereditárias Autoinflamatórias/imunologia , Humanos , Proteínas de Membrana/metabolismo , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Nucleotídeos Cíclicos/biossíntese , Nucleotidiltransferases/metabolismoRESUMO
Super-resolution microscopy methods enable resolution of biological molecules in their cellular or tissue context at the nanoscale. Different methods have their strengths and weaknesses. Here we present a method that enables correlative confocal, structured illumination microscopy (SIM) and single-molecule localization microscopy (SMLM) imaging of structures involved in formation of invadopodia on the same sample. This enables up to four colors to be visualized in three dimensions at a resolution of between 120 and 10 nm for SIM and SMLM, respectively.
Assuntos
Carcinoma de Células Escamosas/patologia , Imageamento Tridimensional/métodos , Iluminação/instrumentação , Microscopia de Fluorescência/métodos , Neoplasias Bucais/patologia , Podossomos/patologia , Imagem Individual de Molécula/métodos , Humanos , Invasividade Neoplásica , Células Tumorais CultivadasRESUMO
The question of whether the distinct isoforms of the family of enzymes phosphoinositide 3-kinases (PI3Ks) play redundant roles within a cell or whether they control distinct cellular processes or distinct steps within the same cellular process has gained considerable importance in the recent years due to the development of inhibitors able to selectively target individual isoforms. It is important to understand whether inhibition of one PI3K can result in compensatory effect from other isoform(s) and therefore whether strategies aimed at simultaneously blocking more than one PI3K may be needed. In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma. Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration. The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2ß and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival. Data further indicate that PI3K-C2ß and p110γ control distinct steps involved in cell migration supporting the hypothesis that different PI3Ks regulate distinct cellular processes.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Classe II de Fosfatidilinositol 3-Quinases , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Isoenzimas/metabolismo , Lisofosfolipídeos/metabolismo , Morfogênese/efeitos dos fármacos , Morfogênese/fisiologia , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.
Assuntos
Microscopia Confocal/métodos , Microscopia/métodos , Algoritmos , Núcleo Celular/metabolismo , Microscopia de Fluorescência/métodosRESUMO
The epidermis and its appendages, the hair follicle and sebaceous gland, have the capacity to constantly regenerate throughout adult life. Postnatal hair follicles undergo a cyclic mode of tissue homeostasis, defined by periods of growth, degeneration, and rest. A multipotent population of stem cells residing within the hair follicle bulge not only generates the hair lineages during each hair cycle, but also transiently contributes to the repair of epidermis following wounding. In this chapter, we provide methods for identifying epidermal stem cells and investigating their proliferative and apoptotic characteristics. We introduce whole-mount and flow cytometry techniques, which complement each other by permitting visualization of the epidermal stem cell compartment in situ and assessment of the phenotype of purified cells. These techniques can easily be adapted to characterize novel putative epidermal stem or progenitor cell populations. By applying whole-mount and flow cytometry techniques to characterize normal and genetically modified mice with skin defects, we expect to learn more about the factors that regulate stem cell self-renewal and differentiation.
Assuntos
Células Epidérmicas , Citometria de Fluxo/métodos , Imagem Molecular/métodos , Fenótipo , Células-Tronco/citologia , Animais , Anexina A5/metabolismo , Apoptose , Ciclo Celular , Imunofluorescência , Camundongos , Microscopia Confocal , Coloração e Rotulagem , Células-Tronco/metabolismoRESUMO
Rho GTPases regulate multiple cellular responses, including cell motility and cell cycle progression. The Rho isoform RhoB represses transformation and affects endosomal trafficking, but its effects on cell adhesion and migration have not been investigated in detail. Here we show that RhoB-null macrophages are more rounded than wild-type macrophages on fibronectin and uncoated glass, and have reduced adhesion to ICAM-1 and glass but not fibronectin. This correlated with lower cell surface expression of beta2 and beta3 integrins but not beta1 integrin. RhoB-null cells migrated faster than Wt cells on fibronectin, consistent with their smaller spread area, but slower than Wt cells on glass, reflecting their reduced adhesion. C3 transferase, which inhibits RhoA, RhoB and RhoC, induced cell spreading but this effect was reduced in RhoB-null cells. However, RhoB is not required for assembly of podosomes, which are integrin-based adhesion sites, whereas C3 transferase induced a decrease in podosomes and defects in tail retraction. Since macrophages do not express RhoC, these effects of C3 transferase are due to inhibition of RhoA rather than RhoB. Our results suggest that RhoB affects cell shape and migration by regulating surface integrin levels.