RESUMO
Endothelial cells respond to mechanical forces exerted by blood flow. Endothelial cell-cell junctions and the sites of endothelial adhesion to the matrix sense and transmit mechanical forces to the cellular cytoskeleton. Here we show that the scaffold protein AmotL2 connects junctional VE-cadherin and actin filaments to the nuclear lamina. AmotL2 is essential for the formation of radial actin filaments and the alignment of endothelial cells, and, in its absence, nuclear integrity and positioning are altered. Molecular analysis demonstrated that VE-cadherin binds to AmotL2 and actin, resulting in a cascade that transmits extracellular mechanical signals to the nuclear membrane. Furthermore, the endothelial deficit of AmotL2 in mice fed normal diet provoked a pro-inflammatory response and abdominal aortic aneurysms (AAAs). Transcriptome analysis of human AAA samples revealed a negative correlation between AmotL2 and inflammation of the aortic intima. These findings offer insight into the link between junctional mechanotransduction and vascular disease.
Assuntos
Antígenos CD , Aneurisma da Aorta Abdominal , Caderinas , Mecanotransdução Celular , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Animais , Humanos , Caderinas/metabolismo , Caderinas/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Angiomotinas , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Aortite/patologia , Aortite/metabolismo , Masculino , Citoesqueleto de Actina/metabolismo , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Actinas/metabolismo , Actinas/genéticaRESUMO
BACKGROUND: The spinal cord is composed of a large number of cells that interact to allow the organism to function. To perform detail studies of cellular processes involved in spinal cord injury (SCI), one must use repeatable and specific methods to target and injure restricted areas of the spinal cord. NEW METHOD: We propose a robust method to induce SCI in zebrafish by laser light. With a 2-photon microscope equipped with a femtosecond near-infrared pump laser, we explored the effects of laser beam exposure time, area, and intensity to induce precise and repeatable SCI with minimized collateral damage to neighboring cells. RESULTS: Through behavioral studies in zebrafish larvae, we assessed the functional outcome of intensive laser light directed at the spinal cord. Our experiments revealed that a laser pulse with wavelength 800 nm, duration 2.6 ms, and light intensity 390 mW was sufficient to induce controlled cell death in a single cell or a spinal cord segment. Collateral damage was observed if cells were exposed to laser pulses exceeding 470 mW. With these settings, we could induce precise and repeatable SCI in zebrafish larvae, resulting in loss of motor and sensory function. COMPARISON WITH EXISTING METHOD(S): Our method offers a simple and more controlled setting to induce SCI in zebrafish. We describe how the near-infrared femtosecond laser should be adjusted for achieving optimal results with minimal collateral damage. CONCLUSIONS: We present a precise and robust method for inducing SCI in zebrafish with single-cell resolution using femtosecond near-infrared laser pulses.
Assuntos
Modelos Animais de Doenças , Lasers , Procedimentos Neurocirúrgicos/instrumentação , Procedimentos Neurocirúrgicos/métodos , Traumatismos da Medula Espinal/fisiopatologia , Animais , Larva , Locomoção , Microscopia Confocal/métodos , Microcirurgia/instrumentação , Microcirurgia/métodos , Traumatismos da Medula Espinal/patologia , Peixe-ZebraRESUMO
The assembly of individual epithelial or endothelial cells into a tight cellular sheet requires stringent control of cell packing and organization. These processes are dependent on the establishment and further integration of cellular junctions, the cytoskeleton and the formation of apical-basal polarity. However, little is known how these subcellular events are coordinated. The (Angiomotin) Amot protein family consists of scaffold proteins that interact with junctional cadherins, polarity proteins and the cytoskeleton. In this report, we have studied how these protein complexes integrate to control cellular shapes consistent with organ function. Using gene-inactivating studies in zebrafish and cell culture systems in vitro, we show that Par3 to be essential for localization of AmotL2 to cellular junctions to associate with VE/E-cadherin and subsequently the organization of radial actin filaments. Our data provide mechanistic insight in how critical processes such as aortic lumen expansion as well as epithelial packing into hexagonal shapes are controlled.
Assuntos
Junções Aderentes/metabolismo , Proteínas de Transporte/genética , Polaridade Celular/genética , Forma Celular/genética , Proteínas de Membrana/genética , Proteínas de Peixe-Zebra/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Angiomotinas , Animais , Animais Geneticamente Modificados , Caderinas/genética , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Membrana/metabolismo , Microscopia Confocal , Interferência de RNA , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Epithelial cells connect via cell-cell junctions to form sheets of cells with separate cellular compartments. These cellular connections are essential for the generation of cellular forms and shapes consistent with organ function. Tissue modulation is dependent on the fine-tuning of mechanical forces that are transmitted in part through the actin connection to E-cadherin as well as other components in the adherens junctions. In this report we show that p100 amotL2 forms a complex with E-cadherin that associates with radial actin filaments connecting cells over multiple layers. Genetic inactivation or depletion of amotL2 in epithelial cells in vitro or zebrafish and mouse in vivo, resulted in the loss of contractile actin filaments and perturbed epithelial packing geometry. We further showed that AMOTL2 mRNA and protein was expressed in the trophectoderm of human and mouse blastocysts. Genetic inactivation of amotL2 did not affect cellular differentiation but blocked hatching of the blastocysts from the zona pellucida. These results were mimicked by treatment with the myosin II inhibitor blebbistatin. We propose that the tension generated by the E-cadherin/AmotL2/actin filaments plays a crucial role in developmental processes such as epithelial geometrical packing as well as generation of forces required for blastocyst hatching.
Assuntos
Citoesqueleto de Actina/metabolismo , Blastocisto/metabolismo , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Angiomotinas , Animais , Blastocisto/citologia , Proteínas de Transporte/genética , Linhagem Celular , Células Epiteliais/citologia , Epitélio/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Junções Intercelulares/metabolismo , Camundongos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Pele/citologia , Pele/metabolismo , Estresse Mecânico , Peixe-ZebraRESUMO
The assembly of individual endothelial cells into multicellular tubes is a complex morphogenetic event in vascular development. Extracellular matrix cues and cell-cell junctional communication are fundamental to tube formation. Together they determine the shape of endothelial cells and the tubular structures that they ultimately form. Little is known regarding how mechanical signals are transmitted between cells to control cell shape changes during morphogenesis. Here we provide evidence that the scaffold protein amotL2 is needed for aortic vessel lumen expansion. Using gene inactivation strategies in zebrafish, mouse and endothelial cell culture systems, we show that amotL2 associates to the VE-cadherin adhesion complex where it couples adherens junctions to contractile actin fibres. Inactivation of amotL2 dissociates VE-cadherin from cytoskeletal tensile forces that affect endothelial cell shape. We propose that the VE-cadherin/amotL2 complex is responsible for transmitting mechanical force between endothelial cells for the coordination of cellular morphogenesis consistent with aortic lumen expansion and function.
Assuntos
Antígenos CD/metabolismo , Aorta/crescimento & desenvolvimento , Caderinas/metabolismo , Proteínas Contráteis/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Citoesqueleto de Actina/metabolismo , Junções Aderentes/metabolismo , Angiomotinas , Animais , Aorta/citologia , Comunicação Celular , Forma Celular , Células Endoteliais/citologia , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Morfolinos/genética , Interferência de RNA , RNA Interferente Pequeno , Peixe-ZebraRESUMO
The establishment and maintenance of apical-basal cell polarity is essential for the functionality of glandular epithelia. Cell polarity is often lost in advanced tumours correlating with acquisition of invasive and malignant properties. Despite extensive knowledge regarding the formation and maintenance of polarity, the mechanisms that deregulate polarity in metastasizing cells remain to be fully characterized. Here we show that AmotL2 expression correlates with loss of tissue architecture in tumours from human breast and colon cancer patients. We further show that hypoxic stress results in activation of c-Fos-dependent expression of AmotL2 leading to loss of polarity. c-Fos/hypoxia-induced p60 AmotL2 interacts with the Crb3 and Par3 polarity complexes retaining them in large vesicles and preventing them from reaching the apical membrane. The resulting loss of polarity potentiates the response to invasive cues in vitro and in vivo in mice. These data provide a molecular mechanism how hypoxic stress deregulates cell polarity during tumour progression.
Assuntos
Neoplasias da Mama/genética , Proteínas de Transporte/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Hipóxia/genética , Proteínas Adaptadoras de Transdução de Sinal , Angiomotinas , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Células CACO-2 , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/cirurgia , Feminino , Células HeLa , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Linfonodos/cirurgia , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Glândulas Mamárias Humanas/cirurgia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Invasividade Neoplásica , Estadiamento de Neoplasias , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais , Vesículas Transportadoras/metabolismoRESUMO
One of the most critical factors in gene expression studies using quantitative real-time PCR is the choice of reference gene. Many of the commonly used reference genes have been shown to vary during a number of biological processes as well as between tissues. It is therefore important to always verify the stability of the gene of choice for all new tissues and experimental conditions. Here, we used two publicly available computer software packages (GeNorm and NormFinder) to investigate the stability of eight potential reference genes in formalin-fixed paraffin-embedded (FFPE) samples from normal oral tissue of different origin as well as from oral squamous cell carcinomas. Both programs found the tubulin α-6 chain (TUBA6) and ribosomal protein S13 (RPS13) to have the most stable expression between malignant and non-malignant tissue. NormFinder also found TUBA6 to be the most stable gene when samples were grouped according to tissue origin. FFPE samples constitute a large research resource, which considerably increases the number of samples available for analysis, leading to more reliable conclusions. Verification of a proper reference gene in oral FFPE tissue is therefore of great importance for future studies.